Physiol Rev 94: 419 – 459, 2014
doi:10.1152/physrev.00039.2011
STRUCTURE AND FUNCTION OF TMEM16
PROTEINS (ANOCTAMINS)
Nicoletta Pedemonte and Luis J. V. Galietta
U.O.C. Genetica Medica, Istituto Giannina Gaslini, 16147 Genova, Italy
Pedemonte N, Galietta LJV. Structure and Function of TMEM16 Proteins (Anoctamins).
Physiol Rev 94: 419 – 459, 2014; doi:10.1152/physrev.00039.2011.—TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion
transport, phospholipid scrambling, and regulation of other membrane proteins. The
first two members of the family, TMEM16A (anoctamin-1, ANO1) and TMEM16B
(anoctamin-2, ANO2), function as Ca2⫹-activated Cl⫺ channels (CaCCs), a type of ion channel that
plays important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and control of neuronal excitability. Genetic ablation of
TMEM16A in mice causes impairment of epithelial Cl⫺ secretion, tracheal abnormalities, and block
of gastrointestinal peristalsis. TMEM16A is directly regulated by cytosolic Ca2⫹ as well as indirectly
by its interaction with calmodulin. Other members of the anoctamin family, such as TMEM16C,
TMEM16D, TMEM16F, TMEM16G, and TMEM16J, may work as phospholipid scramblases
and/or ion channels. In particular, TMEM16F (ANO6) is a major contributor to the process of
phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane.
Intriguingly, TMEM16F is also associated with the appearance of anion/cation channels activated
by very high Ca2⫹ concentrations. Furthermore, a TMEM16 protein expressed in Aspergillus
fumigatus displays both ion channel and lipid scramblase activity. This finding suggests that dual
function is an ancestral characteristic of TMEM16 proteins and that some members, such as
TMEM16A and TMEM16B, have evolved to a pure channel function. Mutations in anoctamin genes
(ANO3, ANO5, ANO6, and ANO10) cause various genetic diseases. These diseases suggest the
involvement of anoctamins in a variety of cell functions whose link with ion transport and/or lipid
scrambling needs to be clarified.
L
I.
II.
III.
IV.
V.
VI.
VII.
TMEM16A AND TMEM16B AS...
REGULATORY MECHANISMS OF...
PHYSIOLOGICAL ROLE OF TMEM16A...
TMEM16F AND OTHER...
LESSONS FROM ANIMAL MODELS...
DRUGGABILITY OF CACCs
CONCLUSIONS
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426
430
438
444
448
450
I. TMEM16A AND TMEM16B AS
Ca2ⴙ-ACTIVATED Clⴚ CHANNELS
A. Identification
Of the more than 20,000 genes contained in the human
genome, a significant fraction codes for membrane proteins
with unknown function (4, 57). In 2008, three independent
research teams found that one of these orphan proteins,
TMEM16A, is a component of the Ca2⫹-activated Cl⫺
channel, CaCC (29, 195, 256). This type of Cl⫺ channel
had been known at the functional level for more than 25
years, but there was no clear understanding of its molecular
identity (48, 80). Typical features of this channel are as
follows: 1) outward rectification of the steady-state current-
voltage relationship due to activation and deactivation at
positive and negative membrane potentials, respectively;
and 2) activation by cytosolic Ca2⫹ with an apparent affinity that is increased by a change of the membrane potential
to positive values. CaCC activity may be detected with a
series of techniques including patch-clamp, short-circuit
current recordings, and fluorescent measurements of I⫺/Cl⫺
fluxes (FIGURE 1). As discussed later, TMEM16 proteins
can also be studied after purification and reconstitution in
artificial membranes (135, 219).
Many studies previously proposed a panel of possible molecular candidates for CaCCs, including CLCA, ClC-3, and
bestrophins (48, 80, 116). However, the identification of
these proteins as components of CaCCs was not totally
convincing.
Yang et al. (256) found TMEM16A as a CaCC by searching
among unknown proteins with multiple transmembrane
segments. Expression of TMEM16A in heterologous expression systems generated Cl⫺ currents activated by receptor-mediated intracellular Ca2⫹ mobilization. Coexpression of TMEM16A with endothelin, angiotensin, muscarinic, histamine, or purinergic receptors, followed by acute
0031-9333/14 Copyright © 2014 the American Physiological Society
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
A
Ca2+
1 nA
Cl-
200 ms
Ca2+
Cl-
Clwhole-cell
inside-out
B
Ca2+ agonists
ionomycin
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
10 µA
5 min
+/- amphotericin B
C
I-
fluorescence
iodide
I-
Ca2+ agonists
HS-YFP
10 s
FIGURE 1. Methods to study Ca2⫹-activated Cl⫺ channels (CaCCs). A: whole cell and inside-out configurations of the patch-clamp technique. The tip of a glass micropipette is sealed onto the plasma membrane. In the
whole cell configuration, the intracellular Ca2⫹ concentration is controlled by equilibration with the pipette
solution. Voltage steps elicit macroscopic CaCC currents (representative traces on the left). CaCC show
increasing activity as the membrane potential is made more positive. In the inside-out configuration, a small
membrane patch is excised and the solution facing the intracellular side is controlled by perfusion.
B: transepithelial Cl⫺ transport measured as short-circuit current. An epithelial layer with high electrical
resistance is mounted in a Ussing chamber-like system. The transepithelial potential is clamped to zero and the
resulting current is measured. Opening of CaCCs on the apical membrane, due to intracellular Ca2⫹ elevation,
results in Cl⫺ secretion, measured as a positive current. C: I⫺ influx detection with the halide-sensitive yellow
fluorescent protein (HS-YFP). Cells expressing HS-YFP and CaCCs are challenged with an I⫺-rich solution plus
a Ca2⫹ agonist. The resulting I⫺ influx through CaCCs causes a rapid quenching of HS-YFP fluorescence. The
rate of fluorescence quenching is proportional to CaCC activity.
stimulation with the corresponding receptor agonist, resulted in appearance of large CaCC currents. TMEM16Adependent currents could also be activated by direct Ca2⫹
application to the cytosolic side of excised membrane
patches. The properties of such currents resembled very
closely those of native CaCCs in terms of Ca2⫹ sensitivity,
voltage dependence, and response to pharmacological inhibitors. In particular, TMEM16A channels showed a typical feature of native CaCCs: the apparent Ca2⫹ affinity was
increased by membrane depolarization (EC50 of 2.6 and 0.4
␮M at ⫺60 and ⫹60 mV, respectively). The single-channel
420
conductance of TMEM16A channels (8.3 pS) appeared
larger than that reported for native CaCCs (1.8 –3.5 pS)
(174). However, in a more recent study, the single-channel
conductance of TMEM16A, calculated by noise analysis,
was found to be 3.5 pS (2). With regard to expression in
native tissues, TMEM16A was found particularly expressed in pancreatic acinar cells, retinal cell layers, proximal renal tubules, sensory neurons of dorsal root ganglia,
submandibular glands, and Leydig cells (256). Knockdown
in vivo of TMEM16A by injected siRNA resulted in reduced fluid secretion by salivary glands.
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
To identify the CaCC protein, Schroeder et al. (195) decided to adopt an expression cloning approach after realizing that the oocytes of Axolotl salamander (Ambystoma
mexicanum), unlike those of Xenopus laevis frogs, do not
have endogenous activity of CaCCs. Interestingly, this difference is due to an important physiological reason. CaCC
currents are used in Xenopus oocytes to generate the fertilization potential that prevents polyspermia. In contrast,
CaCC currents are not needed in the oocytes of Axolotl
salamanders, which are physiologically polyspermic. Injection of Axolotl oocytes with mRNA extracted from Xenopus oocytes generated robust CaCC currents. To identify
the cDNA underlying CaCC activity, Schroeder et al. (195)
generated a cDNA library. Axolotl oocytes were then utilized to express pools of cDNA clones (195). After cycles of
pool fractionation and functional evaluation, a single active
cDNA clone containing the coding sequence of TMEM16A
was identified. Expression of TMEM16A generated Cl⫺
currents with voltage and Ca2⫹ dependence resembling
those of Xenopus CaCC (195). The TMEM16A-dependent
channels were sensitive to classical CaCC blockers such as
DIDS, niflumic acid, and NPPB. Similar currents were also
found when TMEM16A was expressed in HEK-293, a cell
line devoid of endogenous CaCC activity. Interestingly, the
relative permeability of TMEM16A channels to various anions was not fixed but changed dynamically with the status
of channel activation. Schroeder et al. (195) also investigated the activity of TMEM1B protein, a close paralog of
TMEM16A. TMEM16B was also endowed with CaCC activity (195).
Caputo et al. (29) identified TMEM16A using a third type
of approach. These authors and others had previously
found that human bronchial epithelial cells respond to prolonged (24 h) treatment with interleukin-4 (IL-4) or with
interleukin-13 (IL-13) by increasing the activity of CaCCs
(40, 67), an effect that appeared to be mediated by upregulation of the corresponding gene(s). Therefore, a functional
genomics strategy was adopted to identify the molecular
identity of CaCCs. Gene expression microarrays were used
to generate a short list of genes upregulated by IL-4 and
coding for membrane proteins with unknown function
(29). These candidates were then screened using functional
assays combined with siRNA-based gene silencing.
TMEM16A was the only gene whose downregulation resulted in a significant CaCC inhibition measured by a series
of functional assays (iodide influx, short-circuit current,
patch clamp) in different cell systems, including bronchial
CFBE41o- cells, pancreatic CFPAC-1 cells, and primary
cultures of human bronchial epithelial cells (29). Importantly, the effect of TMEM16A knockdown on CaCC activity was not affected by the type of activation (purinergic
receptor stimulation versus Ca2⫹ ionophore). Furthermore,
TMEM16A knockdown did not alter the increase in Ca2⫹
concentration caused by Ca2⫹ mobilizing agents. These re-
sults indicated that TMEM16A is not an indirect regulator
of CaCCs (29).
TMEM16A belongs to a protein family whose members (10
proteins, labeled with letters from A to K, excluding I) have
a length between 800 and 1,000 amino acid residues (106).
TMEM16 proteins have a similar putative structure consisting of eight transmembrane domains and the NH2 and
COOH termini protruding into the cytosol (FIGURE 2). This
structure is not only based on computer programs predicting number and localization of transmembrane domains
but is also supported by experiments done on TMEM16G,
also known as NGEP (41). Insertion of epitope tags at different positions of TMEM16G sequence was used to define
the protein topology. This study also proposed that the
region between the fifth and sixth transmembrane domain
partially crosses the membrane similarly to the reentrant
loop that forms the pore of other types of ion channels.
However, as discussed later and shown in FIGURE 2A, this
topology has been recently questioned (259).
Alignment of amino acid sequence of TMEM16 proteins
reveals a high identity in the predicted transmembrane segments. In particular, a relatively high level of sequence conservation is present within a region in the COOH-terminal
half that was labeled as domain of unknown function 590
(DUF590). This region is now called Anoctamin domain
(http://pfam.sanger.ac.uk/family/Anoctamin).
“Blasting” of the protein sequences available in databases
shows that anoctamin family is relatively ancient, with several homologs detected in animal species outside the subphylum Vertebrata. For example, there are proteins in insects (Drosophila genus, Aedes aegypti, Tribolium castaneum, Bombus terrestris) with more than 40% amino acid
identity to human TMEM16A. A similar degree of identity
is also found in proteins from sea anemones (Nematostella
vectensis), in nematodes (Brugia malayi), and in flatworms
(Schistosoma mansoni). In Caenorhabditis elegans, two
anoctamins, ANOH-1 and ANOH-2, were recently identified (240). ANOH-1 appears to be expressed in sensory
neurons with chemoreceptor function and in intestinal cells.
Inhibition of ANOH-1 function by RNA interference reduced the ability of worms to avoid high osmolarity but not
the response to other stimuli, either mechanical or chemical.
ANOH-2 was expressed in mechanoreceptors, motor neurons, and spermatheca, but its silencing did not generate a
clear phenotype (240). Interestingly, there is also a distant
TMEM16 homolog in yeast, Ist2p, that is involved in tolerance to high extracellular salt concentration (109). Recently, Ist2 was found as an important tethering protein
between the endoplasmic reticulum (ER) and the plasma
membrane (136, 247). Ist2 is localized in the ER, but comes
into contact with the plasma membrane by means of a
highly basic COOH terminus. Deletion of Ist2 in yeast de-
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
lays growth, alters intracellular pH, changes ER organization, and triggers the unfolded protein response.
Analysis of amino acid sequence similarity within TMEM16
proteins allows the generation of a phylogenetic tree (150,
240). Given their high amino acid identity (62%),
TMEM16A and TMEM16B appear as closely related members belonging to the same subfamily. TMEM16C/D/J, and
TMEM16E/F may form two separate subgroups, probably
confined to chordates (240). TMEM16G/H/K are instead
distant paralogs of TMEM16A and as ancient as Eumetazoa (240).
A
824
Re-entrant loop model
824
Revised model (?)
672
614
1
2
3
4
5
614
700
6
7
8
1
2
3
4
5
6
7
8
700
570
570
672
B
Glu702 and Glu705
mediate direct Ca2+
binding
Calmodulin binding,
essential for activity
E
E
CBM1
RCBM
CBM2
Calmodulin binding
domains, regulate
bicarbonate permeability
Segment (b),
calmodulin binding
modulates channel
activity
Dimerization
domain
422
EEEEEAVK domain modulates
voltage-dependence and
Ca2+ sensitivity
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
As expected from the relatively high sequence identity
shared with TMEM16A, TMEM16B was also found associated with CaCC activity. This result was initially found by
Schroeder et al. (195) and subsequently confirmed by three
other teams (172, 208, 209). As discussed later, TMEM16B
channels have biophysical characteristics different from
those of TMEM16A. Because of the eight putative transmembrane segments and the anion channel function,
TMEM16 proteins were also named anoctamins (ANO1–
10). Although anion currents have been found to be associated with other anoctamins, it is not sure that all TMEM16
proteins have an ion channel function (77, 139, 194, 202,
223).
So far, there has been an increasing amount of evidence
supporting the conclusion that TMEM16A is a CaCCforming protein. First, in all cells devoid of endogenous
CaCC activity, such as HEK-293 or FRT cells, transfection
with TMEM16A is enough to generate Ca2⫹-activated Cl⫺
currents with biophysical properties (e.g., voltage dependence) similar to those of native CaCCs (29, 195, 256). This
may imply that TMEM16A protein alone is sufficient to
form a whole channel or that other subunits of CaCC are
already constitutively expressed in all cells. Second,
TMEM16A knockdown with siRNA causes CaCC inhibition (29, 50, 128, 137, 256). Inhibition is never complete,
but this partial effect may be explained by the suboptimal
efficacy of siRNA transfection methods. Also, site-specific
mutagenesis of highly conserved amino acid residues in the
TMEM16A sequence leads to specific changes in CaCC
properties, such as ion selectivity and voltage dependence.
For example, replacement of arginine and lysine residues
with glutamate in the putative pore-forming loop between
transmembrane segments 5 and 6 altered ion selectivity
(256). In addition, mutations of conserved residues in transmembrane segments altered channel gating kinetics (29).
Finally, in a very recent study, TMEM16A protein was
purified and reconstituted into proteoliposomes (219). Ion
transport activity was determined by measuring the efflux
of Cl⫺ from the liposomes. The presence of TMEM16A was
associated with a significant Cl⫺ transport controlled by
Ca2⫹ and transmembrane voltage in a way that resembled
that reported for TMEM16A in cells (219). For example,
the half effective concentration of Ca2⫹ at a given membrane potential was 210 nM, a value close to that measured
in cells. Furthermore, the TMEM16A-dependent Cl⫺ transport was sensitive to classical CaCC blockers (219). All
these experiments strongly indicate that TMEM16A is indeed a CaCC-forming protein but do not obviously rule out
that other proteins are involved in the composition and
regulation of the native channel.
B. Oligomeric Organization and Interactome
of TMEM16A
Two studies indicated that TMEM16A protein is structured
as a homodimer. In one study, TMEM16A protein was
tagged with two different fluorescent proteins, mCherry
and eGFP (201). Coexpression of the green and red
TMEM16A within the same cell demonstrated a significant
level of fluorescence resonance energy transfer (FRET),
which is indicative of a close physical interaction. Chemical
cross-linking and coimmunoprecipitation was also used to
demonstrate dimer formation. In the second study, blue
native polyacrylamide gel electrophoresis (BN-PAGE) was
applied to demonstrate the presence of high-molecularweight complexes of TMEM16A protein consistent with
homodimer formation (58). Interestingly, the dimers are
not abolished by reducing agents and not altered by intracellular Ca2⫹ concentration changes (58, 201). It is not
clear yet whether two TMEM16A subunits form a single- or
a double-pore channel.
In a more recent study, the formation of TMEM16A dimers
was found to involve a region of the protein localized at the
NH2 terminus (225). By analyzing a panel of truncation and
deletion mutants, the authors of this study identified a small
segment of 19 amino acids (position 161–179) essential for
dimerization (by homotypic interaction) and channel function (FIGURE 2B). The authors also introduced a mutation,
A169P, which was predicted to alter the ␣-helix structure
of the dimerization domain. The mutation abolished
dimerization, protein trafficking to the plasma membrane, and
channel activity (225). Interestingly, TMEM16A coimmunoprecipitated with TMEM16B but not with TMEM16F in heterologous expression systems. These results suggest that
TMEM16A and TMEM16B heterodimers may form in cells
coexpressing both genes. However, the real existence of
these hybrid channels in native cells and their biophysical
properties remain to be studied.
FIGURE 2. Structure-function of TMEM16A protein. A: topology of TMEM16A protein. The figure shows two different topologies proposed for
TMEM16A protein. In the first model (left), the region between transmembrane domains 5 and 6 constitutes a reentrant loop that faces the
extracellular environment and is important for the formation of the ion pore (256). In the second model, this region crosses completely the
membrane (forming the sixth transmembrane domain) and then generates a cytosolic loop important for Ca2⫹ binding (259). The red dots and
numbers show the sites where hemagglutinin (TAGs) were inserted to study the topology. [From Yu et al. (259).] B: mechanisms regulating
TMEM16A ion channel function. The figure shows the hypothetical structure of TMEM16A protein (based on the topology proposed in Ref. 259)
with the sites involved in the regulation of ion channel function. Interaction of calmodulin to segment (b), corresponding to the alternatively spliced
exon 6b, modulates channel activity (222). Additional interaction of calmodulin at two other sites (CBM1 and CBM2) increases the permeability
of the channel pore to bicarbonate (101). Another regulatory calmodulin binding site (RCBM) was also identified (237). In the third intracellular
loop, two glumatic acid residues (702 and 705) appear to be the major Ca2⫹ binding sites that mediate channel activation (259). In the first
intracellular loop, the sequence EEEEEAVK is important in the coupling of voltage and Ca2⫹-dependent gating mechanisms (250). A small region
in the NH2 terminus, the dimerization domain, is important for the homotypic interaction between TMEM16A proteins.
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
The interactome of TMEM16A has been recently investigated using a proteomics approach (171). HEK-293 cells
were stably transfected with TMEM16A labeled with a triple FLAG epitope. To stabilize the interaction between
TMEM16A and its possible partners, Perez-Cornejo et al.
(171) used a membrane permeable bifunctional crosslinker, DSP, which has a 12 Å spacer. After cell lysis, the
cross-linked macromolecular complexes were immunoprecipitated with an anti-FLAG antibody and analyzed by
mass spectrometry. Among the hundreds of proteins identified, 73 proteins appeared to be enriched more than threefold in cells transfected with TMEM16A. The most enriched proteins were ezrin, munc18c, radixin, and moesin.
Bioinformatic analysis of the results identified two major
protein networks for TMEM16A interactome: SNARE
proteins, which control vesicle trafficking, and the ezrinradixin-moesin (ERM) scaffolding complex, which links
plasma-membrane associated proteins with the cytoskeleton. The authors focused on the latter group of proteins.
The interaction with TMEM16A/ANO1 was confirmed
by coimmunoprecipitation in lysates of mouse salivary
glands and by bimolecular fluorescence complementation
in transfected cells (171). Furthermore, immunofluorescence experiments demonstrated colocalization of ANO1
with ERM proteins in the apical membrane of cells in the
acini and ducts of salivary glands. Interestingly, knockdown of moesin expression with shRNA caused inhibition
of TMEM16A currents by more than 50% without altering
TMEM16A protein localization in the plasma membrane.
This finding may indicate a role of ERM proteins in the
regulation of TMEM16A activity. In this respect, it is interesting to note that, in another study, modulators of actin
polymerization state such as cytochalasin D and phalloidin
inhibited TMEM16A activity (222). As discussed by PerezCornejo et al. (171), the mass spectrometry analysis did not
identify other integral membrane proteins besides ANO1,
nor known Ca2⫹-binding proteins such as calmodulin. This
may suggest that the native CaCC is not composed of other
subunits providing Ca2⫹ sensitivity or forming the channel
pore. However, as described in subsequent sections of this
review, calmodulin was actually found, with nonproteomics approaches, to contribute to TMEM16A regulation
(101, 222, 237). More information about the TMEM16A/
ANO1 interactome will certainly derive from future experiments using cross-linkers with different reactive groups
and spacer arms, and by using cells with endogenous expression of CaCCs.
Recently, the inositol 1,4,5-triphosphate receptor 1
(IP3R1) was discovered as another important interactor
of TMEM16A, at least in nociceptive sensory neurons (99).
This finding arose from the observation that TMEM16A
can be activated in small neurons of dorsal root ganglia by
Ca2⫹ signals dependent on G protein-coupled receptors but
not by those deriving from voltage-dependent Ca2⫹ channels. Indeed, Jin et al. (99) found that TMEM16A is acti-
424
vated by bradykinin receptor 2 and protease-activated receptor 2 through release of Ca2⫹ from intracellular stores.
In contrast, TMEM16A was insensitive to global intracellular Ca2⫹ elevation caused by membrane depolarization
and opening of Ca2⫹ channels. This particular behavior is
probably due to a localization of TMEM16A in regions of
the cell where the plasma membrane and the endoplasmic
reticulum are very close to each other. The close apposition
of the two membranes creates a compartmentalized microenvironment that isolates TMEM16A from Ca2⫹ influx.
As suggested by coimmunoprecipitation and proximity ligation assays, this arrangement may be favored by a direct
interaction between IP3R1 and two regions of TMEM16A:
the first intracellular loop and the COOH terminus (99).
Compartmentalized Ca2⫹ signaling activating TMEM16A
was also observed in other studies (15, 118). In particular,
bestrophin, by representing a pathway for counter ion
transport, was an important factor contributing to Ca2⫹
release from the intracellular store.
C. Alternative Splicing
Another level of complexity in TMEM16A, and possibly in
other anoctamins, arises from alternative splicing. There
are multiple isoforms of TMEM16A proteins generated by
inclusion/skipping of alternative exons and/or by the use of
an alternative promoter (29, 60, 144, 205). TMEM16A
was initially reported to have at least four alternative segments, which were simply labeled as a, b, c, and d. Inclusion/skipping of segments b (22 amino acids), c (4 amino
acids), and d (26 amino acids) is the result of alternative
splicing of exons 6b, 13, and 15, respectively (60). Instead,
skipping of segment a, which corresponds to the first 116
amino acids of TMEM16A protein, is dependent on the
activation of an alternative promoter. In this case, the sequence corresponding to the first 36 codons is replaced with
a sequence lacking an ATG (205). It was initially believed
that translation would then start at the next available ATG
at position 117, thus entirely skipping segment a (29). It has
been now found that noncanonical start codons, possibly
CTG, allow translation of a large part of segment a in the
absence of the first codon (205).
Expression of TMEM16A isoforms is tissue dependent.
While inclusion of microexon 13 appears to occur in most
transcripts, the splicing of exons 6b and 15 shows a variety
of situations. Interestingly, preferential inclusion of exon 6b
in some tissues seems associated with exclusion of exon 15,
and vice versa (60, 228). In a recent study, an altered expression of TMEM16A isoforms was found in the stomach
of diabetic patients affected by gastroparesis (144). These
individuals showed an increased abundance of transcripts
coding for TMEM16A proteins with shortened NH2 terminus (144).
More recently, another study reported additional exons undergoing alternative splicing in the heart (161). Some tran-
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
scripts were found to miss exon 10, which resulted in deletion of 45 amino acids that include the first putative transmembrane domain. Since this TMEM16A variant would
have an altered transmembrane topology, it is not expected
to be functional. Another variant, without exon 14, would
lack 24 amino acids in the first intracellular loop possibly
containing a phosphorylation site for protein kinase C
(161). The same intracellular loop could also be affected by
inclusion of the alternative exon 13b causing the insertion
of another 40 amino acids.
Expression of different isoforms of TMEM16A protein has
functional implications. Inclusion of exon 6b, resulting in a
protein with additional 22 amino acids (segment b) in the
region preceding the first transmembrane domain, was
found associated with Cl⫺ channels having a decreased sensitivity to cytosolic Ca2⫹ (60). This phenomenon was also
observed in another study (223). At the cytosolic Ca2⫹ concentration of 100 nM, the isoform (ac) is significantly active
in contrast to the isoform (abc) (60, 223). However, the
isoform (ac) is also more prone to inactivation at high Ca2⫹
concentrations (223). The molecular basis for the observed
influence of segment b is not clear. However, future experiments will need to consider that this domain is highly basic
(8 arginine/lysines out of 22 amino acid residues), it has
potential phosphorylation sites, and has a noncanonical
calmodulin-binding site. As discussed later, binding of calmodulin to this domain modulates channel activity (222).
Skipping of microexon 13, an event that appears to be rare
in human tissues (60), also produces channels with altered
characteristics. In a first study, deletion of the four amino
acids (Glu-Ala-Val-Lys, EAVK in the single letter code) corresponding to segment c resulted in channels with reduced
activation at positive membrane potentials (60). More recently, this TMEM16A variant was found to have significantly reduced Ca2⫹ sensitivity (250). Normally, the effect
of cytosolic Ca2⫹ on CaCC channels is a shift of the relationship between normalized conductance and membrane
potential towards more negative values. Xiao et al. (250)
found that an increase in the Ca2⫹ concentration from 1 to
2 ␮M dramatically shifted the voltage dependence of wildtype TMEM16A channels from ⫹64 to ⫺145 mV (250). In
contrast, the ⌬EAVK mutant showed a significant impairment of sensitivity to Ca2⫹: a concentration of 25 ␮M was
required to obtain an activity comparable to that recorded
at 1 ␮M for the wild-type protein (250). By measuring the
kinetics of activation and deactivation of the channel upon
fast Ca2⫹ concentration changes, Xiao et al. (250) concluded that ⌬EAVK increases the dissociation rate of Ca2⫹
from the channel (250). The differences between the two
studies may stem from different experimental conditions
and/or the use of different TMEM16A backbones, with and
without segment b (60, 250). Indeed, the binding of calmodulin at segment b (222) may influence the net response
of the channel to cytosolic Ca2⫹. Despite the different con-
clusions, these studies point to the EAVK segment, and to
the first intracellular loop in general, as an important domain for the regulation of TMEM16A channel activity.
Another isoform of TMEM16A stems from the combined
absence of segments a, b, c, and d (29). This isoform, defined as TMEM16A(0) and mostly expressed in testis (205),
has altered voltage dependence and ion selectivity (61). Recently, it has been found that skipping of segment a is not
complete (205). Actually, the absence of the first ATG does
not result in translation from the second ATG as previously
thought. The presence of non-canonical start codons allow
a large part of segment a to be included in TMEM16A(0)
(205).
So far, there have been no results on the role of other alternative regions in TMEM16A (161). However, it may be
speculated in general that the expression of different
TMEM16A isoforms, even within the same cell and with
the formation of heterooligomers, enhances tremendously
the complexity of CaCC at the molecular level. This variety
of CaCC channels may have different biophysical properties, regulatory mechanisms, and subcellular localization.
The information available so far suggests that alternative
splicing may be a common process within the anoctamin
family. In particular, alternative splicing of TMEM16B
transcripts generates multiple variants. TMEM16B transcripts missing exon 14 (formerly annotated as exon 13)
were found in the murine olfactory epithelium but not in the
retina (208).
Importantly, the four amino acids (ERSQ), that in
TMEM16B correspond to segment c of TMEM16A
(EAVK), prevent channel inactivation at high Ca2⫹ concentrations (237). Noninactivating TMEM16B channels containing this sequence were expressed in retina and pineal
gland. Inactivating channels, devoid of segment c, were instead expressed in the olfactory epithelium and in most
regions of the central nervous system (237).
Other variants of TMEM16B were generated by alternative
splicing of exon 4 and by the use of another starting exon
that resulted in a shortened NH2 terminus (175). Expression of TMEM16B proteins missing the region corresponding to exon 4 resulted in inactive channels. However, coexpression of proteins with and without exon 4 generated
functional channels but with altered properties, particularly
regarding the inactivation process and channel rundown
(175). Interestingly, exon 4 codes for a region that is homologous to that containing the dimerization domain identified in TMEM16A (225).
Inspection of sequences deposited in databases reveals that
multiple isoforms also exist for all TMEM16 proteins. In
TMEM16F, the inclusion of an alternative exon coding for
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21 amino acids within the NH2-terminal region increased
protein function (200).
D. Topology
Predictive computer programs propose for TMEM16A and
other anoctamins a structure consisting of eight transmembrane domains with both the NH2 and COOH termini exposed to the cytosolic environment (29, 195, 256). Most
algorithms predicting transmembrane domains agree in
identifying the position of transmembrane helices 1– 4 and
7– 8. Such regions have an amino acid composition that is
canonical for a protein segment entirely spanning the
plasma membrane. In contrast, the topology of the region
between transmembrane helix 4 and 7 appears more uncertain. It was proposed that a reentrant loop may exist between transmembrane segments 5 and 6 (FIGURE 2A). In this
region, the protein would not cross entirely the membrane
but would exit again on the extracellular side forming a
hairpin. This model was based on a topology study performed on TMEM16G (ANO-7) by inserting an epitope at
different regions of the protein and then looking at the
accessibility of the epitope from the extracellular or intracellular side of the membrane (41). The reentrant loop,
corresponding to a sequence of 103 amino acids, was proposed as a region constituting the channel pore of
TMEM16A and therefore important for ion selectivity
(256). Indeed, mutagenesis to glutamate in this region of
three highly conserved basic amino acids (R621, K645, and
K668) resulted in a significant alteration in ion permeability. In particular, the mutant R621E showed a dramatic loss
of selectivity because the relative permeability for Na⫹, PNa/
PCl, was 0.93 instead of the 0.03 value of the wild-type
protein (256). The orientation of this region was also investigated by exploiting three cysteines localized at positions
625, 630, and 635. A cysteine-modifying reagent, MTSET,
which is unable to cross the membrane, blocked the activity
of wild-type TMEM16A channels when applied from the
extracellular side. There was no block when MTSET was
applied to TMEM16A channels in which the three cysteines
were mutated to alanine. These results indicated that the
amino acids in the 620 – 635 segment are part of the channel
pore and are accessible to the aqueous solution on the extracellular side (256).
The reentrant loop model has been recently questioned in
another study (259). These authors considered the possibility that part of the putative reentrant loop actually crosses
totally the membrane forming the sixth transmembrane domain (FIGURE 2A). The authors introduced hemaglutinin
(HA) epitopes at different sites between residues 570 and
824. The epitope was accessible from the outside at positions 614 and 824 but not at positions 672 and 700. These
results indicated that the region 650 –706 is actually intracellular and not extracellular. As discussed in the subsequent section, this finding represented the basis for the dis-
426
covery of a novel Ca2⫹-binding site. Yu et al. (259) also
explored the issue of ion selectivity by studying the mutant
R621E. In contrast to previous results (256), they found no
change in ion selectivity for this mutant. They also adopted
a scanning mutagenesis approach by replacing each amino
acid between F620 and Q646 with cysteine and then testing
the resulting mutants with extracellular MTSET. Changes
in ion selectivity were not detected. The authors concluded
that amino acid residues beyond position 635 are not accessible from the extracellular side (FIGURE 2A).
In conclusion, determination of TMEM16A structure in the
region between transmembrane segments 4 and 7 appears
particularly difficult and requires further studies. However,
elucidation of this issue is essential to understand the function and regulation of the different anoctamins.
II. REGULATORY MECHANISMS
OF TMEM16A/B CHANNELS
A. Calcium/Calmodulin and Membrane
Potential
Native CaCCs in many cell types are activated by cytosolic
Ca2⫹ in the 100 – 600 nM range, although cases with lower
(micromolar) sensitivity have also been reported (7, 22, 71,
80, 111, 177). However, Ca2⫹ is not alone in activating
CaCCs since, in most cases, these channels are also influenced by membrane potential (6, 7, 22, 177). More precisely, membrane depolarization enhances the Ca2⫹ sensitivity of the channels (7, 120). This behavior may be explained by a Ca2⫹-binding site that is localized within the
transmembrane region of the channel and that is therefore
sensitive to transmembrane potential (7). Similar characteristics have also been reported for the Cl⫺ currents elicited by
TMEM16A and TMEM16B expression. Both proteins are
associated with voltage- and Ca2⫹-dependent Cl⫺ channels.
The half effective Ca2⫹ concentration required for
TMEM16A channels is 0.4 – 0.6 ␮M at positive membrane
potentials (60, 256). Such values increase significantly as
the membrane potential becomes more negative, although
the extent of change may differ among studies (60, 256). In
contrast, TMEM16B channels are less Ca2⫹ sensitive, with
the half effective Ca2⫹ concentration being around 1–3 ␮M
at positive membrane potentials (172, 208). Furthermore,
the kinetics of activation and deactivation of TMEM16B
channels is much faster than that of TMEM16A, with time
constants differing by more than 10-fold (29, 172, 208,
256). There are also significant differences regarding singlechannel conductance: 8 pS for TMEM16A (256) and nearly
1 pS for TMEM16B (172, 208). TMEM16A and
TMEM16B channels are also activated, with various potency and efficacy, by other divalent cations, such as Ba2⫹,
Sr2⫹, and Ni2⫹ (208, 250, 260). Zn2⫹ has instead an inhibitory effect (260).
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
The molecular mechanisms underlying the channel gating
by Ca2⫹ and voltage are unknown. It has been reported that
some native CaCCs and TMEM16A/B channels can be directly activated by a Ca2⫹ increase on the cytosolic side of
isolated membrane patches (250, 256). These findings may
be interpreted as evidence that soluble cytosolic components are dispensable for channel activation. However, a
slow and irreversible rundown of channel activity has also
been reported in another study (222). Therefore, it is probable that unknown proteins with a loose binding to the
plasma membrane control TMEM16A and TMEM16B activity. For example, a significant fraction of TMEM16A
activity in excised patches was restored by adding ATP and
calmodulin to the cytosolic solution (222).
Inspection of TMEM16A and TMEM16B primary structure does not reveal the presence of canonical binding sites
for Ca2⫹ (e.g., EF-hands) or calmodulin, which could be
expected for a Ca2⫹-gated channel. However, there are
multiple regions with negatively charged amino acid residues that may represent potential Ca2⫹-binding sites. In
particular, there is a cluster of five glutamic acid residues
(EEEEE) in the first intracellular loop that is highly conserved among orthologs and close paralogs of TMEM16A
(FIGURE 2B). This negatively charged cluster may be a binding pocket for Ca2⫹, as proposed for the “Ca2⫹ bowl” of
Ca2⫹-dependent K⫹ channels (12, 261). Interestingly, the
fifth glutamate of the EEEEE cluster is actually dependent
on the inclusion of segment c (EAVK) in the TMEM16A
transcript (in the absence of segment c, the fifth glutamic
acid is replaced by an aspartic acid residue from exon 14). If
the EEEEE cluster is indeed involved in Ca2⫹ binding, removal of the EAVK alternative segment could directly affect
the Ca2⫹ sensitivity of the TMEM16A protein, as shown by
Xiao et al. (250). However, deletion of EEEEE abolished
the intrinsic voltage dependence without altering Ca2⫹ sensitivity (250). Therefore, the EEEEEAVK domain may have
an allosteric role in modulating Ca2⫹ sensitivity and not a
direct participation in Ca2⫹ binding. Other regions with
negatively charged residues could be more directly involved.
2⫹
Recently, an important candidate site for direct Ca binding has been identified in the TMEM16A sequence (259).
As discussed in a previous section of the review, these authors found that the region between amino acids 650 and
706 is actually intracellular and not extracellular (FIGURE
2A). This protein region contains two highly conserved glutamic acid residues, E702 and E705, which were hypothesized to interact with Ca2⫹ (FIGURE 2B). Mutagenesis of
both residues to glutamine shifted the apparent Ca2⫹ sensitivity of TMEM16A/ANO1 channels from the submicromolar to the millimolar range (259). The authors also mutagenized the glutamic acid 702 to cysteine and tested the
effect of the cysteine-modifying reagents MTSET and
MTSES⫺ applied to the cytosolic side of the membrane. The
two reagents had the opposite effect, consisting of a decrease and an increase of apparent Ca2⫹ affinity, respectively, relative to the unmodified E702C mutant. These results support the role of E702/E705 in direct binding to
Ca2⫹ for the opening of the TMEM16A channel (259).
However, additional studies are needed to demonstrate direct Ca2⫹ binding. Indeed, an alternative interpretation of
results by Yu et al. (259) is that mutagenesis of E702/E705
alters Ca2⫹ binding at another site by an allosteric mechanism.
It is important to note that E702 and E705 are conserved in
the TMEM16B protein despite a nearly 10-fold lower Ca2⫹
sensitivity relative to TMEM16A. In a recent study, replacement of the third intracellular loop of TMEM16A, which
contains E702 and E705, with that of TMEM16B lowered
Ca2⫹ sensitivity (199). The resulting chimeric channel behaved in part like the wild-type TMEM16B. Therefore,
other amino acids in the third intracellular loop are also
important in determining the Ca2⫹ sensitivity. Such amino
acids may influence the position and orientation of the glutamates 702 and 705 and therefore their binding to Ca2⫹.
Recently, this region was also found important for
TMEM16A trafficking to the plasma membrane (2).
In addition to the third intracellular loop and the EEEEAVK
sequence, other TMEM16A/TMEM16B domains may participate in direct Ca2⫹ binding or in the allosteric modulation of Ca2⫹ sensitivity. In the search for these sites, we
must consider that, in addition to glutamic and aspartic
acid, other amino acid residues with oxygen in the side
chain, or even the carbonyls of the peptidic backbone, could
participate in Ca2⫹ coordination. Furthermore, Ca2⫹-binding site(s) may include noncontiguous amino acid residues.
Indeed, these sites could be generated in the folded
TMEM16A protein by residues that reside in different regions of the primary structure. Finally, it is also theoretically possible that Ca2⫹-binding sites are created at the
interface of TMEM16A subunits during the formation of
dimers.
Calmodulin may be another factor contributing to the Ca2⫹
dependence of TMEM16A channels, as also suggested by
studies on native CaCCs. For example, a study carried out
in an olfactory cell line concluded that calmodulin was absolutely required for endogenous CaCC activity. Transfection of calmodulin mutants having a dominant negative
effect inhibited CaCC currents (102). As stated before,
TMEM16A protein has a noncanonical binding site in segment b (coded by exon 6b). In a recent report, segment b of
TMEM16A was found to be important for binding to calmodulin (222). Calmodulin coimmunoprecipitated with
the TMEM16A(abc) but not with the TMEM16A(ac) variant. Furthermore, TMEM16A currents were strongly reduced by calmodulin inhibitors (trifluoperazine and J-8) or
by inhibitory peptides (222). However, the TMEM16A(ac)
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
isoform (unable to bind calmodulin according to immunoprecipitation results) was still activated by Ca2⫹. Therefore,
multiple Ca2⫹-dependent mechanisms, including calmodulin binding, may be involved in the gating of TMEM16A
channels (FIGURE 2B).
Different results were found in another study (237). These
authors identified a different site for calmodulin binding
(termed RCBM, i.e., regulatory calmodulin-binding motif),
localized in the NH2-terminal region, before the first transmembrane domain, and more than 30 amino acid residues
after the insertion of segment b (FIGURE 2B). The RCBM is
present in TMEM16A and TMEM16B, but it is much less
conserved in other anoctamins. Vocke et al. (237) showed
evidence of direct interaction between calmodulin and peptides containing the RCBM sequence. Mutagenesis of specific residues within the RCBM revealed the existence of
two regions or lobes. Both regions were important for binding of TMEM16A RCBM to calmodulin and for the Ca2⫹dependent channel activity of TMEM16A in cells. It is important to note that mutagenesis of RCBM also reduced the
trafficking of TMEM16A protein to the plasma membrane.
However, activation of currents at ⫹200 mV suggested that
a fraction of the mutant protein could reach membrane and
be activated by extreme voltages but not by Ca2⫹, presumably because of impaired binding to calmodulin (237).
The first part of RCBM was also important for the activation of TMEM16B by Ca2⫹ (237). However, in this protein,
the second half of RCBM had another role. When this segment was mutated, TMEM16B was activated normally by
Ca2⫹ but did not inactivate. In contrast, the wild-type
TMEM16B initially studied by Vocke et al., cloned from rat
olfactory epithelium, showed a process of slow inactivation
that, after the peak of activity triggered by Ca2⫹, closed the
channels within 30 – 40 s (237). Interestingly, these authors
found another TMEM16B variant, derived from the retina,
that did not inactivate. When the sequences of the two
variants were compared, Vocke et al. (237) found that both
TMEM16B channels contained the RCBM, but the retinal
variant contained four additional amino acids, ERSQ,
which correspond to segment c of TMEM16A. This finding
suggests that segment c controls the inactivation of
TMEM16B channels.
Another role for calmodulin in the modulation of
TMEM16A channel function was recently reported (101).
It was found that the bicarbonate permeability of
TMEM16A channels expressed in HEK-293 cells is
strongly increased in a calmodulin-dependent way by high
cytosolic Ca2⫹ concentrations. With a submaximal Ca2⫹
concentration (0.4 ␮M), the relative permeability to bicarbonate, expressed as PHCO3/PCl was 0.35. At a maximal
concentration (3 ␮M), this value became 1.04. The high
Ca2⫹ concentration affects permeability also to other anions so that the pore appears less selective. The authors
428
found that this phenomenon was not observed in outsideout membrane patches, which indicates the involvement of
a diffusible factor that is lost during excision of the membrane. This factor was identified as calmodulin because 1)
the increase in bicarbonate permeability was blocked by a
pharmacological inhibitor of calmodulin binding (J-8), 2)
inhibited by transfection of anti-calmodulin siRNAs, and 3)
restored in excised patches by adding exogenous calmodulin (101). Direct interaction was demonstrated in pull-down
experiments: precipitation of calmodulin fused to GST resulted in coprecipitation of TMEM16A protein. The authors identified two candidate domains for calmodulin
binding. The first one, named CBM1, was at the NH2 terminus right before transmembrane domain 1 (FIGURE 2B).
This domain partially overlaps the RCBM identified by
Vocke et al. (237). The second one, CBM2, was localized
before transmembrane domain 7. Two critical isoleucines,
at positions 317 and 762, were mutagenized to impair calmodulin binding at CBM1 and CBM2, respectively. The
single mutants and, more markedly, the double mutant displayed a significantly reduced bicarbonate permeability at 3
␮M Ca2⫹ and decreased interaction between calmodulin
and TMEM16A in pull-down experiments (101). The role
of Ca2⫹ concentration in the regulation of TMEM16A ion
permeability was confirmed in salivary gland cells. Importantly, the study by Jung et al. (101) was done with the (ac)
isoform of TMEM16A. Therefore, this isoform lacks the
segment b shown by Tian et al. (222) to be a site of interaction with calmodulin. This may explain, at least in part,
why, in the study by Jung et al. (101), calmodulin inhibition
by J-8 or siRNA resulted in alteration of permeability to
bicarbonate but not in a change in channel activity, as instead shown by Tian et al. for the isoform (abc) (222).
To sum up, the role of calmodulin in the regulation of
TMEM16A/B channels appears complex and requires further investigation. By binding to segment b, calmodulin
may influence positively channel activity (222). Vocke et al.
(237) found instead that calmodulin binds to a close but
separate region, RCBM. According to these authors, calmodulin binding to RCBM is essential for TMEM16A activation and, in TMEM16B, for inactivation. In the third
study, by binding to CBM1 and CBM2 (FIGURE 2B), calmodulin transduces changes in Ca2⫹ concentrations to
changes in bicarbonate permeability (101), a modification
that has a high physiological importance. As discussed by
Jung et al. (101), high micromolar cytosolic Ca2⫹ concentrations can be reached in nanodomains at the apical membrane of secretory cells in salivary glands and in pancreas
during stimulation. This condition can switch CaCCs to a
state highly permeable to bicarbonate, a characteristic that
may support the secretion of a bicarbonate-rich fluid (101).
It is important to note that other investigators have also
reported a dynamic ion permeability of TMEM16A and
TMEM16B (189, 195). In these studies, the ion selectivity
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
was not constant during the recordings but changed according to the levels of cytosolic Ca2⫹ concentration.
Ca2⫹ may also have inhibitory activity on TMEM16A
channels (61, 223, 256). High Ca2⫹ concentrations, particularly in the micromolar range, can lead to channel inhibition. The mechanism is unknown but may involve phosphorylation by Ca2⫹/calmodulin kinase II (CaMKII) since
the phenomenon is attenuated by the inhibitor KN-62
(223). As discussed in a previous paragraph, in a specific
TMEM16B variant, the relatively slow rundown caused by
Ca2⫹ increase seems to be mediated by direct binding to
calmodulin (237).
Recently, results obtained with purified TMEM16A in proteoliposomes suggest that calmodulin is not needed for
TMEM16A activity (219). In purification steps, calmodulin
did not co-elute with TMEM16A, and addition of calmodulin to liposomes did not change Cl⫺ transport. However,
as discussed by the authors of this study, experimental conditions (e.g., the use of detergents) could affect binding of
calmodulin. Therefore, it can be concluded that
TMEM16A is by itself able to transport Cl⫺ and to be
activated by Ca2⫹ and voltage. However, additional proteins, such as calmodulin, may modulate channel activity in
a native system.
In addition to Ca2⫹ and calmodulin, TMEM16A channel
activity is also regulated by temperature (34). The role of
TMEM16A and TMEM16B as “heat sensors” is discussed
in the section on nociceptive neurons.
B. Phosphorylation
Another possible mechanism involved in the regulation/gating of TMEM16A/B channels is phosphorylation. In fact,
some native CaCCs, particularly in epithelial cells, appear
to be activated by Ca2⫹/calmodulin-dependent kinases
(CaMKs) (85, 251). Such results contrast with those obtained for CaCCs in other tissues in which phosphorylation
by CaMK has actually an inhibitory effect. For example,
CaMK in smooth muscle causes a desensitization of CaCCs
so that they close in spite of a prolonged intracellular Ca2⫹
increase (6, 73, 241). In contrast, the Ca2⫹-dependent phosphatase activity of calcineurin has a stimulatory effect (74).
So far, the molecular basis explaining these different behaviors of native CaCCs has not been elucidated. However, it is
important to note that Cl⫺ channels activated by CaMK are
voltage independent (251), whereas channels inhibited by
CaMK show activation at depolarizing membrane potentials that is typical of classical CaCCs (73). It is expected
that the discovery of TMEM16A and TMEM16B will help
to resolve this issue.
There are multiple putative phosphorylation sites in the
primary structure of these proteins as indicated by predic-
tion programs such as GPS 2.1, NetPhosK 1.0, Disphos 1.3,
Prosite, and pkaPS (20, 95, 158, 203, 253). Analysis of
TMEM16A amino acid sequence identifies a number of
potential phosphorylation sites ranging between 8 and 48
depending on the program. However, there are five sites
[S74, S94, T216, S280, T530; residues numbered according
to the TMEM16A(abcd) isoform] that have been recognized as putative PKC sites by at least three prediction programs. The phosphorylation sites in TMEM16A and the
corresponding kinase(s) may have a distinct regulatory effect on channel activity. However, experiments are needed
to demonstrate that these sites are indeed phosphorylated
and that this results in a significant effect on protein function. In a recent study, the role of TMEM16A phosphorylation was investigated using various kinase inhibitors
(222). None of these compounds decreased TMEM16Adependent Cl⫺ currents. Actually, inhibition of CaMKII
resulted in increased activation of CaCC currents by ATP
(but not by ionomycin). The issue of TMEM16A phosphorylation was further investigated by mutagenesis in the
COOH terminus of two serines that are putative phosphorylation sites for the Erk1,2 kinase. The TMEM16A mutant
showed a decreased channel activity when cells were stimulated with extracellular ATP, therefore through purinergic
receptors, but not with the Ca2⫹ ionophore ionomycin
(222). The partial or null effect of maneuvers aimed at
inhibiting phosphorylation (kinase inhibitors or mutagenesis of putative phosphorylation sites) contrasts with the
strong CaCC current inhibition observed when experiments on TMEM16A-expressing cells were carried out in
the absence of intracellular ATP (222). This may suggest the
involvement of specific kinases that are insensitive to the
inhibitors used in the study or the need for ATP to keep
TMEM16A active through mechanisms other than phosphorylation.
Another mechanism of regulation of native CaCCs involves
changes in the metabolism of inositol polyphosphates. DMyo-inositol 3,4,5,6-tetrakiphosphate [Ins(3,4,5,6)P4] uncouples Ca2⫹-dependent Cl⫺ secretion and CaCC activity
from intracellular Ca2⫹ elevation (30, 86, 229). As well
established in many studies, stimulation of cells with phospholipase C-based stimuli (e.g., stimulation with purinergic
agonists) causes the production of Ins(1,4,5)P3, which triggers the release of Ca2⫹ from intracellular stores. However,
this CaCC-activating stimulus is followed by a more delayed increase in Ins(3,4,5,6)P4, which acts on CaCC as a
feedback inhibitory signal (30, 86, 229). The inhibitory
effect of Ins(3,4,5,6)P4, which may involve an okadaic acidsensitive phosphatase (252), can be antagonized by high
intracellular Ca2⫹ concentrations (85). Therefore, the specific effect of this inositol polyphosphate could be the reduction of the Ca2⫹ sensitivity of the channel. In a recent
study, a synthetic membrane-permeable inositol polyphosphate analog, INO-4995, was used on human epithelial
cells (224). Incubation with INO-4995 increased
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
TMEM16A-dependent Cl⫺ transport without altering intracellular Ca2⫹ levels. This result reveals the possibility to
modulate TMEM16A pharmacologically by intervening on
regulatory mechanisms.
III. PHYSIOLOGICAL ROLE OF TMEM16A
AND TMEM16B
A. Epithelial Cells
Ca2⫹-activated Cl⫺ currents are a common finding in electrophysiological studies carried out in epithelial cells. Shortcircuit current recordings in different types of polarized
epithelia reveal the presence of Ca2⫹-activated Cl⫺ secretion that may be triggered by Ca2⫹ mobilizing agents such
as purinergic receptor agonists (ATP, UTP) or Ca2⫹ ionophores (ionomycin, A23187) (67, 107, 140). The mechanism of Cl⫺ secretion requires the coordinated activity of
various channels and transporters localized in the apical
and basolateral membranes of the epithelial cells. In particular, the Na⫹ gradient across the basolateral membrane,
maintained by the activity of the Na⫹-K⫹-ATPase, is utilized by the Na⫹/K⫹/2Cl⫺ cotransporter to generate an intracellular accumulation of Cl⫺ (75, 76). This condition
favors the exit of the anion through apical channels activated by different second messengers, including cytosolic
cAMP or Ca2⫹.
There are at least two main lines of evidence proving that
cAMP- and Ca2⫹-dependent Cl⫺ secretion is mediated by
separate apical Cl⫺ channels, namely, CFTR and CaCC, at
least in the airway epithelium. First, selective pharmacological inhibitors allow the separation of the two components.
For example, the thiazolidinone CFTRinh-172 blocks CFTR
Cl⫺ channels with high selectivity (235). In the presence of
this compound, Ca2⫹-elevating agents are still able to induce Cl⫺ secretion, whereas the cAMP-dependent Cl⫺ secretion is totally inhibited (29). The second important proof
derives from studies on epithelial cells from cystic fibrosis
(CF) patients. CF is a genetic disease caused by the loss of
function of the CFTR protein (181). Transepithelial ion
transport recordings on CF airway epithelia show a severe
reduction of cAMP-dependent but not of Ca2⫹-dependent
Cl⫺ secretion, thus demonstrating that the latter is dependent on another type of protein different from CFTR (5,
66). The CF defect is particularly important in the airways
where the deficit in Cl⫺ secretion causes dehydration of the
epithelial surface and impairment of mucociliary transport
(143). This favors the colonization of the airways by pathogenic bacteria. In CF epithelia, the presence of a second type
of Cl⫺ channel that is not altered by CFTR gene mutations
is interesting for the development of therapeutic strategies.
Actually, in vitro activation of CaCC activity in CF epithelia
increases the thickness of the airway surface fluid, thus compensating for the CFTR defect (218).
430
In 2008, the discovery of TMEM16A as a Cl⫺ channel
protein provided a molecular basis for epithelial CaCCs
(29, 195, 256). In fact, TMEM16A was found by searching
the genes upregulated by IL-4 in human bronchial epithelial
cells (29). Knockdown of TMEM16A expression by transfection with siRNAs caused a significant inhibition of Ca2⫹dependent Cl⫺ secretion. Silencing was effective not only in
cells treated with IL-4 but also in untreated cells (29). These
results indicate that TMEM16A is an important component
of CaCC in airway epithelia under resting conditions and
that stimulation with IL-4 or IL-13 (40, 67), which simulates asthma-like inflammatory conditions in vivo, further
enhances the epithelium ability to secrete Cl⫺ by increasing
TMEM16A expression. The physiological role of the upregulation of TMEM16A by cytokines is unknown. However, two recent studies have provided a possible answer
(89, 198). TMEM16A was found specifically expressed in
mucus-producing goblet cells with little expression in ciliated cells (FIGURE 3). More precisely, there was a uniform
low expression in the airway epithelium under resting conditions, even in goblet cells. However, induction of goblet
cell hyperplasia by treatment of human bronchial epithelial
cells with Th-2 cytokines or by sensitization of mice with
ovalbumin resulted in an increased number of cells coexpressing at high levels TMEM16A and the mucin 5AC
(MUC5AC, a marker of goblet cells). Importantly, specific
coexpression of TMEM16A and MUC5AC was also confirmed in the airways of asthmatic patients (89). By analyzing the time course of TMEM16A and MUC5AC expression during treatment with IL-4, Scudieri et al. (198) found
that, in the first 24 h, TMEM16A appeared mainly in cells
that were nonciliated and devoid of mucin expression.
However, after 72 h of treatment, the percentage of cells
coexpressing TMEM16A and MUC5AC had largely increased. These results suggest that, during the process of
goblet cell metaplasia induced by Th-2 cytokines,
TMEM16A overexpression is an early event of differentiation to goblet cells.
IL-4 and IL-13 are potent inducers of mucus secretion and
goblet cell metaplasia in the airways (121). TMEM16A
overexpression in goblet cells could be a mechanism favoring the secretion and fluidity of mucus (FIGURE 3). This
mechanism could be related to the recent discovery that
TMEM16A is highly permeable to bicarbonate, particularly under conditions of high Ca2⫹ mobilization (101).
It has been shown that HCO3⫺ is particularly important in
allowing maximal expansion of mucus granules upon
secretion (69). Therefore, physiological activation of
TMEM16A in goblet cells during mucin release could be
a way to provide the required concentration of bicarbonate. In this respect, it should be noted that, in goblet cells,
the secretory vesicles contain high levels of ATP in addition to mucin (114). Exocytic mucin release would then
occur simultaneously with autocrine stimulation of puri-
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
Normal epithelium
IL-4, IL-13, asthma
Mucous metaplasia
ATP
Cl–
–
HCO3
TMEM16A
Ca2+
MUC5AC
FIGURE 3. Role of TMEM16A in the airway epithelium. Mucous metaplasia, occurring in the airways of asthmatic patients
and induced in vitro by treatment with Th-2
cytokines (IL-4 and IL-13), is characterized
by an increased number of goblet cells.
Under these conditions, TMEM16A is specifically expressed in the apical membrane
of goblet cells (89, 198). The permeability
of TMEM16A channels to bicarbonate
(101) may be important for the release of
mucins. The high content of ATP in mucin
granules may couple mucin release to
TMEM16A activation by means of purinergic receptors (114).
Goblet cell
nergic receptors. The resulting Ca2⫹ increase would activate TMEM16A/CaCC, thus generating a pulse of anion secretion synchronized with mucin release (FIGURE
3). It is also possible that TMEM16A is directly linked to
the mechanism of mucin release itself. Actually, Huang et
al. (89) have tested the effect of TMEM16A inhibitors,
and these compounds caused a significant reduction of
mucin release.
Interestingly, IL-4 not only affects TMEM16A but also
strongly upregulates the expression of pendrin (SLC26A4),
a transporter for Cl⫺, I⫺, SCN⫺, and bicarbonate (70, 170).
This effect suggests that inflammatory conditions and mucus hypersecretion in the airways are generally associated
with a boost in anion transport capacity.
The elucidation of the role of TMEM16A in the airways
needs further studies. Its association with goblet cell hyper-
plasia/metaplasia is particularly important in the pathogenesis and treatment of several diseases characterized by mucus hypersecretion, including asthma, cystic fibrosis, and
chronic obstructive pulmonary disease (185). With regard
to cystic fibrosis, it is important to remark that TMEM16A
overexpression occurs in goblet cells, whereas CFTR is localized in ciliated cells (198). It is not clear if this finding
implies that these two channels have actually different physiological roles. It is also important to assess TMEM16A
expression in cystic fibrosis airways. In primary cultures,
cells from normal and cystic fibrosis individuals have similar TMEM16A expression (198). However, the severe inflammatory response in the airways, triggered by bacterial
colonization, and characterized by massive neutrophil infiltration and mucus hypersecretion, could affect TMEM16A
expression in a positive or a negative way. This issue requires further studies to assess the suitability of TMEM16A
as a drug target in cystic fibrosis.
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Recently, three studies have added further interest on
TMEM16A function in airway epithelial cells. It has been
shown that TMEM16A affects cell proliferation and influences repair of the bronchial epithelium in cystic fibrosis
(187). In another study, it was found that TMEM16Adependent Cl⫺ transport inhibits, with an unknown mechanism, the release of the pro-inflammatory cytokine IL-8
(233). Therefore, activation of TMEM16A could have antiinflammatory effects. Finally, it has been found that a
TMEM16 protein in Drosophila melanogaster is involved
in host defense activity against the pathogenic bacterium
Serratia marcescens (248). It is tempting to speculate that a
similar role could be played by TMEM16A in the airways.
The relationship between TMEM16A and CaCCs has also
been demonstrated in other epithelia outside the airways.
For example, TMEM16A protein is expressed in the apical
membrane of rodent cholangiocytes, where it is probably
involved in the Ca2⫹-dependent fluid secretion of biliary
ducts, possibly mediated by mechano-sensitive ATP release
(50, 51). The properties of CaCCs in cultured cholangiocytes resembled those of TMEM16A-dependent channels
and silencing of TMEM16A expression in such cells inhibited Ca2⫹-dependent Cl⫺ transport (50). In another study,
the endogenous Ca2⫹-activated Cl⫺ currents of salivary
gland cells were compared with the currents arising from
heterologous expression in HEK-293 cells of TMEM16A or
bestrophin-2 (186). The currents were all strikingly similar
in terms of Ca2⫹ sensitivity and time/voltage dependence.
However, Ca2⫹-dependent Cl⫺ transport and fluid secretion were normal in Bestrophin-2 knockout mice. In contrast, Ca2⫹-dependent Cl⫺ transport was significantly reduced in TMEM16A⫺/⫺ animals (186).
A large TMEM16A expression was also revealed in serous
and mucous cells of airway submucosal glands (62). Actually, the TMEM16A mRNA was 10 and 100 times more
abundant than CFTR mRNA in serous and mucous cells,
respectively. The high TMEM16A expression was in agreement with a large size of the Ca2⫹-dependent Cl⫺ currents
measured in whole cell patch-clamp recordings. Also, measurement of Cl⫺ secretion by the short-circuit technique
revealed a large Ca2⫹-dependent component (62).
Despite strong lines of evidence in favor of TMEM16A as
an epithelial Cl⫺ channel, there are indications that other
types of CaCCs may also exist. In a recent study, a selective
TMEM16A channel inhibitor (T16Ainh-A01) was used in
different epithelial cell types (155). In differentiated bronchial epithelia, the inhibitor caused little effect on the Cl⫺
secretion induced by Ca2⫹-elevating agents. A similar negative finding was also obtained in intestinal epithelia. In
contrast, T16Ainh-A01 abolished Cl⫺ secretion in salivary
glands. The conclusion of the study was that CaCCs are
heterogeneous and TMEM16A is a minor contributor to
Ca2⫹-dependent Cl⫺ secretion in airway and intestinal ep-
432
ithelia (155). This conclusion was also supported by the low
amounts of TMEM16A protein detected by western blots
(although a large TMEM16A protein increase was observed upon treatment with IL-4). The issue of TMEM16A
role in the airway epithelium needs further consideration. In
fact, other types of studies, based on gene silencing with
siRNA and on gene knockout animals, are in favor of a
more significant involvement of TMEM16A in airway Cl⫺
secretion (29, 184, 167). It is possible that TMEM16A is
indeed expressed at low levels in the airway epithelium
under resting conditions and that another type of CaCC
exists. In this respect, it is important to note that a previous
study had reported that CaCCs in polarized and nonpolarized airway epithelial cells have different properties (5).
Nonpolarized cells show Ca2⫹-dependent Cl⫺ currents
with the typical voltage dependence of classical CaCCs. In
contrast, the currents of polarized cells are less voltage dependent. If another CaCC protein exists, the role of
TMEM16A may be more important under inflammatory
conditions when an increase in the ability to secrete Cl⫺ is
needed. Interestingly, TMEM16A is upregulated by IL-4
also in biliary duct epithelial cells (50), which suggests that
stimulation of Cl⫺ secretion by proinflammatory stimuli is a
general phenomenon in various organs.
The lack of TMEM16A function in the intestine resulting
from the T16inh-A01 inhibitor study (155) deals with a
quite controversial issue regarding the presence of one or
more types of Cl⫺ channels in the apical membrane of intestinal epithelial cells. In many studies, CFTR appears as
the most important Cl⫺ conductive pathway in the apical
membrane, responsible for both cAMP- and Ca2⫹-activated
Cl⫺ secretion (5, 45, 134, 211). The conclusion of these
studies is that cAMP-dependent Cl⫺ secretion is directly due
to CFTR activation, whereas the response to Ca2⫹ would be
more indirect. Indeed, Ca2⫹ activates basolateral K⫹ channels and the resulting hyperpolarization of the membrane
potential would favor the exit of Cl⫺ through open CFTR
Cl⫺ channels. On the other hand, there are also studies
indicating that a separate Ca2⫹-activated Cl⫺ conductance
exists in the apical membrane of intestinal epithelial cells
(146, 148). Furthermore, the carbachol-induced Cl⫺ secretion was severely reduced in the colon of TMEM16A⫺/⫺
mice (167). It is possible that expression of a CaCC conductance in the intestine is not constitutive, such as CFTR, but
influenced by age or other physiological and pathological
conditions. For example, TMEM16A expression in the intestinal epithelium seems regulated by the epidermal
growth factor (152). Another factor influencing the contribution of TMEM16A/CaCC to intestinal Cl⫺ secretion is its
subcellular localization. Actually, in a recent study,
TMEM16A protein was detected by immunofluorescence
in the lateral membrane of colonic epithelial cells (81). In
agreement with this localization, CaCC inhibitors increased
Cl⫺ secretion.
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
Interestingly, expression and function of TMEM16A/
CaCC in the apical membrane of intestinal epithelial cells
could be the basis for secretory diarrhea induced by rotaviral infection (113, 168). Indeed, the rotaviral toxin NSP4
induces Cl⫺ secretion through activation of CaCCs. Therefore, pharmacological inhibition of CaCCs could represent
a useful antidiarrheal approach (113, 168).
Another site of expression of TMEM16A is the kidney. A
recent study (26) showed expression of TMEM16A in
MDCK, a canine cell line resembling the principal cells of
the collecting duct, that is used as an in vitro model to study
the growth of kidney cysts. TMEM16A was also expressed
in the epithelium lining the cysts of autosomal polycystic
kidney disease patients (26). Silencing of TMEM16A, but
not of TMEM16F, with siRNAs inhibited Ca2⫹-dependent
Cl⫺ secretion in MDCK cells. Similar inhibition was obtained with TMEM16A blockers. Cyst growth was measured in two ways: with MDCK cells suspended in a collagen gel and with an embryonic kidney cultured model.
When TMEM16A activity was antagonized with pharmacological agents or with siRNAs, cyst growth was significantly inhibited (26). These results suggest that TMEM16A
is an important protein contributing to the progression of
polycystic kidney disease.
B. Smooth Muscle
Smooth muscle cells from different organs, particularly
from blood vessels, show Ca2⫹-activated Cl⫺ currents
(124). Actually, there seems to be two different CaCCs: a
“classical” one, with voltage-dependent gating (6, 73, 74),
and a second one that is instead modulated by intracellular
cGMP and has a different sensitivity to niflumic acid and
zinc (141, 142). The two types of channels have a different
pattern of distribution in the smooth muscle cells from
blood vessels and colon (142).
CaCCs in smooth muscle are believed to stimulate contraction. This occurs because Cl⫺ is accumulated intracellularly
through the activity of cotransporters like NKCC1. Indeed,
NKCC1 knockout mice have reduced blood pressure and
vascular smooth muscle tone (149). Because of the favorable electrochemical gradient, the opening of CaCCs results
in Cl⫺ exit and membrane depolarization, that in turn activates voltage-dependent Ca2⫹ channels. There may be different sources for Ca2⫹ increase leading to CaCC activation. Stimulation of G protein-coupled receptors for norepinephrine, acetylcholine, and histamine causes Ca2⫹ release
from intracellular stores (27, 98, 169, 239). CaCC activation may also derive from a positive feedback loop involving Ca2⫹ entry through voltage-dependent Ca2⫹ channels
and Ca2⫹-induced Ca2⫹ release through ryanodine receptors (124, 191).
CaCC inhibitors like niflumic acid have been widely used to
support the role of CaCC in smooth muscle. CaCC inhibi-
tion with such compounds causes smooth muscle relaxation
(36, 37, 100, 122, 191, 262). However, the classical CaCC
inhibitors used in many studies, like niflumic acid or NPPB,
have a series of different activities on channel and non-channel
proteins (124). For example, they may block CFTR (197, 264)
or Ca2⫹ channels (11), activate K⫹ channels (165), or cause
emptying of Ca2⫹ stores (38, 176). The development of novel
and more specific CaCC inhibitors may help to elucidate the
role of CaCCs in smooth muscle (43).
The link between CaCCs and TMEM16A protein in
smooth muscle cells is currently under investigation in
many laboratories. So far, it is still unclear whether
TMEM16A is expressed and associated with CaCCs in all
smooth muscle cell types. With the use of specific antibodies
on murine tissues, TMEM16A protein appears highly expressed in the smooth muscle of the airways and reproductive tract but, intriguingly, not at all in the smooth muscle of
blood vessels (87). In contrast, other studies have detected
mRNA and protein TMEM16A in freshly isolated vascular
smooth muscle cells from rodents (42, 137, 206, 238). Silencing of TMEM16A by siRNA resulted in CaCC inhibition (137, 238). Analysis of TMEM16A mRNA splicing
showed the coexpression in these cells of multiple variants
mostly due to alternative splicing of segments b and d (42,
137, 238).
Interestingly, TMEM16A expression and function in
smooth muscle cells are altered under pathological conditions. In one study, performed on smooth muscle cells isolated from the basilar arteries of hypertensive rats,
TMEM16A protein expression was downregulated compared with that of normal rats (238). In particular,
TMEM16A expression appeared to correlate inversely with
cell proliferation by regulating the levels of cyclin D1 and
cyclin E (238). Therefore, downregulation of TMEM16A
appears to favor remodeling of the wall of the basal artery,
which supplies blood to the brain.
In another study, TMEM16A was involved in the hyperreactivity of airway smooth muscle cells (ASMCs) under asthma-like conditions (263). Mouse sensitized with ovalbumin
to induce a chronic inflammation showed increased contractility along with increased TMEM16A expression.
Contractility of ASMCs isolated from TMEM16A knockout mice was significantly impaired compared with wildtype animals (263). These results indicate that TMEM16A
may be a drug target to prevent bronchoconstriction in
asthma. This conclusion also arises from the results of another study in which CaCC inhibitors had a relaxant activity on ASMCs (89).
Chronic hypoxia was found to upregulate TMEM16A expression and function. In particular, rats were exposed for
3– 4 wk to low oxygen levels. In these animals, pulmonary
arterial smooth muscle cells (PASMCs) showed larger
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CaCC currents, increased TMEM16A protein and mRNA,
and increased contractile responses (213).
TMEM16A-dependent CaCC activity is also upregulated in
pulmonary hypertension (64). A rat model of pulmonary
hypertension was generated by injection of monocrotalin.
PASMCs from these animals showed an increase in the
expression of TMEM16A at the mRNA and protein levels,
and a parallel increase in Ca2⫹-activated Cl⫺ currents. Contractility of PASMCs was antagonized by CaCC inhibitors,
including niflumic acid and the TMEM16A-selective
T16Ainh-A01 (64). Interestingly, the PASMCs from animals
with pulmonary hypertension also showed an altered pattern of TMEM16A splicing: the transcripts with inclusion
of segments b were more abundant than in control animals.
It would be interesting to assess if there are changes in the
pattern of splicing also in other physiological or pathological conditions. There is evidence that the alternative splicing of many smooth muscle genes is affected by the phenotypic state of the cells (e.g., phasic vs. tonic smooth muscle;
reviewed in Ref. 63). Since alterations in the splicing machinery of a cell have pleiotropic effects on multiple genes,
we may also expect a change in the relative abundance of
TMEM16A isoforms.
Another source of complexity influencing the role of
TMEM16A in smooth muscle cells is phosphorylation. As
mentioned before, native CaCCs in smooth muscle cells are
regulated by a complex mechanism involving Ca2⫹-dependent kinases and phosphatases such as CaMKII and calcineurin, respectively (6, 8, 74, 126, 241). The efficacy of
kinase and phosphatase inhibitors is dependent on the cytosolic free Ca2⫹ concentration (126). For example, cyclosporin A, a calcineurin inhibitor, decreases CaCC currents
at 500 nM but not at 1 ␮M Ca2⫹. Therefore, the net balance
between activation and inactivation of CaCC is probably
dependent on the amplitude and kinetics of intracellular
Ca2⫹ transients. It is still not clear whether the activity of
kinases and phosphatases on CaCCs directly involves phosphorylation of TMEM16A or of a regulatory subunit.
C. Interstitial Cells of Cajal
In the gastrointestinal tract, the contraction of smooth muscle layers is controlled by the electrical activity of a specialized cell type, the interstitial cells of Cajal, ICCs (92, 242).
ICCs are interposed between the terminations of enteric
neurons and smooth muscle cells, thus mediating the transduction of motor neurotransmitters such as acetylcholine.
Rhythmic depolarizations of ICCs are transferred electrically to smooth muscle cells, by gap junctions, triggering
contraction. Interestingly, TMEM16A, known as DOG1,
was found as a strong marker for gastrointestinal stromal
tumors, GISTs (55), and also useful to identify ICCs in the
gastrointestinal tract because of its high and specific expres-
434
sion in these cells (72). A microarray analysis had actually
identified TMEM16A as a gene highly expressed in ICCs
before its identification as a CaCC-forming protein (33).
The reason for expression of TMEM16A/DOG1/ANO1 in
GISTs may lie in the possible development of these tumors
from ICCs.
For years, the ionic basis of membrane depolarization in
ICCs was unclear. The discovery of TMEM16A/ANO1/
DOG1 as a Cl⫺ channel protein and its high expression in
ICCs (72, 87) strongly suggested that a Cl⫺ conductance
could be an important component of ICC electrical activity.
Indeed, the slow wave depolarization of ICCs appeared to
be caused by activation of niflumic acid-sensitive CaCCs
(266). To reach this conclusion, Zhu et al. (266) used mice
in which ICCs were labeled by specific expression of GFP.
This characteristic allowed the identification of ICCs within
the mixed cell population isolated from the intestine. Electrophysiological recordings demonstrated the presence of a
Ca2⫹-activated Cl⫺ conductance that was responsible for
the spontaneous depolarizations shown by the isolated
ICCs, as demonstrated by its sensitivity to niflumic acid.
The relationship between TMEM16A and the slow wave
depolariziations in ICCs was further demonstrated in
knockout animals. TMEM16A knockout animals displayed a drastic decrease in the electrical activity and contractility in the intestine and in the stomach (87, 94). Importantly, the impairment of gastrointestinal motility was
not due to a loss of ICCs (since these cells were normally
distributed, as demonstrated by staining for the C-kit
marker), but to a loss of TMEM16A expression (87). Indeed, slow waves were totally absent in the gastric and
intestinal muscles of TMEM16A knockout mice (94).
These findings strongly demonstrate the importance of
TMEM16A in the peristalsis of the gastrointestinal tract
and suggest that alterations in the function and expression
of TMEM16A protein may be a feature of gastrointestinal
motility disorders. In this respect, as previously discussed,
the pattern of TMEM16A splicing appeared altered in the
stomach of patients with diabetic gastroparesis (144). The
resulting TMEM16A variants, with shortened NH2 terminus, showed a change in CaCC gating kinetics. In another
study, reduction in immunostaining for TMEM16A revealed a depletion of ICCs as a possible cause of slow transit
constipation (103).
ICCs are not important only in the gastrointestinal tract.
Slow electrical waves are also present in the oviduct of
female mice to control the transport of oocytes. These
rhythmic changes in membrane potential and the resulting
contraction of myosalpinx were absent in Tmem16a knockout mice (46).
It is interesting to note that the urethra appears as an exception to the rule of specific expression of TMEM16A in
ICCs. In urethras from sheep, mouse, and rat, TMEM16A
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
was highly expressed in smooth muscle cells but not in ICCs
(192).
D. TMEM16B in Olfactory Neurons
Ca2⫹-activated Cl⫺ currents have been described in many
types of neurons where they may be involved in the triggering of action potentials and in the modulation of excitability. CaCCs have been particularly studied as mediators of
olfactory stimuli. The cilia of receptor neurons in the nasal
olfactory epithelium respond to odorants by increasing intracellular cAMP (193). This messenger opens in turn cyclic
nucleotide-gated channels (CNGCs) that are permeable to
Na⫹ and Ca2⫹ (154). The cation influx causes per se membrane depolarization, but Ca2⫹ also activates CaCCs that
mediate Cl⫺ efflux (21, 110, 119, 130). As in epithelial cells,
the efflux of Cl⫺ is favored by the activity of the bumetanide-sensitive Na⫹/K⫹/2Cl⫺ cotransporter, which generates the outwardly directed gradient (82, 159, 180). The
depolarization arising from Cl⫺ efflux through CaCCs represents a mechanism of electrical amplification of the initial
olfactory stimulus. This mechanism is favored by the specific compartmentalization of odorant receptors, CNGCs,
and CaCCs on the membrane of olfactory cilia (65, 179).
This special organization allows the intracellular messengers generated by olfactory stimuli (cAMP, Ca2⫹) to reach
high concentrations within the small volume of cilia and to
reach rapidly the downstream actuators (CNGCs and
CaCCs).
In 2009, TMEM16B (ANO2) protein was proposed as the
olfactory CaCC. TMEM16B was found as an abundant
membrane protein in the proteome of mouse olfactory cilia
(208). This finding was confirmed in another study in which
quantitative analysis detected TMEM16B protein expression at levels higher than those of CNGC subunits (178).
The link between olfactory CaCCs and TMEM16B was
also supported by functional studies. Transfection of
TMEM16B in HEK-293 cells generated Cl⫺ currents whose
properties resembled those of native olfactory CaCCs (172,
208).
CaCCs have been identified also in vomeronasal sensory
neurons (VSNs). The vomeronasal organ is a specialized
structure devoted to the detection of pheromones. In VSNs,
the mechanism of transduction has evolved differently from
that of the main olfactory epithelium neurons. G proteincoupled odorant receptors in VSNs do not increase cAMP
but trigger the activation of phospholipase C and therefore
the production of diacylglycerol. This molecule stimulates
TRPC2 causing Ca2⫹ influx and CaCC activation (108,
254). As evidenced by various studies, TMEM16B/ANO2
may be the main component of CaCC also in VSNs (19,
254).
Recently, a study has seriously questioned the role of
CaCCs in olfactory stimulus transduction (19). A knockout
mouse was generated to investigate the relationship of
TMEM16B/ANO2 with olfaction. This animal showed no
CaCC currents in olfactory neurons, thus demonstrating
that TMEM16B protein is indeed the main CaCC-forming
protein in these cells. However, electro-olfactograms
showed that TMEM16B⫺/⫺ animals had only a partial
(40%) reduction of the electrical response to odorants. Furthermore, olfactory tests showed that TMEM16B⫺/⫺
mice, in contrast to CNGA2⫺/⫺ animals, showed no impairment in the ability to detect odorants (19). These intriguing results contradict the common belief, supported by
many studies, that CaCCs are indeed required in olfaction.
It is not clear at the moment whether CaCCs are completely
dispensable for olfaction or if they may serve under particular circumstances. In this respect, it should be noted that
the wide use of nonspecific Cl⫺ channels inhibitors, such as
niflumic acid, may have led to an overestimation of the
actual role of CaCCs in olfaction. The development of more
selective TMEM16B pharmacological inhibitors could be
important to elucidate the physiological role of CaCCs in
olfactory receptors as well as in other cells.
E. TMEM16B in Photoreceptors
In vertebrate photoreceptors (cones and rods), CaCCs are
believed to regulate the release of glutamate at the synaptic
terminal by local feedback mechanisms. In the dark, there is
a continuous influx of Na⫹ through cGMP-gated cation
channels that depolarize the membrane potential. This depolarization keeps the voltage-dependent Ca2⫹ channels at
the synaptic terminal in the open state causing an influx of
Ca2⫹ and hence a steady release of glutamate through fusion of vesicles. The influx of Ca2⫹ also activates CaCCs
with a consequent effect on the electrical properties of the
photoreceptor membrane (13, 14, 35, 133). In cones, which
have an equilibrium potential for Cl⫺ close to the resting
membrane potential in the dark (approximately ⫺36 mV),
opening of CaCCs tends to stabilize the membrane potential (220). In rods, where the Cl⫺ equilibrium potential
(⫺20 mV) is more positive than the resting potential, CaCC
opening causes Cl⫺ efflux. This may have a feedback regulatory role on voltage-dependent Ca2⫹ channels through an
anion-binding site (10, 221).
TMEM16B was found highly expressed in the photoreceptor synaptic terminals of mouse retina (209). Stöhr et al.
(209) found that the TMEM16B COOH terminus has a
PDZ class I binding motif through which it forms a macromolecular complex with PSD95, VELI3, and MPP4 adaptor
proteins. This interaction appeared functionally important
since TMEM16B was lost from the photoreceptor membrane in mice defective for MPP4 protein. When expressed
in HEK-293 cells, TMEM16B generated Cl⫺ currents very
similar to those of native CaCCs previously described in
photoreceptors. Subcellular localization and functional
properties suggested that TMEM16B is a main CaCCforming protein of photoreceptors (209).
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In another study, carried out on salamander retina, Mercer
et al. (147) found evidence for TMEM16A expression and
function at the photoreceptor ribbon synapse. Interestingly,
these authors analyzed the distribution of voltage-dependent Ca2⫹ channels (VDCCs), CaCCs, and transmitter release sites by comparing the response to depolarizing voltage steps in the presence of BAPTA or EGTA, two chelating
agents with different kinetics for Ca2⫹ binding. Synaptic
release of transmitter was not blocked when the intracellular solution in the photoreceptor contained the slow EGTA
agent. Furthermore, the release was only partially reduced by using the fast BAPTA agent. These results indicate that the distance between VDCCs and the site of
transmitter release is very short, below 100 nm. In contrast, CaCCs appeared more distant from VDCCs since
their activation by a depolarizing pulse was markedly
reduced by EGTA in a concentration-dependent way
(147). The estimated average distance between CaCCs
and VDCCs was 300 – 600 nm in cones and 200 – 450 nm
in rods. This more diffuse distribution, estimated by
functional measurements, appeared consistent with the
results obtained by immunostaining of the CaV1.4 Ca2⫹
channel subunit and of TMEM16A (147).
Future studies are needed to clarify the relative contribution
of TMEM16A and TMEM16B to photoreceptor function
and whether they may coexist in the same cells. This may
require the development of conditional knockout mice with
selective gene ablation in retinal cells.
F. TMEM16A in Nociceptive Neurons
The role of CaCCs in nociceptive neurons was elucidated by
Liu et al. (128) by investigating the mechanism of action of
bradykinin, an inflammatory mediator and a potent inducer
of pain sensation (FIGURE 4). In small nociceptive neurons
of dorsal root ganglia, bradykinin elicited phospholipase
C-dependent inhibition of M-type K⫹ channels and activation of a Ca2⫹-dependent Cl⫺ conductance (128). The resulting membrane depolarization triggered firing of action
potentials by neurons. These neurons were found to express
TMEM16A protein, as also shown previously by Yang et
al. (256), and knockdown of TMEM16A expression by
transfection of siRNA decreased the amplitude of CaCC
currents (128). Recently, TMEM16A was found to directly
interact with the inositol 1,4,5-triphosphate receptor 1
(99). This interaction may be important in nociceptive neurons for the selective activation of TMEM16A by bradykinin and not by voltage-dependent Ca2⫹ channels (see also
paragraph on TMEM16A interactome).
Recently, another important mechanism of activation of
TMEM16A, highly relevant to its expression in sensory
neurons, was discovered (34). TMEM16A/ANO1 and
TMEM16B/ANO2 were found to be highly sensitive to
temperature when expressed in HEK-293 cells (FIGURE 4).
436
In particular, temperatures above 44°C activated
TMEM16A Cl⫺ currents even in the absence of Ca2⫹ (high
BAPTA concentration on the cytosolic side of the membrane). At more physiological temperatures, heat and Ca2⫹
appeared to act in a synergistic way on channel activity:
increases in cytosolic free Ca2⫹ concentrations decreased
the temperature threshold required for TMEM16A activation (34). The role of TMEM16A as a heat sensor was also
demonstrated in sensory neurons of dorsal root ganglia
(DRG). Actually, DRG neurons were already known to
express other heat sensors such as the TRPV1 channel, the
capsaicin receptor (16). However, the presence of other
temperature-sensitive mechanisms in these cells was expected because Trpv1 knockout mice keep a response to
high temperature (249). Cho et al. (34) demonstrated that
DRG neurons respond to high temperature with activation
of Cl⫺ currents and that such currents are preserved in cells
from Trpv1 knockout mice. The temperature-sensitive Cl⫺
currents were dependent on the expression of TMEM16A
because 1) they were inhibited in neurons isolated from
mice injected with anti-TMEM16A siRNA, and 2) they
were strongly reduced in neurons derived from a conditional knockout mouse (TMEM16A deleted only in sensory
neurons). When TMEM16A was downregulated in vivo by
siRNA silencing or by gene knockout, mice showed a decreased response to noxious high temperature (⬎50°C).
The mechanism of action of TMEM16A is based on neuron
membrane depolarization, which in turn triggers firing of
action potentials. Indeed, the relatively high intracellular
Cl⫺ concentration in sensory neurons (⬎30 mM) causes
Cl⫺ efflux and, hence, depolarization when TMEM16A
channels open. Cho et al. (34) demonstrated that both
TRPV1 and TMEM16A contribute to the heat sensitivity of
neurons because a combination of capsazepine (TRPV1 inhibitor) and mefloquine (CaCC inhibitor) was required to
fully block the depolarization induced by high temperature.
In conclusion, the expression and function of TMEM16A in
nociceptive neurons indicate this protein as an important
target for analgesic therapies. However, the role of
TMEM16A in the transduction of painful stimuli also implies that pharmacological activators of this channel, developed for the treatment of other diseases (e.g., cystic fibrosis), may cause serious pain as a side effect.
G. TMEM16B in Hippocampal Neurons
A recent study has revealed the importance of CaCCs in
general, and of TMEM16B in particular, in controlling action potential firing and frequency in the somatodendritic
region of hippocampal neurons (90; FIGURE 4). The authors
found that the application of depolarizing stimuli in murine
cells triggered a conductance activated by the voltage-dependent influx of Ca2⫹. Indeed, this conductance was removed by blocking Ca2⫹ channels with Cd2⫹ or by chelating intracellular Ca2⫹ with BAPTA. The Ca2⫹-dependent
conductance was identified as a CaCC component because
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
A
DRG cell
TMEM16A
BK
Cl–
Ca2+
Heat
30 mM Cl–
Ca2+
TRPV1
B
CA3
CA1
Ca2+
Cl–
TMEM16B
Ca2+
Ca2+
Ca2+
Cl–
Ca2+
5 mM Cl–
FIGURE 4. TMEM16A and TMEM16B function in neurons. Expression of TMEM16A in dorsal root ganglion
cells is important in nociception (top). TMEM16A is activated by the intracellular Ca2⫹ increase evoked by
bradykinin (BK) and/or directly by heat (34, 128). Because of the relatively high intracellular Cl⫺ concentration,
opening of TMEM16A channels causes depolarization and therefore the generation of action potentials that
are transmitted from the periphery to the central nervous system. In the hippocampus (bottom), TMEM16B
channels modulate the synaptic transmission between CA3 and CA1 neurons. Ca2⫹ elevation induced by the
neurotransmitter in the postsynaptic compartment activates TMEM16B channels. Because of the low intracellular Cl⫺ concentration, TMEM16B channel opening hyperpolarizes the cell thus causing an inhibitory effect
on neuronal excitability (90).
of the following lines of evidence: 1) it was abolished by
CaCC inhibitors like niflumic acid or NPPB and 2) its reversal potential was dependent on the Cl⫺ concentration in
experimental solutions. To understand the molecular identity of CaCCs in hippocampal neurons, the authors analyzed the expression of TMEM16A and TMEM16B. Only
TMEM16B was expressed at mRNA and protein levels.
Knockdown of TMEM16B by shRNA significantly inhibited the neuronal CaCC current. Furthermore, this current
was unaltered in neurons from TMEM16A knockout
mice (90).
Interestingly, CaCC inihibitors caused a broadening of action potentials in CA1 and CA3 pyramidal neurons (by
nearly 30% with 100 ␮M niflumic acid). This finding reveals that the role of TMEM16B is to act as a brake on
neuronal excitability. The increase in spike width caused by
niflumic acid persisted even when the intracellular solution
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437
NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
contained EGTA instead of BAPTA (90). Since EGTA is a
slow chelating agent, this result indicates that TMEM16B
channels are close to the site of Ca2⫹ entry (i.e., voltagedependent Ca2⫹ channels).
The excitatory synaptic response was studied by recording
from CA1 neurons while stimulating the axons of CA3
neurons (90). It was found that CaCCs act in the somatodendritic region of CA1 neurons by raising the threshold of
action potentials and contrasting the summation of excitatory postsynaptic potentials. Obviously, the inhibitory
role of CaCC on action potential firing is dependent on the
normal low Cl⫺ concentration (5–10 mM) in mature neurons. As discussed by Huang et al. (90), increases in Cl⫺
concentration, as those that may occur during prolonged
neuronal activity or under pathological conditions, are expected to change the influence of CaCCs from inhibitory to
excitatory.
H. TMEM16A Role in Cell Volume
Regulation
All cells are able to regulate their own volume by means of
a complex array of channels and transporters that mediate
the efflux or uptake of electrolytes and organic osmolytes.
For example, when cells are exposed to a hypotonic environment, they initially swell because of osmotic uptake of
water. However, they subsequently shrink, returning to
their initial volume, by activating in parallel K⫹ and Cl⫺
channels (212). The resulting net efflux of KCl drives water
outside the cells, thus causing the so-called “regulatory volume decrease” (RVD). RVD is also favored by the characteristic permeability of volume-sensitive Cl⫺ channels to
organic osmolytes, such as taurine and myo-inositol, that
are also important in determining the cell shrinkage (96).
Cell volume decrease by activation of K⫹ and Cl⫺ channels
may also occur in isotonic conditions. Isotonic shrinkage
can be the mechanism underlying the apoptotic cell volume
decrease (123). Volume decrease, particularly if localized to
a region of the cell, can also be important in cell shape
changes, a process associated with cell migration. Furthermore, cell volume regulation may be continuosly needed to
compensate for changes in intracellular osmolarity due to
uptake and metabolism of nutrients and macromolecules
(123).
The anion channel that is most frequently associated with
cell volume changes, particularly with activation by hypotonic shock, is the volume-regulated anion channel
(VRAC), also known as volume-sensitive organic osmolyteanion channel (VSOAC). This channel has biophysical
characteristics differing from those of CaCCs. It has an
intrinsic outwardly rectifying current-voltage relationship
(i.e., the channel has a higher conductance at positive membrane potentials) and a typical time-dependent inactivation
when the membrane is depolarized (97, 115, 163). These
438
characteristics indicate that VRAC and CaCCs are due to
different molecular entities. Actually, the protein(s) constituting VRAC are still unknown. However, this does not
exclude TMEM16A/CaCC and other anoctamins as other
important physiological determinants of cell volume regulation. In cells expressing TMEM16A, mobilization of intracellular Ca2⫹ could induce anion efflux, thus contributing to cell volume decrease. This mechanism is supported by
experimental data: knockdown of TMEM16A by siRNA in
pancreatic CFPAC-1 and intestinal HT-29 cells decreased
the amplitude of Cl⫺ currents evoked by a hypotonic shock
(3). Furthermore, RVD was impaired in cells treated with
siRNAs against TMEM16A and in epithelial cells from the
TMEM16A knockout mouse. Intriguingly, the currents activated by cell swelling and RVD appear to depend also on
other anoctamins, particularly TMEM16F, TMEM16H,
and TMEM16J (3).
In future studies, it will be important to determine the relationship between anoctamins and VRAC and how the activity of different Cl⫺ channel proteins are controlled during regulation of cell volume.
IV. TMEM16F AND OTHER ANOCTAMINS
A. TMEM16F as a Multifunctional
Membrane Protein
Among anoctamins, TMEM16F (ANO6) is one of the most
highly expressed in different tissues and cell types (194,
202). In mouse, TMEM16F, like TMEM16H and
TMEM16K, shows a high expression in several tissues,
a characteristic that may imply important housekeeping
roles (194). In contrast, TMEM16B, TMEM16C, and
TMEM16D appear mainly expressed in neuronal tissues. In
particular, while TMEM16B is highly expressed in the eye,
TMEM16C and TMEM16D are expressed in spinal cord,
brain stem, and cerebellum. Instead, TMEM16E is specifically localized in skeletal muscle and thyroid (194).
In terms of phylogenetic analysis, TMEM16F is a close
relative of TMEM16E/ANO5 and a distant relative of
TMEM16A and TMEM16B (68). In 2010, Suzuki et al.
(215) reported the unexpected association of TMEM16F
with phospholipid scramblase activity.
The plasma membrane is characterized by an asymmetric
distribution of lipids in the bilayer, being the negatively
charged phosphatidylserine (PS) much more abundant in
the inner leaflet (18). This asymmetrical distribution is
maintained by the activity of a protein called “flippase”
(aminophospholipid translocase, a P-type ATPase). However, under physiological and pathological conditions, activation of a Ca2⫹-dependent “scramblase” dissipates the
asymmetry by catalyzing the rapid externalization of PS on
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
Externalized
PS detected by
annexin V binding
Ba/F3 cells
TMEM16F
identification
FACS analysis and
sequencing of cells
with enhanced PS
binding
Iteration
cDNA library and
transfection in
native Ba/F3 cells
Fluorescence-activated
cells sorting (FACS)
FIGURE 5. Strategy for the identification of
TMEM16F as a phospholipid scramblase. The
Ca2⫹-dependent externalization of phosphatidylserine (PS) in the Ba/F3 cell line was quantified
by measuring the binding of annexin V to cell
surface (215). Cycles of fluorescence-activated
cell sorting allowed the isolation of a cell clone
characterized by large scramblase activity. A
cDNA library generated from these cells was
transfected in native Ba/F3 cells to identify the
coding sequence inducing scramblase activity
(215).
Ba/F3 cells
with enhanced
scramblase
activity
the cell surface (18). This process has a variety of important
roles in different cells. In platelets, exposed PS triggers the
coagulation cascade. PS externalization is also a general
feature of cells undergoing apoptosis. Indeed, exposed PS is
a signal for the removal of dying cells by phagocytes (18).
Furthermore, transient and localized PS scrambling may be
a general signaling phenomenon associated with various
cell processes including exocytosis, membrane fusion, and
synaptic transmission (1, 93, 105, 210, 231, 258).
The molecular identity of scramblases was unclear. There
are four proteins belonging to the same family, which still
have the official name of phospholipid scramblases:
PLSCR1– 4 (18). However, the direct role of these proteins
in the translocation of phospholipids is uncertain. Upregulation or knockdown of PLSCR proteins does not change
the rate of phospholipid scrambling (1, 18, 56, 267). It
appears now that PLSCR proteins are signaling molecules
with a possible function in the nucleus as gene activators
(190).
Suzuki et al. (215) used an expression cloning approach to
look for proteins directly involved in phosholipid scrambling. The authors used a mouse B-cell line, Ba/F3, with
endogenous Ca2⫹-dependent scramblase activity, as detected by binding of fluorescent annexin V to the cell surface
(FIGURE 5). Repeated rounds of fluorescence-activated cell
sorting (FACS) were performed to select a population of
cells with increased PS externalization upon treatment with
a Ca2⫹ ionophore. At the end of the process, Suzuki et al.
(215) generated a subline (Ba/F3-PS19) with nearly 100fold higher staining with annexin V compared with the
original cell line. The authors found that this characteristic
was the result of scramblase upregulation and not of flippase inactivation. To identify the underlying scramblase, a
cDNA library, generated from the mRNA of Ba/F3-PS19
cells, was transfected in the parental Ba/F3 cells (FIGURE 5).
FACS analysis of stable transfected cells identified a clone
characterized by high PS on the surface even without treatment with the Ca2⫹ ionophore. These cells appeared to
express a TMEM16F protein with a mutation, a replace-
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439
NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
ment of the aspartic acid at position 409 with a glycine, in
the first intracellular loop (215). The results indicated that
TMEM16F is associated with scramblase activity and that
the D409G mutant, spontaneously arisen in Ba/F3 cells,
increased the sensitivity of the scramblase to intracellular
Ca2⫹, so that PS was externalized even under resting conditions. Of note, no scramblase activity was found for
TMEM16A. Suzuki et al. (215) also found a loss-of-function mutation in the TMEM16F gene of a patient with Scott
syndrome. This is a genetic disease in which platelets have a
defect in PS scrambling leading to a coagulation deficit and
bleeding. The patient had a G-to-T homozygous mutation
at the splice-acceptor site in intron 12 causing skipping of
exon 13 with sequence frameshift and generation of truncated TMEM16F protein (215).
The results obtained by Suzuki et al. (215) showed that cells
with constitutive exposure of PS on the cell surface, as those
expressing the mutant TMEM16F-D409G, were viable.
This finding was intriguing because PS externalization was
considered an important signal of apoptosis. In a subsequent study, the same research group, by further screening
of the original cDNA library, identified another version of
TMEM16F with a gain of function (200). This TMEM16F
version, named D430G-L, corresponded to a splice variant
having 21 additional amino acids at position 24 plus the
previously identified aspartic-to-glycine mutation. Interestingly, D430G-L protein was even more active than the
D409G mutant. Cells expressing D430G-L showed a high
and constitutive level of PS on the cell surface, were viable,
and grew in response to IL-3 with the same rate as parental
cells. Also, there was no difference in terms of apoptosis
induced by FasL (200). Finally, cells with high PS on the
surface were not engulfed by macrophages in vitro and in
vivo. These results indicate that PS externalization is not
sufficient per se as a signal to trigger phagocytosis and further emphasize the information that PS scrambling may
occur in nonapoptotic cells.
Interestingly, TMEM16F is also endowed with ion channel
activity, although the biophysical properties of TMEM16Fdependent channels differ among studies. In the first report
on this topic (139), TMEM16F appeared as an essential
component of the outwardly rectifying Cl⫺ channel
(ORCC), a type of anion channel characterized by intermediate single-channel conductance (⬃50 pS) and nonlinear
current-voltage relationship. ORCCs are typically activated
in different types of cells when excised membrane patches
are treated with strong and prolonged membrane depolarization (117). They are also activated in intact Jurkat lymphocytes during apoptosis induced by FasL (216). Martins
et al. (139) found that knockdown of TMEM16F/ANO6
(but not of TMEM16E/ANO5 nor of TMEM16H/ANO8)
by siRNA strongly inhibited the FasL-induced activation of
ORCC chloride currents. A similar inhibitory effect of
TMEM16F gene silencing was found when ORCCs were
440
induced by other apoptotic stimuli such as ceramide and
staurosporine. Silencing of TMEM16F downregulated
staurosporine-induced ORCC also in epithelial cell lines
(A549 and 9HTEo-). Overexpression of TMEM16F in
A549 cells resulted in ORCC Cl⫺ currents even in the absence of staurosporine (139). The direct role of TMEM16F
in forming the ORCC channel was supported by the finding
of an altered halide permeability caused by mutations
(Y405F, K553A, and K834A) introduced in the putative
pore-forming region (139).
It has been previously reported that ORCC activity is modulated by CFTR (196). Martins et al. (139) found that
CFTR expression increased the activity of ORCC triggered
by staurosporine and that this effect was inhibited by antiTMEM16F siRNAs (139). Interestingly, the TMEM16Fdependent ORCC activity was not Ca2⫹ dependent, since it
persisted in the presence of the BAPTA chelating agent.
Martins et al. (139) also found that the activity of
TMEM16F as an ORCC facilitates apoptosis. This effect
may be due to the efflux of Cl⫺ favoring cell shrinkage, a
typical phenomenon occurring during apoptosis.
Activity of TMEM16F/ANO6 as a Cl⫺ channel has also
been recently detected in dendritic cells (217). In contrast to
epithelial and Jurkat cells, TMEM16F in dendritic cells appears to be Ca2⫹ dependent. Indeed, elevation of intracellular Ca2⫹ by ionomycin or IP3 triggered the activation of
outwardly rectifying Cl⫺ currents. Such currents were also
activated, in a Ca2⫹-dependent way, by CCL21, a physiological agonist of the chemokine receptor CCR7 that triggers chemotactic activity in dendritic cells. Knockdown of
TMEM16F by siRNA resulted in reduction of Ca2⫹-activated Cl⫺ currents and, importantly, in inhibition of dendritic cell migration (217).
Two recent studies have also established an association between TMEM16F and Ca2⫹-activated Cl⫺ channels (77,
202). In both studies, intracellular Ca2⫹ elevation elicited
outwardly rectifying Cl⫺ currents, although after a delay of
several minutes. In contrast to TMEM16A, the strong rectification of currents was maintained even at the highest
Ca2⫹ concentrations. However, the two studies reported
different values of Ca2⫹ sensitivity, with an estimated EC50
of 9.6 ␮M in one study (202) and of nearly 100 ␮M in the
other (77). As discussed by Grubb et al. (77), this discrepancy may be related to experimental conditions. The use of
EGTA, a buffer that is not appropriate to control Ca2⫹ in
the micromolar range, may be an important factor. The two
studies on TMEM16F also reported differences in ion selectivity. Shimizu et al. (202) found that TMEM16F currents
are carried by Cl⫺ with no significant contribution of cations, as demonstrated by the lack of effect following replacement of Na⫹ with the large cation NMDG⫹. In contrast, Grubb et al. (77) found that TMEM16F expression
generated Ca2⫹-dependent Cl⫺ channels that were also per-
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
meable to Na⫹. In particular, the relative permeability PNa/
PCl was 0.3 (77). Shimizu et al. (202) also found that the
expression of TMEM16F was not associated with the appearance of Cl⫺ channels activated by staurosporine, as
instead shown by others (139). They also showed no relationship between TMEM16F and the volume-sensitive Cl⫺
channel (202).
The identification of TMEM16F as a Cl⫺ channel contrasts
with the results of another recent study in which
TMEM16F was actually found to mediate a cation conductance (255). The authors generated a TMEM16F knockout
mouse. The platelets, erythrocytes, and B-cells from these
animals showed reduced Ca2⫹-dependent PS exposure, in
agreement with the function of TMEM16F as a scramblase
component. Importantly, TMEM16F ⫺/⫺ mice showed
impaired coagulation, as measured by thrombin generation, and prolonged bleeding, thus resembling the phenotype of Scott syndrome patients. The knockout animals
were also markedly less prone to induced carotid artery
thrombosis compared with wild-type mice. Megakaryocytes, the precursors of platelets, were studied with patchclamp recordings. Cells from wild-type mice showed a
Ca2⫹-dependent outwardly rectifying conductance that
was absent from the cells of TMEM16F ⫺/⫺ animals.
TMEM16F channel properties were also studied by its expression in Axolotl oocytes and HEK-293 cells (255). Unlike TMEM16A, the conductance generated by TMEM16F
expression did not become linear at high Ca2⫹ concentrations but remained outwardly rectifying. Surprisingly, the
TMEM16F currents were found to carry cations and not
Cl⫺ (255). Changes in the ion composition of the experimental solutions during recordings caused shifts in reversal
potential of the currents consistent with PNa/PCl of ⬃7. In
the same type of experiments, the PNa/PCl of TMEM16A
was ⬃0.14. The cation selectivity of TMEM16F channels
was confirmed in megakaryocytes of wild-type mice.
TMEM16F channels were almost equally permeable to
Na⫹, K⫹, Li⫹, Rb⫹, and Cs⫹. Importantly, TMEM16F
were more permeable to Ca2⫹ and Ba2⫹ than to monovalent cations (255). Measurements of single-channel conductance gave a very small value of nearly 0.5 pS. Because of
these characteristics, TMEM16F channels were called
SCAN channels (for “small-conductance Ca2⫹-activated
cation channels”). The SCAN acronym was chosen to distinguish the new type of channels from the better known
CAN channels, whose activity is instead mediated by
TRPM4 and TRPM5 (78). TMEM16F-SCAN channels
showed a peculiar pharmacological sensitivity. They were
insensitive to NPPB, niflumic acid, flufenamic acid, and
classical CaCC blockers, but were sensitive to CAN inhibitors such as ruthenium red and 2APB.
Interestingly, Yang et al. (255) identified an amino acid
residue in the fifth transmembrane domain of TMEM16F
that is critical for ion selectivity. They noted that
TMEM16F has a glutamine residue (Q559) in a position
that is instead occupied by a lysine in TMEM16A and
TMEM16B. Mutagenesis of Q559 to lysine transformed
TMEM16F in a channel more permeable to Cl⫺ (PNa/PCl ⫽
2.2), whereas mutagenesis of K584 to glutamine converted
TMEM16A channels to a less anion-permeable state (PCl/
PNa ⫽ 4.1 instead of 7.0).
Yang et al. (255) also introduced mutations at the glutamic
acid residues that in TMEM16F are equivalent to those
(E702 and E705) mediating Ca2⫹ sensitivity in TMEM16A
(259). Mutation E670Q in TMEM16F (equivalent to
E705Q in TMEM16A) reduced TMEM16F surface expression, probably due to destabilization of the protein. In contrast, mutation E667Q (corresponding to E702Q in
TMEM16A) decreased 200-fold (from ⬃14 ␮M to ⬃ 3 mM
at ⫹60 mV) the Ca2⫹ sensitivity of TMEM16F channels
without perturbing TMEM16F targeting to cell surface
(255). This finding indicates that TMEM16A and
TMEM16F share the same putative Ca2⫹-binding site.
Surprisingly, expression in HEK-293 cells of wild-type
TMEM16F or TMEM16F with the D409G mutation identified by Suzuki et al. (215) did not cause any increase in
Ca2⫹-induced PS exposure.
The results by Yang et al. (255) are intriguing because they
suggest that anion and cation channels may coexist within
the anoctamin family. However, the discrepancy between
these results and those obtained by other authors (77, 139,
202), who identified TMEM16F as an ORCC and/or a
CaCC, remains to be clarified.
Another puzzling result is the finding that overexpression of
TMEM16F (wild-type or D409G mutant) is able to induce
PS scrambling in Ba/F3 cells (215) but not in HEK-293 cells
(255). These results would indicate that TMEM16F is required but not sufficient for scramblase activity.
Yang et al. (255) propose three alternative hypotheses to
explain TMEM16F involvement in scramblase activity
2⫹
(FIGURE 6). According to the first hypothesis, Ca
influx
through TMEM16F may activate the real (still unknown)
scramblase. However, this hypothesis implies that
TMEM16F-dependent Ca2⫹ influx could be bypassed by
directly increasing cytosolic Ca2⫹ with ionomycin. This
contrasts with the results obtained by Kmit et al. (112).
Ca2⫹ elevation by ionomycin did not induce PS scrambling
in lymphocytes obtained from two patients with Scott syndrome. In the second hypothesis, TMEM16F may work as
a Ca2⫹ sensor of the scramblase. Finally, the third hypothesis postulates that the same TMEM16F protein mediates
both cation transport and phospholipid scrambling. In one
possible model, Ca2⫹ binding to TMEM16F changes its
conformation leading to formation of a cleft through which
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441
NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
A
the hydrophilic headgroups of phospholipids may translocate from one side to the other of the membrane. This
change in conformation could also create a pore for cations
(or anions).
TMEM16F has both channel and scramblase activities
16F
B. Activity of Other Anoctamins as
Phospholipid Scramblases
B
TMEM16F as a lipid scramblase regulates the activity of a channel
16F
C
TMEM16F as a Ca2+-permeable channel activates a scramblase
Ca2+
16F
FIGURE 6. Possible models for TMEM16F function. TMEM16F
may work as a dual function protein, carrying out both ion transport and phospholipid scrambling (top). Alternatively, TMEM16F
scramblase activity may regulate the activity of other channels,
possibly by modifying the local lipid environment (middle). Finally,
TMEM16F may be essentially a channel that regulates a separate
scramblase (bottom). This regulation may involve TMEM16F-dependent Ca2⫹ influx.
442
TMEM16F as a scramblase is not alone within the
anoctamin family. In a recent study, Suzuki et al. (214)
generated a murine cell line devoid of TMEM16F expression. These cells were used as a recipient for the expression
of anoctamins. Scramblase activity was detected using fluorescently labeled lipids. Suzuki et al. (214) found that
TMEM16D, TMEM16F, TMEM16G, and TMEM16J mediate Ca2⫹-dependent scrambling of phosphatidylserine
(PS), phosphatidylcholine (PC), and galactosylceramide
(GalCer) (TABLE 1). Instead, TMEM16C expression elicited
scrambling of PC and GalCer but not of PS. Interestingly,
TMEM16D-expressing cells showed a significant level of
scramblase activity under resting conditions, in the absence
of intracellular Ca2⫹ elevation (214).
Suzuki et al. (214) also looked for ion channel activity. Only
TMEM16A and TMEM16B generated CaCC currents
(214). In particular, in contrast to other studies (77, 139,
202), TMEM16F did not appear to function as a channel
(TABLE 1). Suzuki et al. (214) also noted that some
anoctamins, namely, TMEM16E, TMEM16H, and
TMEM16K, had neither scramblase nor channel activity.
The authors proposed that the lack of function may be due
to a prevalent intracellular localization of these proteins.
The subcellular localization of some anoctamins is actually
a matter of controversy. In one study, various anoctamins
including TMEM16F were found to be intracellular and to
lack any ion channel activity (47). This finding is in contrast
to the activity of TMEM16F as an anion/cation channel
and/or a scramblase component (77, 139, 202, 215, 255).
Furthemore, other anoctamins also showed a detectable
plasma membrane localization and ion transport activity
(194, 223), although TMEM16H-K (ANO8 –10) were
more highly localized in intracellular compartments. The
discrepancy among different studies may be due to experimental conditions. It is important to note that TMEM16F
and other anoctamins appear to require high micromolar
Ca2⫹ concentrations (77, 202, 216, 255). Therefore, concentrations normally used to activate TMEM16A and
TMEM16B may not allow detection of the activity of the
other anoctamins. Other factors, possibly related to a cell
type-dependent regulation of subcellular localization, may
also be involved. It is possible that some anoctamins traffic
from intracellular compartments to plasma membrane and
back on the basis of regulatory signals that are still to be
defined.
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
Table 1. Channel/scramblase activity of anoctamins
Scramblase
PS
PC
GalCer
Channel activity
16A
16B
16C
16D
16E
16F
16G
16H
16J
16K
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
Table 1 reports the ability of the various anoctamins to perform the scrambling of phosphatidylserine (PS),
phosphatidylcholine (PC), and galactosylceramide (GalCer) as determined by Suzuki et al. (214). Table 1 also
reports channel activity for TMEM16A, TMEM16B, and TMEM16F as determined in multiple studies (29, 77,
139, 195, 202, 255, 256). However, channel activity of TMEM16F has not been confirmed in other studies
(47, 214). Other reports suggest Cl⫺ channel function also for other anoctamins.
Another source of variability may arise from different regulatory mechanisms responsible for anoctamin activation.
Some anoctamins, such as TMEM16A and TMEM16B,
may be more directly activated by Ca2⫹, whereas others
may require more indirect mechanisms based on Ca2⫹-dependent phosphorylation or interaction with other regulatory subunits (194).
It is also interesting to consider the possibility that some
anoctamins form heterooligomers. For example, coexpression of TMEM16A and TMEM16J elicited a level of anion
transport activity that was smaller than that elicited by
TMEM16A alone (194). This inhibition may arise from a
direct interaction between the two types of proteins. Such a
possibility indicates that the results obtained from heterologous expression of TMEM16A in some null systems such
as HEK-293 or FRT cells may be significantly affected by
the endogenous expression of other anoctamins, particularly TMEM16F, TMEM16H, and TMEM16K (194).
C. AfTMEM16, an Ancestral
Channel/Scramblase
Recently, Malvezzi et al. (135) succeeded in purifying a
TMEM16 protein of the fungus Aspergillus fumigatus. This
protein (afTMEM16) was reconstituted into artificial vesicles. Ion channel activity was detected when vesicles with
afTMEM16 were fused to planar lipid bilayers. These channels showed very large single-channel conductance (⬃300
pS), poor ion selectivity (PK/PCl ⫽ 1.5), and activation by
Ca2⫹ and depolarizing membrane potentials (135). When
reconstituted into liposomes, afTMEM16 showed scramblase activity induced by micromolar Ca2⫹ concentrations.
The assay was based on fluorescently labeled phospholipids
(PS, PC, PE) and quenching by dithionite (FIGURE 7). The
same liposomes also showed ion channel activity, as demonstrated with an ion flux assay. Side by side comparison of
the results obtained with the two methods allowed the study
of afTMEM16 dual function. For example, neutralization
by mutagenesis of a di-acidic motif corresponding to the
two glutamic acid residues important for Ca2⫹ sensitivity in
TMEM16A and TMEM16F resulted in afTMEM16 protein with reduced scramblase and channel activity (135).
This finding was interpreted as the proof of a common
Ca2⫹-binding site regulating both functions of afTMEM16.
The authors also concluded that separate pathways mediate
ion transport and lipid scrambling within afTMEM16. Indeed, replacement of K⫹ with an impermeant cation,
NMDG⫹, blocked ion flux but did not prevent phospholipid scrambling. Moreover, the use of a particular lipid
composition in the generation of artificial membranes also
blocked channel but not scramblase activity (135).
D. TMEM16C, a Kⴙ Channel Regulator
TMEM16C/ANO3 is an anoctamin with a particular expression in the central and peripheral nervous systems of
human, mouse, and rat (32, 88, 194). Immunostaining
demonstrated a high TMEM16C expression in a subset of
nociceptive neurons in dorsal root ganglia (88). To assess
the physiological role of TMEM16C, Huang et al. (88)
generated TMEM16C knockout rats. Behavioral tests revealed that animals without TMEM16C were hypersensitive to high temperatures (ⱖ50°C). Electrophysiological recordings from DRG neurons of mutant rats demonstrated a
broadened shape of action potentials and a decreased
threshold for action potential firing (88). When the electrical properties of neurons were studied in detail under voltage-clamp conditions, Huang et al. (88) found, unexpectedly, that a Na⫹-activated K⫹ current was strongly reduced
in knockout animals. Interestingly, TMEM16C was found
to colocalize and directly interact with Slack, the protein
forming the Na⫹-activated K⫹ channel. In a heterologous
expression system, HEK-293 cells, TMEM16C did not
form ion channels by itself but enhanced the activity of
Slack when both proteins were expressed together (88). In
particular, the presence of TMEM16C enhanced the Na⫹
sensitivity and single-channel conductance of Slack. This
finding may explain how TMEM16C regulates the excitability of nociceptive neurons. Prolonged trains of action
potentials caused by pain stimuli increase intracellular Na⫹
concentration. In the presence of TMEM16C, Na⫹ increase
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443
NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
A
Ca2+
Ion-sensitive
electrode
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
FIGURE 7. Detection of afTMEM16 as a
dual function protein. Purification and reconstitution of Aspergillus fumigatus TMEM16
protein (afTMEM16) in liposomes elicits Ca2⫹dependent ion transport, as detected with an
ion-sensitive electrode (135). Liposomes with
afTMEM16 also show scramblase activity.
This was measured with fluorescent lipids that
are transported by afTMEM16 from the inner
to the outer leaflet of liposome membrane.
The fluorescence of the externalized lipid is
quenched by dithionite. The rate of fluorescence quenching is proportional to scramblase activity.
B
Dithionite
afTMEM16
Ca2+
afTMEM16
upregulates Slack function, thus causing hyperpolarization
of the cell membrane.
V. LESSONS FROM ANIMAL MODELS
AND HUMAN DISEASES
A. Animal Models
Despite the physiological importance of CaCCs in various
tissues, until now there was no evidence of human pathologies caused by defective Ca2⫹-dependent Cl⫺ transport.
However, information deriving from knockout mice indicates that a deficit in CaCC activity/expression could have
serious consequences in various organs. At the time of the
identification of TMEM16A as a channel protein, the effects of the gene knockout were already known. Indeed,
Rock et al. (182) published in 2008 an article reporting the
importance of TMEM16A protein in murine trachea development. Actually, these authors had initially identified
TMEM16A in a screen for genes expressed in the zone of
polarizing activity (ZPA) of the developing limb (183). Interestingly, mice homozygous for a null allele of
TMEM16A did not show any alteration of limb development but died within 1 mo of birth (182). The most appar-
444
afTMEM16
ent abnormality was the presence of ventral gaps in the
cartilage rings of the trachea and an abnormal trachealis
muscle, which caused the trachea to collapse, thus mimicking human tracheomalacia (182). The authors also suggested that the cartilage defect is actually secondary to defects in the epithelium or trachealis muscle, where
TMEM16A is normally expressed (182). Interestingly, similar congenital cartilagineous defects can also be observed in
CFTR knockout mice (24), suggesting the presence of a
common defect associated with Cl⫺ transport and mediated
by separate Cl⫺ channels, TMEM16A and CFTR (184).
TMEM16A ⫺/⫺ animals also show an accumulation of
mucus in the airways (184) and an impairment of mucociliary transport (167) that mimic the CF phenotype. In addition to the trachea, TMEM16A plays a fundamental role
also in the pacemaker activity (slow waves) generated by
interstitial cells of Cajal (ICC) in gastrointestinal smooth
muscles (87, 94). Indeed, slow waves fail to develop in
muscles of TMEM16A ⫺/⫺ mice, although ICC networks
develop normally (87, 94). It is highly probable that the lack
of gastrointestinal peristalsis also contributes to the lethal phenotype of TMEM16A ⫺/⫺ animals. Given the very severe and
complex phenotype of conventional knockout mice, a more
refined analysis of the role of TMEM16A in each organ requires the development of conditional knockout animals. For
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
example, the use of mice with selective TMEM16A ablation in
nociceptive neurons demonstrated TMEM16A involvement
in the detection of thermal stimuli (34).
As discussed before, another important animal model was
created by knocking out the TMEM16B gene (19). The
results from this mouse confirmed the identification of
TMEM16B as the cilial CaCC (208). In the mouse,
TMEM16B protein is expressed on the apical surface of the
main olfactory epithelium (where it colocalizes with the
ciliary marker acetylated tubulin), in vomeronasal sensory
neurons (colocalized with TMEM16A), and on the axons of
the olfactory sensory neurons (19). TMEM16B ⫺/⫺ mice
lack CaCC currents in the main olfactory epithelium and in
the vomeronasal organ and show a 40% reduction in the
fluid-phase electro-olfactogram responses. However, as
stated previously, they have no detectable impairment
of olfaction (19). Future studies will need to address this
issue by generating conditional knockout mice for the
TMEM16B gene. In this way, by knocking out the gene at
the desired location and at a specific time, it will be possible
to rule out possible compensatory mechanisms that may
intervene in the conventional knockout mouse by minimizing the effect of TMEM16B gene loss.
Recently, two knockout mice defective for TMEM16F have
been also generated. One of these animal models (255) has
been discussed in the previous paragraph. This animal
shows a significant impairment in the coagulation process
and in the PS scramblase activity of platelets, erythrocytes,
and B-cells. The authors reported no other obvious phenotype stating that the knockout mice were viable and fertile
(255). In a subsequent study, another group of investigators
reported instead a severe defect in mineral deposition in the
bone of TMEM16F animals resulting in reduced skeleton
size and skeletal deformities (53). This defect was consistent
with a particular expression of TMEM16F/ANO6 in ossifying tissues of endochondral and intramembranous bones.
In particular, TMEM16F appeared constantly expressed in
osteoblast and, less markedly, in osteocytes. Ehlen et al.
(53) reported a high (60%) mortality of TMEM16F-deficient mice at birth. This may be caused by asphyxation since
the mutant animals showed a narrow ribcage due to deformed ribs. Abnormalities at birth also involved the limb
and the skull bones. Analysis of the embryos demonstrated
no alteration of osteoblast differentation but a strongly reduced bone mineralization ability. This defect was confirmed in vitro on osteoblast cultures from wild-type and
mutant animals. Osteoblasts from TMEM16F ⫺/⫺ mice
showed a 50 –70% reduction in mineral deposition but no
alteration of proliferation or apoptosis (53). Interestingly,
osteoblasts of knockout mice also showed a markedly reduced PS scramblase activity as detected by annexin V binding following treatment of cells with the Ca2⫹ ionophore
ionomycin. The authors investigated whether TMEM16Fdependent scramblase activity in osteoblasts is related to
apoptosis. Therefore, they triggered apoptosis with staurosporine. This treatment resulted in equal PS scrambling in
both wild-type and TMEM16F-defective cells (53). These
results suggest the presence of two types of scramblase activity: one type activated by intracellular Ca2⫹ elevation
and mediated by TMEM16F/ANO6, and the other type
triggered by apoptotic stimuli and due to another unknown
protein. Regarding the role of TMEM16F in bone development, Ehlen et al. (53) commented that PS scrambling is
important for bone mineralization. TMEM16F may be important for the release of PS-rich vesicles. These vesicles,
carrying high concentrations of Ca2⫹ and phosphate, initiate the mineralization process.
Comparison of the two studies done on TMEM16F knockout mice (53, 255) evokes intriguing questions. What is the
reason for the skeletal abnormalities and the high mortality
in one study and not in the other? In this respect, it has to be
noted that Scott syndrome patients show no obvious skeletal defects. On the other hand, Ehlen et al. (53) did not
observe a marked defect in coagulation in their animals,
although they did not perform specific tests. It is possible to
hypothesize that other anoctamins may differently compensate for the loss of TMEM16F in humans compared with
mice. Furthermore, differences in the strategy for the generation of the knockout animals may also explain the different phenotypes (53, 255).
B. Human Genetic Diseases
Precious information on the role of anoctamins may also
derive from human genetic diseases (TABLE 2). Among the
members of the anoctamin gene family, TMEM16E/ANO5
was the first to be associated with human diseases. In 2004,
Tsutsumi et al. (227) proposed that gnathodiaphyseal dysplasia (GDD), a rare dominant skeletal syndrome, could be
due to dominant mutations occurring in the so-called
GDD1 gene, that corresponds to TMEM16E. A linkage
analysis of two independent families allowed the authors to
find two different missense mutations, both at codon 356,
which corresponds to an evolutionarily conserved cysteine
in human, mouse, zebrafish, fruit fly, and mosquito (227).
Recently, a novel mutation, T513I, was found in another
family with GDD (138).
Topology prediction performed using the TMpred software
suggested that GDD1 contains eight transmembrane domains, with cytosolic NH2 and COOH termini (227). Immunolocalization studies demonstrated that wild-type
GDD1 protein is localized in the ER and that overexpression of the mutant proteins decreased cell adhesion and
changed cell morphology (227). Because of protein localization and topology, the authors hypothesized that GDD1
might function as an intracellular channel of the ER membrane involved in the Ca2⫹-dependent signaling pathway,
and that mutations induce excess Ca2⫹ release from the ER
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
Table 2. Anoctamins in physiology and pathology
Relationship With Human Genetic
and Nongenetic Diseases
TMEM16A/ANO1
Upregulation in gastrointestinal
stromal tumors (GISTs), in breast
cancer, and in head and neck
squamous cell carcinomas
(HNSCCs); upregulated in asthma
TMEM16B/ANO2
TMEM16C/ANO3
Mutated in cervical dystonia
TMEM16E/ANO5
Mutated in gnathodyaphyseal dysplasia
(GDD), limb-girdle muscular
dystrophy (LGMD2L), and Miyoshi
myopathy (MMD3)
TMEM16F/ANO6
Mutated in Scott syndrome
Phenotype in Knockout Mouse
Physiological Role
Tracheal malformation, impaired
mucociliary clearance, block of
gastrointestinal peristalsis
Transepithelial ion transport, smooth muscle
contraction, nociception, cell proliferation,
and migration
Absence of CaCCs of olfactory receptors
but no impairment of olfaction
Modulation of olfactory transduction,
phototransduction, and neuronal
excitability
Increased sensitivity to heat
Regulation of Na⫹-dependent K⫹ channel
(Slack)
Unknown (membrane repair?)
Impaired coagulation and skeleton
abnormalities (in 2 different models)
Phospholipid scramblase? role as a Cl⫺
(ORCC/CaCC) and/or cation channel
(SCAN)
TMEM16G/ANO7
Upregulation in prostate cancers
Unknown
TMEM16K/ANO10
Mutated in cerebellar ataxia
Unknown
(227). In 2007, the same authors demonstrated that, in the
adult mouse, GDD1 protein is highly expressed in cardiac
and skeletal muscle tissues, and in growth plate chondrocytes and osteoblasts in bone, suggesting diverse cellular
roles of GDD1 in the musculoskeletal system (151). In this
respect, recessive mutations in TMEM16E/ANO5 were associated with two different muscular dystrophies, proximal
limb-girdle muscular dystrophy (LGMD2L) and distal Miyoshi myopathy (MMD3), with characteristics that resemble dysferlinopathies caused by mutations in the dysferlin
gene (23, 84). Dysferlin is a component of the sarcolemmal
repair machinery, predicted to function as a fusogen in the
formation of the patch membrane required for membrane
resealing (79). Therefore, it was proposed that TMEM16E
may play a role in the dysferlin-dependent muscle membrane repair pathway (23). In this respect, Cl⫺ currents
were recorded after membrane wounding in sea urchin embryos (59), suggesting that TMEM16E may be responsible
for a chloride current needed during membrane repair in
muscle (23). Loss-of-function TMEM16E mutations may
cause skeletal muscle demise because of defective membrane repair (23).
A second anoctamin gene causing a human genetic disease is
TMEM16K. Mutations in TMEM16K/ANO10 were found
to cause autosomal-recessive cerebellar ataxia (236).
TMEM16K has the highest expression in the adult brain,
particularly in the cerebral cortex and in the cerebellum,
consistently with the relatively late onset of ataxia (236).
Although it has not been elucidated yet whether
TMEM16K codes for a CaCC, this hypothesis is suggestive,
because these channels participate in the regulation of neuronal excitability. Indeed, one of the most important mechanisms causing cerebellar ataxia is deranged Ca2⫹ signaling
in Purkinje cells (104), which is the consequence of mutations of several known ataxia-associated genes, like Ca2⫹
pumps or voltage-gated Na⫹ or K⫹ channels (226, 230,
446
243). If TMEM16K protein is a (or a subunit of a) CaCC, it
might play a role in influencing Ca2⫹ signaling in Purkinje
cells, and an abnormal function of TMEM16K could lead
to cerebellar ataxia through this mechanism (236).
Recently, a study reported the identification of a third disease-causing gene in the anoctamin family. Linkage analysis
combined with whole-exome sequencing identified mutations in the TMEM16C/ANO3 gene that cause a dominant
form of craniocervical dystonia (32). Five missense mutations were identified in different families: T54I, W490C,
R494W, S685G, and K862N. A sixth mutation was a C to
T base change in the 5=-untranslated region of the gene. The
mechanism of action of mutations is unknown since the
function of TMEM16C is not well defined. However,
TMEM16C seems particularly expressed in striatum and,
as discussed previously, appears to regulate a neuronal K⫹
channel (88). Therefore, it is possible that alterations in
TMEM16C function caused by the dominant mutations
perturb the excitability of striatal neurons. It would be interesting to assess if the causal relationship between
TMEM16C and craniocervical dystonia in humans is related with the regulatory role of TMEM16C on Na⫹-dependent K⫹ channels discovered in rats (88).
As discussed in previous sections of this review, another
anoctamin gene, TMEM16F, was found to be mutated in
Scott syndrome (215). Scott syndrome is a rare congenital
bleeding disorder characterized by a deficiency in platelet
procoagulant activity that is not associated with decreased
levels of coagulation factors (244, 268). More precisely,
Scott syndrome is due to an aberrant phospholipid composition of the outer membrane leaflet of platelets, namely, a
defective surface exposure of PS upon Ca2⫹-dependent activation by injured blood vessels (267). Defective function
of the Ca2⫹-dependent scramblase protein is the cause
of Scott syndrome (127, 267). Direct sequencing of
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
TMEM16F gene of a subject affected by Scott syndrome revealed that the patient carried a homozygous mutation at the
splice-acceptor site in intron 12, which caused skipping of
exon 13 with a frameshift resulting in the premature termination of the TMEM16F protein in exon 14, at the third transmembrane segment (215). More recently, Castoldi et al. (31)
identified two novel mutations in the TMEM16F gene in a
patient with Scott syndrome: a transition at the first nucleotide of intron 6 (IVS6⫹1G¡A), disrupting the donor
splice site consensus sequence of intron 6, and a singlenucleotide insertion in exon 11 (c.1219insT), predicting a
frameshift and premature termination of translation at
codon 411.
types of human cancers were analyzed (188). TMEM16A
was mostly expressed in head and neck squamous cell carcinomas (HNSCCs) and in gastrointestinal stromal tumors
(GISTs). In another study, TMEM16A/ANO1 was found to
be overexpressed in breast cancers (25). TMEM16A overexpression is in many cases due to amplification of the corresponding chromosomal region in 11q13 (9, 188). Interestingly, ANO1 protein expression is associated with reduced survival and increased metastatic potential (9, 52,
188). In particular, individuals with cancers having
TMEM16A expression were characterized by a median survival time of 23 mo compared with 56 mo for individuals
without TMEM16A expresssion (188).
In a study carried out on the platelets of a Scott syndrome
patient and from control individuals, the results provided
evidence for the existence of at least two mechanisms of
phospholipid scrambling (232). Stimulation of platelets
with the Ca2⫹-mobilizing agonists convulxin and thrombin
generated a high level of phosphatidylserine externalization
in normal platelets. This process was totally absent in the
platelets of the patient, which indicated the total dependence on a functional TMEM16F protein. In contrast, the
PS exposure induced by a pro-apoptotic stimulus was only
partially inhibited in the platelets of the Scott syndrome
patient (232). If TMEM16F is a Ca2⫹-dependent scramblase, these results would indicate the existence of a second
protein involved in the externalization of PS. Alternatively,
it can be hypothesized that TMEM16F is one of the multiple regulators (e.g., the Ca2⫹ sensor) of a still unknown
scramblase.
In some in vitro studies, done on cultured cancer cells,
TMEM16A expression correlated with the rate of cell proliferation. Indeed, TMEM16A knockdown by RNA interference (25, 52, 129) or inhibition with CaCC inhibitors
(25, 145, 207) resulted in reduced cell growth in vitro.
TMEM16A was also found to influence cell migration, as
determined by wound healing assays. The rate of cell migration was significantly reduced by TMEM16A downregulation (9, 129, 188). In some studies, TMEM16A appeared to affect migration but not proliferation (9, 188). In
another study, TMEM16A influenced GIST cell growth in
vivo but not in vitro (204). TMEM16A role in tumor
growth in vivo was also demonstrated for HNSCC, prostate
carcinoma, and breast cancer (25, 52, 129).
C. TMEM16 Proteins and Cancer
Even before the discovery of TMEM16A and TMEM16B
as possible Cl⫺ channels, it was found that many anoctamins are highly expressed in human cancers (TABLE 2).
TMEM16A, named DOG1 (discovered on gastrointestinal
stromal tumor 1), was highly expressed in gastrointestinal
stromal tumors (245). Known also as TAOS2 (tumor amplified and overexpressed sequence 2), TMEM16A gene
was amplified and overexpressed in oral squamous cell carcinomas (91). TMEM16G, also termed NGEP, is particularly expressed in prostate cancers (17). Furthermore, the
pattern of TMEM16F RNA splicing is associated with the
metastatic potential of mammary cancers in mouse and
with the malignancy of breast cancers in humans (49).
The reason for the high expression of some anoctamins,
particularly TMEM16A, in tumors is unclear. However,
various studies have recently started to investigate the relationship between TMEM16A and cancer.
It appears that TMEM16A/ANO1 overexpression is not a
general phenomenon but is related to specific types of tumors. In a recent study, 4,000 samples belonging to 80
The differences among studies, particularly regarding the
effect of TMEM16A in vitro, may imply cell type-dependent mechanisms. For example, Ubby et al. (228) expressed
various TMEM16A isoforms in HEK-293 cells using an
inducible system. TMEM16A expression did not correlate
with cell migration or proliferation in vitro. Therefore, the
role of TMEM16A in cell proliferation and migration is not
universal but dependent on the particular intracellular and
extracellular environment of cancer cells.
The link between TMEM16A/ANO1 and cancer cell
growth is still unclear. However, it may be related to altered
cell signaling. For example, downregulation of TMEM16A
expression in GIST cells resulted in strong upregulation of
insulin-like growth factor-binding protein 5 (IGFBP5), a
potent antiangiogenic factor (204). In breast cancer cells,
TMEM16A expression/function instead correlated with
signaling through the EGF receptor and the calmodulindependent kinase (25). Furthermore, it was found that
TMEM16A overexpression increases the phosphorylation
of ERK1/2 and the levels of cyclin D1 (52). In this study,
overexpression of TMEM16A with a mutation (K610A)
that abolishes Cl⫺ transport failed to induce phosphorylated ERK1/2 (52). This finding indicates that Cl⫺ transport
is important for the effect on cell cycle and proliferation, a
mechanism that may involve a change in intracellular Cl⫺
concentration. It is important to remark that, in non-cancer
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
cells (basilar artery smooth muscle cells), TMEM16A expression appears to have an inverse relationship with proliferation (238). Wang et al. (238) found that TMEM16A
downregulation induced by angiotensin II stimulation was
associated with increased cell proliferation and increased
levels of cyclins D1 and E.
Another mechanism that may explain the role of TMEM16A
in cell proliferation and, particularly, in cell migration is cell
volume regulation. Coordinated efflux of Cl⫺ and K⫹
causes cell shrinkage. This may favor the migration of cancer cells through tissues. Indeed, swelling and shrinkage
occur at the front end and rear end of a migrating cell,
respectively. The role of TMEM16A in cell volume regulation was demonstrated in an overexpressing cancer cell line
(BHY). Application of a hypotonic shock did not cause the
expected cell swelling (188). Actually, cell swelling was observed only when TMEM16A was silenced with siRNA.
This result may be explained by a fast activation of
TMEM16A, in nonsilenced cells, that rapidly shrinks the
cells, thus preventing the detection of the transient cell
swelling.
In summary, several studies indicate an important role of
TMEM16A in some types of human cancers. TMEM16A is
therefore an important diagnostic and prognostic marker
and may represent an important drug target.
The other anoctamins may have additional and even more
important roles in cancer cell biology. For example,
TMEM16F, with its involvement in cell migration, cell volume regulation, and apoptosis (3, 139, 217), could have a
particular impact on the growth and metastatization of human cancers.
VI. DRUGGABILITY OF CACCs
For many years, the lack of selective inhibitors has limited
the studies on CaCCs. Selective and potent blockers are
valuable research tools to identify ion channels, to analyze
tissue distribution and contribution to physiological processes, and also to explore the properties of the channel
pore. Available CaCC inhibitors were neither selective nor
potent, requiring high concentrations to completely block
Cl⫺ currents with undesirable side effects. Niflumic acid
(FIGURE 8), often considered a specific blocker and even
used to identify membrane currents as CaCCs in different
tissues, can also affect other channels or cause emptying of
intracellular Ca2⫹ stores (11, 38, 80, 165, 176, 197, 264).
In addition to being valuable research tools, more selective
CaCC inhibitors could also have important applications as
therapeutic agents (234, 235).
When TMEM16A was discovered as a CaCC component, it
was found that the anion transport associated with its expression is sensitive to a large panel of general Cl⫺ chan-
448
nel inhibitors (FIGURE 8), including niflumic acid, NPPB,
DIDS, mefloquine, and fluoxetine in the 5–20 ␮M range
(29, 195, 256). DPC, another nonspecific Cl⫺ channel
inhibitor, required millimolar concentrations to be effective (195). CFTRinh-172, a compound widely used as a
selective CFTR inhibitor (28, 132), was ineffective on
TMEM16A currents (29). In contrast, GlyH-101, which is
also used to block CFTR (153), showed a significant activity at 20 –50 ␮M (29).
Other CaCC/TMEM16A inhibitors have been identified by
screening chemical libraries using a high-throughput functional assay. In one of the first studies, the molecular identity of CaCCs was still unknown. Therefore, the authors
used an intestinal epithelial cell line, HT-29, with endogenous activity of CaCC, to perform a high-throughput
screening of a library of 50,000 compounds (44). The authors reported the identification of six new classes of CaCC
inhibitors, two of which (classes A and B) considered particularly interesting, on the basis of multiple criteria, including potency, water solubility, drug-like properties, identification of active analogs, chemical stability, and CaCC targeting (44). Among these inhibitors, the compound labeled
CaCCinh-A01 (FIGURE 8) is one of the most used to test the
contribution of CaCCs to physiological processes (81, 166).
After the identification of TMEM16A as a CaCC, Verkman
et al. (155) performed a second high-throughput screening
aimed at the discovery of new classes of small molecule
TMEM16A inhibitors, using a rat epithelial cell line, FRT,
with heterologous expression of TMEM16A, and a library
of 110,000 compounds. The authors found four novel
classes of inhibitors that seem to target directly TMEM16A
protein, rather than upstream processes, such as extracellular agonist binding or Ca2⫹ signaling. In addition, they
compared the effect of one of the newly discovered
TMEM16A inhibitors, T16inh-A01 (FIGURE 8), with that of
CaCCinh-A01 (44) in various tissues, concluding that, while
in salivary gland epithelium TMEM16A is responsible for
all CaCC activity, in airway and intestinal epithelia it is only
a minor contributor to total CaCC current (155). T16inhA01 has also been used by different research groups as a
pharmacological probe to demonstrate the involvement of
TMEM16A channel in a series of physiological and pathological processes including transepithelial ion transport,
smooth muscle function, and cancer (43, 52, 152, 213).
T16inh-A01 was also found to inhibit acrosomal reaction in
human spermatozoa, thus revealing a possible role of
TMEM16A in the fertilization process (164).
In another study, the screening of a relatively small chemical
library (2,000 compounds) identified benzbromarone (FIGURE 8), dichlorophen, and hexachlorophen as TMEM16A
inhibitors (89). These compounds inhibited airway smooth
muscle contraction and mucin release from goblet cells,
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
F
F
OH
O
O2N
H
N
HO
COOH
O
S
N
O
C
S
F
N
H
N
S
C
O S
N
OH
O
niflumic acid
NPPB
tannic acid
OH
DIDS
CH3O
HO
Br
HO
O
O
OH
HO
O
OH
eugenol
O
OH
HO
O
O
Br
OH
O
CH3
O
HO
OH
O OH
OH
HO
benzbromarone
O
OH
N
O
HO
OH
N
H
N
S
O
N
Eact
O
O
S
O
O
NH
O
S
T16inh-01
N
N
O
O
S
CaCCinh-01
O
FIGURE 8. Pharmacological modulators of CaCCs. Structure of classical CaCC blockers (niflumic acid,
NPPB, DIDS), of new TMEM16A inhibitors (T16inh-A01, CaCCinh-A01, tannic acid, eugenol, benzbromarone),
and of a TMEM16A activator (Eact).
which underlines the potential of TMEM16A as a drug
target in asthma.
Natural compounds appear as another interesting source of
TMEM16A/CaCC inhibitors. In one study, by screening a
chemical library with a high representation of natural compounds, the authors found that tannic acid (FIGURE 8) is an
effective inhibitor of TMEM16A Cl⫺ currents (156). Tannic acids and other related compounds also had an inhibitory effect on the contractility of arterial smooth muscle.
The authors concluded that the gallotannin content of red
wine and green tea could be the molecular basis for their
beneficial effect on the cardiovascular system (156). In another study, the antidiarrheal effect of a Thai herbal preparation was investigated (257). Fractionation of the preparation by HPLC led to the identification of eugenol as a
TMEM16A inhibitor, although with potency in the high
micromolar range.
In a recent study, the evaluation of a selected panel of anthranilic acid derivatives allowed the identification of a potent TMEM16A inhibitor, termed MONNA (162). This
compound acted with an IC50 of 0.08 and 1.3 ␮M on Xenopus and human TMEM16A, respectively. MONNA appeared to be quite selective regarding other anion channels,
since it did not block CFTR, ClC2, and bestrophin 1 (162).
As for other TMEM16A inhibitors, it is not clear yet if
MONNA acts directly on the channel or indirectly through
interaction with a regulatory protein.
Verkman et al. (157) have also looked for activators of
TMEM16A, which could be useful as research tools and as
potential drugs for the treatment of salivary gland dysfunction, cystic fibrosis, dry eye syndrome, intestinal hypomotility, and other disorders associated with Cl⫺ channel dysfunction. The chosen strategy was once again the screening
of a library of 110,000 compounds using FRT cells with
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449
NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
heterologous expression of TMEM16A (157). The authors
reported the identification of six classes of TMEM16A activators, two of which (classes E and F) considered particularly interesting, because they do not cause elevation of
cytoplasmic Ca2⫹ and produce maximal TMEM16A activation, although class E activator (FIGURE 8) can activate
TMEM16B as well (157). Looking at a panel of analogs, the
authors also realized that minor chemical structural
changes can convert an activator into an inhibitor, since
they found compounds belonging to classes B and E that
fully inhibited TMEM16A Cl⫺ conductance, which supported the conclusion that these compounds target
TMEM16A directly (157). Interestingly, a similar behavior,
i.e., compounds having both activator and inhibitor properties, had also been previously reported for native CaCCs
(125, 173). In this respect, it should be noted that the properties of CaCC pharmacological modulators may be affected by phosphorylation (246).
Pharmacological sensitivity of TMEM16A channels may
also be affected by cell environment and extent of expression. Indeed, it was found that, in heterologous expression
systems, but not in native cells, TMEM16A channels are
stimulated by openers of Ca2⫹-activated K⫹ channels such
as 1-EBIO (223, 224).
In summary, a large panel of pharmacological inhibitors
and, to a lesser extent, of activators of the TMEM16A
channel is now available. In contrast, the pharmacology of
the other anoctamins is still in its infancy. TMEM16A pharmacological modulators may have broad therapeutic applications. By antagonizing the contractility of smooth muscle
cells (43), TMEM16A inhibitors may be useful in hypertension, asthma, and as spasmolithic agents. The inhibition of
mucin release shown by some TMEM16A inhibitors (89)
suggests that they may be beneficial in asthma not only as
bronchodilators but also to prevent mucus hypersecretion.
However, it is not clear whether inhibition of mucin release
from goblet cells is indeed a good thing for asthmatic patients. Actually, activators of TMEM16A were considered
useful in cystic fibrosis and other chronic respiratory disease
to promote mucociliary clearance (157). Therefore, the precise role of TMEM16A in the airway epithelium needs to be
clarified. Recently, a study done on a transgenic mouse has
shown that hypersecretion of MUC5AC in the airways actually has protective role (54). The implications of this
study are that the abundant mucin secretion alone is not the
cause of airway obstruction in some respiratory diseases.
Inadequate support of electrolytes and water, in parallel
with mucin release, may be a critical factor.
TMEM16A activators have the potential ability to generate
important side effects (FIGURE 9). If delivered to the airways
by inhalation, they could potentially generate bronchoconstriction by acting on airway smooth muscle. If delivered
systemically, they could induce pain, at least theoretically,
450
by acting on the DRG cells where TMEM16A works as a
heat sensor (34). Furthermore, it is not clear whether activators may also generate problems (e.g., hypertension,
spasms, colics) in the cardiovascular system, gastrointestinal tract, urinary system, and other organs where the activity of TMEM16A favors smooth muscle contraction.
TMEM16A inhibitors may also generate side effects systemically. For example, block of TMEM16A could produce
block of gastrointestinal peristalsis. A better understanding
of TMEM16A function in different tissues, its interaction
with regulatory networks, and the role of splicing variants
will be important for the rational development of selective
drugs.
VII. CONCLUSIONS
The identification of TMEM16A/ANO1 and TMEM16B/
ANO2 apparently ended a search that lasted for several
years: the long sought CaCCs had finally a molecular identity (29, 195, 256). In particular, it appeared that the finding of two different proteins working as CaCCs, each one
available in multiple variants, was able to explain the variety of biophysical characteristics previously reported for
CaCCs in different tissues (48, 80). TMEM16A channels
were similar to native CaCCs expressed in epithelial and
smooth muscle cells, whereas TMEM16B channels were
more representative of CaCCs observed in olfactory receptors and other neurons. Furthermore, the existence of
TMEM16A and TMEM16B paralogs in the anoctamin
family suggested that other types of Cl⫺ channels could be
discovered. Indeed, TMEM16F/ANO6 expression was
found associated with the activity of outwardly rectifying
Cl⫺ channels, ORCCs (139), or with CaCCs having “noncanonical” characteristics (77, 202). However, the intriguing and unexpected discovery of TMEM16F as a membrane
protein linked to the scrambling of membrane phospholipids (215) revealed the possibility that some anoctamins have
additional or alternative functions.
At the moment, it is difficult to reconcile all results obtained
with TMEM16F. One possibility is that TMEM16F is essentially a scramblase that in turn regulates ion channels,
possibly by modification of the local lipid environment (FIGURE 6). In this respect, there are several examples of channels or receptors whose activity is influenced by electrostatic
interaction with the charged head groups of phospholipids
or by changes in the thickness/composition of the lipid bilayer (39, 83, 131). If TMEM16F regulates other channels,
this could explain the discrepant results obtained in different studies. Indeed, TMEM16F has been found to associate
not only with Cl⫺ channels (CaCC or ORCC) but also with
cation channels (255). To explain these conflicting results, it
can be hypothesized that TMEM16F regulates different
types of cation and anion channels and that the expression
of these channels is cell type dependent or affected by other
types of experimental conditions. Other anoctamins may
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TMEM16 PROTEINS AS CHLORIDE CHANNELS
inhibition of
mucociliary clearance
analgesia
epithelial
cells
nociceptive
neurons
stimulation of
anion secretion,
mucolytic therapy
pain
TMEM16A
inhibitors
TMEM16A
activators
bronchial relaxation
anti-hypertension
block of peristalsis
inhibition of cancer cell
proliferation/migration
smooth muscle
cells, ICCs
cancer
cells
bronchoconstriction
stimulation of
cell proliferation (?)
FIGURE 9. TMEM16A as a drug target. Scheme of possible pharmacological effects (blue: therapeutic
effecs; red: potential undesired effects) of TMEM16A activators and inhibitors. Stimulation of TMEM16A
activity in the epithelial cells of the airways could potentially improve mucociliary clearance by enhancing the
secretion of anions (Cl⫺ and bicarbonate) and the fluidification of mucus. However, because of TMEM16A
function in smooth muscle cells and nociceptive neurons, TMEM16A activators could also cause bronchoconstriction and/or increased sensitivity to painful stimuli. Block of TMEM16A function with inhibitors could be
potentially beneficial to induce relaxation of smooth muscle cells in different organs (e.g., to treat hypertension). However, TMEM16A inhibitors, by acting on ICCs, could also cause block of intestinal peristalsis.
also behave in this way. For example, TMEM16C/ANO3,
although apparently devoid of channel activity, is able to
regulate the activity of the Slack K⫹ channel (88). The underlying mechanism is unknown, although there is evidence
of a direct physical interaction between the two proteins
(88). If TMEM16C is a scramblase, as recently proposed
(214), the translocation of lipids in close proximity of the
Slack channel could be the basis of the regulatory mechanism.
Another alternative hypothesis is that TMEM16F is actually a channel that instead regulates a phospholipid scramblase (FIGURE 6). This scheme would be consistent with the
finding of TMEM16F as a cation channel with a particular
permeability to divalent cations (255). Accordingly, the
TMEM16F-dependent Ca2⫹ influx could be the mechanism
through which a separate scramblase is activated. The role
of TMEM16F as a channel and not a scramblase would be
also consistent with the lack of significant phospholipid
scrambling when TMEM16F is overexpressed in HEK-293
cells (255).
At the moment, it is not clear which hypothesis is correct.
However, the recent study on the fungal TMEM16
(afTMEM16) reveals that ion transport and lipid scrambling may be indeed carried out by the same anoctamin
(135). Similar studies, involving purification and reconstitution in an artificial lipid bilayer, need to be replicated on
other anoctamins, particularly on mammalian TMEM16F,
to assess their functions in a cell-free system. If TMEM16F
is indeed a dual activity protein, working also as an ion
channel, it remains to be explained why there are contrasting results about its ion selectivity. Another intriguing aspect of channels associated with TMEM16F is the relatively
long delay (minutes) between cytosolic Ca2⫹ elevation and
channel activation (77, 202). This behavior may imply an
indirect mechanism of activation by Ca2⫹ or a Ca2⫹-induced traffic of the protein from intracellular compartments to the plasma membrane. Actually, there are discrepant results about the subcellular localization of TMEM16F
and other anoctamins (47, 77, 202). The contrasting results
may derive from different experimental conditions that
have an effect on the localization of TMEM16F and other
anoctamins.
In contrast to TMEM16F and other close paralogs,
TMEM16A and TMEM16B expression is not associated
with scramblase activity (214). This could mean, as sug-
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NICOLETTA PEDEMONTE AND LUIS J. V. GALIETTA
gested by results obtained with afTMEM16 (135), that
the dual function, particularly the scramblase activity, is
an ancestral characteristic. Conversely, CaCC activity is
a function that was later acquired by a branch of the
anoctamin family represented by TMEM16A and
TMEM16B.
Despite the variety of functions, a common characteristic of
most, if not all, anoctamins is the regulation by cytosolic
Ca2⫹. The activation seems mediated by direct interaction
of Ca2⫹ with a pair of highly conserved glutamic acid residues (255, 259). Mutagenesis of these acidic residues lowered the Ca2⫹ sensitivity of channels associated with
TMEM16A, TMEM16F, and afTMEM16 (135, 255, 259).
However, since mutagenesis of acidic residues can alter
Ca2⫹-dependent activation in an allosteric way, other
Ca2⫹-binding sites cannot be excluded.
It is also probable that anoctamins, particularly TMEM16A
and TMEM16B, are additionally controlled by calmodulin.
Nevertheless, in this case, the situation is more complex.
Different calmodulin-binding sites have been identified,
each one with a different role on channel activity. For example, one study showed that calmodulin binding essentially affects the ion selectivity of TMEM16A, particularly
its permeability to bicarbonate, but not its gating (101).
Conversely, in other studies calmodulin binding to
TMEM16A influenced its channel activity (222, 237). Understanding the precise role of calmodulin requires further
investigation. However, it is interesting to note that the
different studies have in common the focus on a region
localized in the NH2 terminus before the first transmembrane segment. Interestingly, calmodulin seems also to have
a role on channel inactivation, a process that, at least for
TMEM16B, could desensitize the channel during long-lasting cytosolic Ca2⫹ increases (237).
In summary, the study of anoctamins has started to reveal a
variety of functions, from ion transport to membrane lipid
dynamics. While TMEM16A and TMEM16B may be considered as “pure” channels, full understanding of the main
role played by the other anoctamins needs further investigation. The discovery of TMEM16 proteins also paves the
way for the search of novel pharmacological agents that will
be potentially useful for the treatment of a variety of human
diseases. TMEM16A, as discussed in previous sections of
this review, is a potential target for the treatment of asthma,
cystic fibrosis, hypertension, gastrointestinal motility disorders, and cancer. Instead, TMEM16F, because of its particular role in blood coagulation, could be a target of antithrombotic therapies (160). However, the expression of
TMEM16A, TMEM16F, and other anoctamins in multiple
tissues implies a careful design of drugs to avoid important
side effects. This requires a better comprehension of the
structure-function relationship, physiological role, and regulation of each single anoctamin, a goal that will require a
452
combined effort based on multiple approaches including
gene silencing in native cells, overexpression in null systems,
and conditional knockout in mice.
ACKNOWLEDGMENTS
We thank Dr. Anna Capurro for revising and correcting the
manuscript.
Address for reprint requests and other correspondence:
L. J. V. Galietta, U. O. C. Genetica Medica, Istituto Giannina Gaslini, Largo Gerolamo Gaslini, 5, 16147 Genova,
Italy (e-mail: galietta@unige.it).
GRANTS
Our research is supported by grants from Telethon Foundation (GGP10026), Fondazione Italiana per la Ricerca
sulla Fibrosi Cistica (FFC #2/2012), and Ministero della
Salute (Ricerca Finalizzata: GR-2009-1596824; Ricerca
Corrente: Cinque per mille).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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