Chloride Channels
Yuanting Lu
Cristian Escobar
09/23/2011
Cells actively transport Cl- across the plasma membrane by transporters that accumulate Clintracellularly (Cl- loaders including the Na+-K+-Cl- cotransporters NKCC, Cl--HCO3- exchangers AE, and
Na+-Cl- cotransporters NCC) or pump Cl- out of the cell (K+-Cl- cotransporters KCC and Na+-dependent Cl- HCO3- exchanger NDCBE). Cl- flows passively across a variety of Cl- channels in the plasma membrane
including Ca-activated Cl- channels (CaCC), cAMP-activated Cl- channels (CFTR), cell-volume regulated
anion channels (VRAC), and ligand-gated anion channels (GABAA and glycine receptors). In addition, Clchannels and transporters are found in intracellular membranes, such as the endosomallysosomal
pathway, and play a role in regulating intra-vesicular pH and Cl- concentration. Intravesicular pH and [Cl-]
are important in vesicular trafficking.
Functions
• Stabilization of membrane potential.
ClC-1
Voltage dependent
ClC-2
GABA
Glycine
• Depolarization
Bestrophin
TMEM16A
Ligand gated Cl- channels
Ca2+
• Cell volume regulation.
Volume sensitive Cl- channels
VDCC
• Fluid transport in epithelia.
ClC-Kb
pH
CFTR
cAMP
• Neutralization of H+ in lysosomes.
pH
ClC-3
CLC Chloride Channels
CLC Chloride Channels Structure
CLC Chloride Channels
Stabilization of membrane potential
Influx of Cl- suppresses
depolarizing
inputs
and
stabilizes membrane potential
Myotonia and muscle fiber type changes. Mutations in Na1 (1) or Cl2 (2) channels or the lack of the sarcolemmal chloride channel
(ClC-1; 2) can lead to overexcitability of the sarcolemma (myotonia). Normal muscle responds with single action potentials upon
single stimuli, whereas myotonic muscle often responds with runs of action potentials. Increased membrane excitation can cause
protein kinase C (PKC) activation in the nucleus and changes in the pattern of myogenic regulating factors (MRF). The myogenic
factors control gene transcription and therewith couple membrane excitation to the muscle fiber type. A second signaling pathway
involves cytoplasmic Ca21. The propagation of action potentials into the transverse tubule system (TT) activates the L-type Ca21
channel and stimulates Ca21 release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR). A Ca21 signaling
pathway into the nucleus is suggested.
CLC Chloride Channels Functions
Endosomal acidification
•
H+ concentration inside of
lysosomes require Cl- influx
to
neutralize
positive
charges.
•
Some ClC channels mediate
Cl−:H+ exchange rather than
voltage-dependent anion
channel activity.
CLC Chloride Channels
Epithelial transport
•
ClC-Kb
transport
Clacumulated
by
NKCC
trasporter to the intertisium.
Fluid transport and Cl– channel. Ascending loop of Henle (thick ascending limb, TAL) is an important part
of the tubular segment (left panel), playing a cardinal role in construction of high osmolality in the renal
medulla by unidirectional transport of NaCl. Luminal Na+, K+ and Cl– are transported via the Na/K/2Cl
transporter into cells without water. Na+ and Cl– are transferred into the basolateral space and thereby
concentrated in osmolality. K+ is back-leaked by K+ channels resulting in lumen-positive (compare with
interstitium) transtubular potential. This lumen-positive potential drives cations, Ca2+ or Mg2+, through
the cell junction (claudin) under the electrochemical gradient. Thus, all 3 modes of transport, Na/K/2Cl
transporter, K channel and Cl channel (ClCK) are cardinal in the construction of the high osmolality.
Cystic fibrosis transmembrane conductance regulator (CFTR)
•
Localization on apical
membranes
•
Plays an important role
in fluid secretion in
airways,
intestine,
sweat glands.
•
Their
activity
is
regulated by cAMPdependent
phosphorylation
Model showing proposed domain
structure
of
cystic
fibrosis
transmembrane conductance regulator
(CFTR).
MSD,
membrane-spanning
domain;
NBD,
nucleotide-binding
domain; R, regulatory domain; PKA,
cAMP-dependent protein kinase.
current-voltage relationship of channel
at 357C with symmetrical 140 mM Nmethyl-D-glucamine chloride (n •5 for
each data point).
CFTR gating
CFTR, example of transport
Intestine
Fig. 2. A model of secretory epithelial cell and secretory diarrhea. Cholera toxin or heat-stable enterotoxin can increase the
intracellular cAMP or cGMP levels by activating the membrane-localized adenylate cyclase (AC) or guanylate cyclase (GC). An
increase in the intracellular cAMP or cGMP leads to the phosphorylation of the R-domain of CFTR by PKA or cGMP dependent
protein kinase II (cGKII), which, in turn, activates the CFTR chloride channel, resulting in Cl secretion into the lumen. As a
consequence, Na+ and water are effuxed into the lumen through the paracellular transport mechanism. Therefore, the net
result is the secretion of fluid and lectrolytes across the apical surface into the gut lumen. Cl is taken up from the basolateral
(blood) side by the Na+–K+–2Cl cotransporter (NKCC). K+ recycles through basolateral K+ channels, and Na+ is pumped out of
the cell by the Na+–K+-ATPase.
CFTR, example of transport
Airways
Cell models for electrolyte transport in human airways. CFTR is the dominant pathway for luminal
Cl− exit in human airways, while TMEM16A (ANO1) is probably the major secretory pathway in
mouse airways. (ADE: adenosine).
Ca2+ Activated Chloride Channels
Regulation of membrane potential by CaCCs
Ca2+ Activated Chloride Channels
Functions
Physiological roles of CaCCs. In epithelial cells, activation of CaCCs by intracellular Ca2þ elevation leads to Cl secretion followed by
transepithelial transport of Naþ and water. In smooth muscle cells, activation of CaCCs is part of an amplification echanism.
Intracellular Ca2þ increase by extracellular stimuli activates CaCCs and Cl efflux. The resulting membrane depolarization opens
voltage-dependent Ca2þ channels that cause a further intracellular Ca2þ increase, thus potentiating contraction. Another
amplification mechanism occurs in olfactory receptors, where the initial Ca2þ increase is triggered by cAMP-gated channels. CaCC is
also involved in phototransduction and regulation of neuronal excitability
Ca2+ Activated Chloride Channels
TMEM16A (ANO1)
ANO1 is activated by intracellular Ca2+ in a voltage-dependent manner.
Dose–response relationship of ANO1 activation by Ca2+. Current
responses were normalized versus those observed at 10 mM (circles,
260 mV, n58) or 3 mM Ca2+ (triangles, 160 mV, n58). d, An amplitude
histogram of single-channel currents activated by Ca2+ at 160 mV.
Putative topology of TMEM16A channels. TMEM16A
(NM 018043) has eight putative transmembrane
domains and a p-loop between transmembrane (TM)
domain 5 and 6 but no apparent similarity to other ion
channels. A number of consensus sites for
phosphorylation by kinases are present in the Nterminus, but no binding site for Ca2+ (numbering of
amino acids according to splice product a,c,d [13]).
Three cysteines (at position 651, 656, 661) are located
in the pore forming loop and are accessible to sulfhydryl
reagents.
Ca2+ Activated Chloride Channels
Airways
Contribution of Ca2+-dependent Cl− secretion by TMEM16A
(ANO1) in mouse and human airways. Cell models for
electrolyte transport in mouse airways. CFTR is the dominant
pathway for luminal Cl− exit in human airways, while
TMEM16A (ANO1) is probably the major secretory pathway
in mouse airways. (ADE: adenosine).
Ligand Activated Chloride Channels
Inhibitory Glycine Receptor
•
Glycine is a neurotransmitter
in inhibitory synapses
•
GlyR are pentamers of α
and  subunits (2 α : 3 ).
Schematic
drawing
of
the
pentameric
arrangement of GlyR subunits in homo-oligomeric
a1 (left) and heterooligomeric a1b (right) GlyRs.
Binding sites for glycine are indicated in yellow,
and glycine sites also capable of binding
strychnine are shown by a red surround
Ligand Activated Chloride Channels
GABA Receptor
•
2 types of receptor:
GABAa = LGCC
GABAb = G-protein couple
Volume sensitive Cl- channels
• They have
been
difficult to
identify.
• MCLC
intracelular
Cl- Channel
Mechanism of cell swelling and a possible role of VDCC in tubulo-glomerular feedback. (A) Mechanism
of cell volume regulation and activation of VDCC is shown. The cell under the aniso-osmotic surrounding
is illustrated at the top of the panel. The tonicity of extracellular and intracellular solution is expressed
on the bottom. Cells swell in hypotonic (indicated as a white column) extracellular solution by an influx
of water via aquaporin channels. The influx of water per se or change of volume induces several lines of
signal transduction, which then activate Cl– (VDCC) and K+ channels. Efflux of these ions lowers the
concentration of ions in the cell interior reaching equilibrium to extracellular tonicity, and then cell
volume is restored.
• Two key properties for ion channels:
– Selective ion conduction
– Gating
• ClC channels: “fast gating”
Crystal Structure of the
EcClC Fab complex
FIG.1 A
Red:
Light Fab chain
Orange: Heavy Fab chain
Blue:
ClC subunit
Green: ClC subunit
Each subunit within the dimer
forms its own independent pore for
Cl- ions.
EcCLC Crystal structure
FIG. 1.A
Selectivity filter
FIG. 1.B
FIG. 2.B
Whole-cell oocyte currents expressing ClC-0 from Torpedo ray
+80 mV to -180 mV
WT
-100 mV
E166 mutants
Single channel
measurement
Probability to be open
Mutant EcClC crystal structure
WT
E148A
Close
FIG. 2.B
E148Q
Open
FIG. 4.B
FIG. 4.D
Proposed gating mechanism
FIG. 5
Conclusions:
• When the Sext site is occupied by the
glutamate carboxyl group, the pore is closed;
When the site is occupied by a Cl- ion, the
pore is open.
• Two pores are gated independently.
• The conformational change is local.
Ca2+ - activated Cl- currents are
dispensable for olfaction
G.M. Billing, B. Pal, P. Fidzinski & T. J. Jentsch
Nature Neuroscience, vol 14(6), 2011
Main olfactory epithelium(MOE)
& the vomeronasal organ (VNO)
http://www.neuro.fsu.edu/~mmered/vomer/
Olfactory System
1.
2.
3.
4.
5.
6.
Olfactory bulb
Mitral cells
Bone
Nasal epithelium
Glomerulus
Olfactory receptor
cells
http://en.wikipedia.org/wiki/Olfactory_system
OSN signal transduction pathway
The disruption of Ano2 caused no change in
G-protein, adenylate cyclase.
Ano2 in MOE and VNO
FIG. 1
Ano2 in MOE and VNO
•Ano2 co-localized with the cilia marker protein ac tubulin in
MOE and the Cnga2 subunit of the olfactory CNG channel.
FIG. 2
Ano1 & Ano2
Olfaction is not grossly impaired in
-/Ano2 mice
Tyrosin hydroxylase helps convert tyrosine to dopamine / norepinephrine /epinephrine.
Ca2+ activated Cl- currents
-/are absent from Ano2 neurons
FIG. 5
Disruption of Ano2 moderately
reduces electro-olfactograms(EOGs)
Grey: NFA
Black:Normal
Ringer
No olfactory deficits detected
-/in Ano2 mice
Conclusions
• Disruption of Ano2 in mice virtually abolished
Ca2+ -activated Cl- currents in MOE and VNO.
• Disruption of Ano2 reduced fluid-phase EOG
by only ~40%, did not change air-phase EOG.
• Ca2+ -activated Cl- currents are dispensable for
olfaction.
Experimental system
• Colon Carcinoma epithelial cell line T84
• SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium)
• Fluorescence spectrometer configuration:
SPQ emission and excitation spectra
Ex
Ex
Sample
Sample
Em
SPQ loaded T84 cells
443 nm
Em
10 µM SPQ in water
443 nm
320 nm
344 nm
344 nm
Stern-Volmer constants
2.5 µM SPQ in water
SPQ loaded T84 cells
•
Nigericin K+/H+ antiporter
•
Tributyltin Cl-/OH- antiporter
•
KCl and fluorescein gradient
•
Fluorescein as internal standar
(ex=495 nm em = 519 nm)
SPQ
Fluorescein
F0/F = 1 + Ksv [Q]
Functional Assay on T84 cell
cAMP IBMX isopreterenol
Control
Glibenclamide (100 µM)
CFTR(inh)-172 (5 µM)
Conclusions:
• Using SPQ as Cl- sensing molecule can be
applied to measure it accurately
• This study present an easy method to study
CFTR activity.
Thank
you !!