Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Question: There is belief among scientists researching sensory biology that evolutionarily there is a limited
repertoire of signaling molecules that could be combined or utilized to transduce external signals into electrical
currents, which will in turn encode information for the organism. With this stance in mind, compare inositol
phospholipid signaling across the modalities of sight, taste, and touch. In your response do not be limited to the
molecule IP3, rather include both pathways bifurcating from the enzyme PLC and also incorporate any use of downstream pathways such as the eicosanoid pathway. Your response should be focused at the cell signaling
organization level including both molecular and electrophysiological findings and take you from events at the
membrane to those at the nucleus. After completing your response, make some knowledgeable conjectures as to
which of these pathways could be operational (and how you might test this) in the transduction of pheromone
information in the vomeronasal organ. Likewise, describe why others might be unlikely based on reported
literature. Estimated Length of Response: 15-20 pages ds, with full literature citation section.
Introduction & Background
Sensory systems utilize a restricted array of signaling molecules to transduce external
information into internal, electrical signals that the brain can then interpret. In addition to the
cyclic nucleotide system, the phosphatidylinositol (PI) system has been shown to convey these
external signals to the nervous system in a wide variety of sensory systems. It is thought that
these mechanisms are conserved across sensory modalities. Here I will compare the intracellular
signaling cascades utilizing the phosphatidylinositol system in phototransduction, taste
transduction and mechanosensation.
The PI signaling system has been implicated in a wide variety of cellular signaling
pathways such as metabolism, growth, differentiation, secretion, contraction, as well as in
sensory transduction such as phototransduction. In the classically described scheme,
phosphatidylinositol 4,5-bisphosphate (PIP2), present on the inner leaflet of the plasma
membrane, is hydrolyzed via the action of phospholipase C (PLC) into equimolar amounts of
inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is a unique product of the PI
pathway in that it is water-soluble and able to diffuse throughout the cell and initiate further
signaling cascades, particularly by binding to its receptor, the IP3R, in the membrane of the
endoplasmic reticulum. DAG remains membrane-bound, but has been implicated in several
signaling pathways nonetheless, as it activates protein kinase C in the presence of calcium
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Jessica H. Brann
(Berridge and Irvine, 1984; Majerus et al., 1990). The products of the PI system also have
implications in nuclear signaling. IP3 can enter the nucleus via a nuclear pore and activate IP3Rs
facing the nucleoplasm, mobilizing Ca2+ from the nuclear envelope. In addition, IP3 can be
synthesized in the nucleoplasm, exit through a nuclear pore, and mobilize other Ca2+ stores in the
cytoplasm, which is then free to enter the nucleus. DAG is also present in the nuclear envelope
and subject to degradative pathways as well as interactions with protein kinase C within the
nucleus (Irvine, 2002). Interestingly, interruption of the genes encoding PI pathway proteins also
interrupts mRNA export from the nucleus (Chi and Crabtree, 2000). Thus it appears that the PI
pathway may not only have a role in mediating immediate signaling in sensory cells, but also
function on a gene-regulation level in most, if not all, cells.
It appears that PIP2 is synthesized when needed and is formed by a two-stage
phosphorylation mechanism whereby phosphoinositol (PI) is phosphorylated at the 4-position of
the inositol head group to yield phosphatidylinositol 4-phosphate (PIP). This product is in turn
phosphorylated at the 5-position, yielding PIP2 (Berridge and Irvine, 1984; Zuker 1996). In
addition to synthesis mechanisms, there are degradative pathways consisting of
phosphomonoesterases within the cell to convert PIP2 back to PI. When an agonist binds to and
stimulates a receptor, the signaling pathway diverts PIP2 out of these metabolically expensive
“futile” cycles. Cleavage of PIP2 then yields the second messengers described above, which are
then free to initiate other signaling mechanisms, the details of which will be described below
(Berridge and Irvine, 1984).
Phototransduction
In this section I will discuss the phototransduction cascades in both vertebrates and
invertebrates. I will also discuss the theorized role of the PI system in vertebrate photoreception.
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Jessica H. Brann
Phototransduction cascades are quite different in vertebrates than those described in
invertebrates. Although there are points at which regulation by phosphoinositide derivatives are
likely instrumental in regulation, the central cascade does not utilize the PI pathway. There are
four main stages comprising vertebrate phototransduction. The first is photoisomerization,
where a photon interacts with the chromophore of rhodopsin, 11-cis retinal and isomerizes it to
the all-trans configuration. The protein then undergoes a series of conformational changes, the
final activated product being metarhodopsin II (R*). The second step entails activation of the
heterotrimeric G-protein, transducin, by R*. R* promotes the exchange of guanosine
trisphosphate (GTP) for guanosine diphosphate on the G subunit, thereby promoting rapid
dissociation of G-GTP (or G*). G* then stimulates the activation of cGMP phosphodiesterase
(PDE) in the third step. PDE activation results in the degradation of cGMP, causing an overall
cytoplasmic decrease of this second messenger. This results in the closure of the cGMP-gated
(CNG) cation channels (Na+ and Ca2+ permeable) and a reduction in the constant dark inward
current, causing a hyperpolarization of the membrane and a reduction of glutamate release from
the nerve terminal of the photoreceptor, concluding the phototransduction cascade (Burns and
Baylor, 2001; Arshavsky et al., 2002). Deactivation of R* occurs by the binding of arrestin and
phosphorylation by rhodopsin kinase (Burns and Baylor, 2001).
The role of the PI system in vertebrate phototransduction remains controversial.
Components of this system are expressed in rod photoreceptors from the mouse retina, including
PLC4 and G11, known to activate this specific PLC (Peng et al., 1997). PLC1, PLC1, and
PLC1 are found in bovine retina, with PLC1 expressed in the photoreceptor cell layer. In
addition, light stimulation was found to enrich PLC1 activity in bovine retinal membranes
(Ghalayini et al., 1998). However, application of analogs of IP3, protein kinase C, and inhibitors
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Jessica H. Brann
of phospholipase C failed to affect the phototransduction system in whole-cell voltage clamp
studies (Jindrova and Detwiler, 1998).
Although the main transduction cascade in vertebrate photoreceptors is the cyclic
nucleotide system, deriviatives of lipids play a regulatory role. DAG suppresses the activity of
rod CNG channels in excised oocyte patches in the absence of in the absence of protein kinase
activity. It thus appears that the actions of PKC are unnecessary to the interaction between DAG
and CNG channels (Gordon et al., 1995; Kramer and Molokanova, 2001). The interaction
between DAG and the CNG channel appears to be a direct one and not one mediated by
derivatives of DAG. Instead, it is thought that there is either a binding site for DAG on the
channel or that DAG alters the bilayer surrounding the channel, leading to altered channel
activity (Crary et al., 2000).
An interesting twist to the current dogma is the idea of PIP2 modulation of a wide variety
of ion channels and transporters (see Hilgemann et al., 2001 for a recent review). This
modulation is independent of IP3, DAG, calcium, or PKC. In recent studies, the role of PIP2 in
CNG channel regulation has been questioned. Rod CNG channels are inhibited by the
application of ATP, and tyrosine kinase inhibitors block this effect. The application of ATP also
results in the phosphorylation of PI to generate PIP2. Application of an antibody against PIP2 in
rod cell excised patches from Xenopus blocked the ATP-induced inhibition of CNG channels. In
addition, rod CNG channels are strongly inhibited by PIP2 when expressed in Xenopus oocytes.
Application of U 73122, a phospholipase C inhibitor, did not alter the effect of PIP2. The same
antibody against PIP2 described above was able to potently inhibit phosphodiesterase activity,
which in turn implies that PIP2 is able to activate PDE in photoreceptors, probably via
transducin, and therefore change how the CNG channel is modulated (Womack et al., 2000).
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Jessica H. Brann
In contrast to vertebrates, the mechanism of invertebrate phototransduction is not as well
described. Invertebrates use the phosphatidylinositol system, and the proteins and scaffolding
mechanisms involved are well known. The general scheme is as follows: a photon is absorbed
by rhodopsin, catalyzing conformational changes within the protein and yielding activated
rhodopsin or metarhodopsin (R*). R* activates a heterotrimeric G protein of the Gq family,
which then activates PLC, producing IP3 and DAG from PIP2. However, it is still unclear how
this cascade leads to the opening of cation-selective ion channels called transient receptor
potential (TRP) channels; this in turn leads to depolarization. It is clear this this is the fastest G
protein-coupled cascade described to date (Zuker, 1996; Hardie and Raghu, 2001).
The fundamental experiments demonstrating the importance of the PI system in
photoreception were those investigating the norpA (no receptor potential A) mutant in
Drosophila melanogaster. Although NorpA mutants lacked receptor potentials upon light
stimulation, the rhabdomere was normal in both appearance and in rhodopsin levels. It was
noted that these mutants had several problems in PI metabolism, as seen in the low levels of
several enzymes necessary to maintain this pathway. IP3 levels were also extremely low in the
mutants (Inoue et al., 1985). It was found by thin layer chromatography that the norpA mutant
lacked phospholipase C activity entirely and later that norpA was the gene that encoded a
putative phospholipase C (Yoshioka et al., 1985; Bloomquist et al., 1988). Phospholipase C was
shown to rescue the norpA mutant with chimera technology, where a norpA minigene was
expressed in mutant flies (McKay et al., 1995).
Meanwhile in Limulus photoreceptors, the PI system appeared to have a role in
phototransduction as well. Photoreceptors were found to express the enzymes necessary to
maintain PI metabolism. In addition, light elevated levels of IP3, and intracellular injection of
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Jessica H. Brann
IP3 was able to mimic the normal electrophysiological response to light (Brown et al., 1984).
PLC activity was found to be biphasically regulated by calcium in the cuttlefish Sepia officinalis;
the authors assert that this serves as a negative feedback mechanism that contributes to
adaptation in the visual system (Rack et al., 1994). A PI-specific PLC that was capable of
hydrolyzing PIP2 was cloned from squid and was shown to be necessary for the light response;
squid photoreceptors were now known to express the machinery needed for phototransduction,
namely rhodopsin, G proteins, and PLC (Szuts, 1993; Carne et al., 1995).
Although the mechanism connecting the initiation of the transduction cascade to the
opening of cation channels in the membrane is not known, the identity of those cation channels is
known. The discovery of a drosophila mutant, transient receptor potential or trp, led to the
discovery of large family of TRP proteins that expressed throughout the sensory modalities. In
the original trp Drosophila mutant, the response to prolonged illumination declined over time,
hence the name “transient.” These channels are now described as nonselective cation channels,
but the permeabilities of some channels within the TRP family may be more selective for Ca2+.
TRP and TRPL (TRP-like) channels in Drosophila photoreceptors are known to carry the
depolarizing current. Theories on the activation of TRP channels are varied (Minke and Cook,
2002).
INAD (inactivation no after potential D) is a scaffolding protein that was originally
shown to interact with TRP via a C-terminal PDZ domain. A point mutation at proline 215
eliminated the interaction (Shieh and Zhu, 1996). Shieh et al. also showed that norpA (PLC) and
INAD were in a protein-protein interaction complex by overlay assay and site-directed
mutagenesis. Interruption of this activity-independent interaction disrupted the kinetics of
activation and deactivation (Shieh et al., 1997). INAD is now known to interact via its five PDZ
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Jessica H. Brann
domains with virtually all of the proteins necessary for phototransduction in Drosophila,
including TRP, PLC, PKC, calmodulin and rhodopsin (Xu et al., 1998). This perhaps the best
described scaffolding complex, and scaffolding mechanisms similar to this one are likely to
become a theme in intracellular signaling mechanisms.
To this point, invertebrate phototransduction appears to be relatively straightforward, as
all that seems to be missing is the second messenger between PLC activation and TRP channel
opening. The evidence had implied that IP3 was the second messenger involved; however, with
the publication of a paper by Acharya et al. this assumption had to be questioned. This group
generated IP3 receptor Drosophila mutants. These mutants died in early larval stages, which
demonstrated the necessity of the IP3R for growth and differentiation. The group then generated
mosaic animals; in those photoreceptor cells deficient for IP3R, phototransduction was shown to
be normal. Response kinetics were thoroughly analyzed but deficiencies in Drosophila
phototransduction were not found. Even more disturbing was the finding that TRP channels do
not localize near the intracellular calcium stores, indicating that IP3 activation of the IP3R and
subsequent calcium store release was not connected to TRP activation (Acharya et al., 1997).
However, current prevailing thought is polyunsaturated fatty acids (PUFAs) activate TRP
channels in the Drosophila photoreceptor. PUFAs such as arachidonic acid (AA) and linolenic
acid (LA) are deriviatives of DAG and would thus tie the PI system more solidly to invertebrate
phototransduction. Whole-cell recordings from photorceptors showed that AA or LA were able
to elicit reversible stimulate inward currents. In double trp/trpl mutants, the current elicited by
AA or LA was abolished (Chyb et al., 1999)
Thus the transduction cascades have been roughly sketched for both vertebrate and
invertebrate phototransduction. However, these schemes can be contradictory and a consensus
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Jessica H. Brann
on mechanisms of deactivation has not been met. It is not clear what role the PI system will
ultimately be found to play in phototransduction, particularly in vertebrate systems, but it is clear
that the signaling components are present in photoreceptor cells. It would be unusual for all of
the pieces of a signaling mechanism to be present if they did not have a functional role.
Taste transduction
Taste buds are found within the epithelium lining the tongue, palate, and pharynx. Each
bud is composed of approximately 40-120 cells, including taste receptor cells (TRCs), as well as
supporting, precursor, and basal cell types. The small bipolar TRCs send a thin, dendritic-like
process to the surface of the epithelium; this process expresses the transduction machinery. The
larger basolateral surface of the TRC makes synaptic contacts with with sensory axons
(Lindemann 1996). The TRCs are not neurons themselves, but instead release transmitter
directly onto the cranial nerves innervating the taste buds, including the facial, glossopharyngeal,
and vagus nerves (Herness and Gilbertson, 1999). A single TRC makes contacts with several
nerve fibers, and each fiber has been shown to innervate several different taste buds (Lindemann,
1996).
There are four to five basic tastes: salty, sour, sweet, bitter and umami. Both sour and
bitter compounds are often toxic, and it is thougth that the ability to detect these compounds is
part of a defense mechanism (Herness and Gilbertson, 1999).
Bitter taste. Several transduction cascades have been proposed for bitter taste. In the
first, bitter compounds such as quinine and tetraethylammonium have been shown to block
potassium channels directly, thereby depolarizing the cell (Rosenzweig et al., 1999). Another
cascade is inferred from the expression of the G protein  subunit gustducin, which is very
similar to the G protein transducin found in photoreceptor cells (McLaughlin et al., 1992). In a
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Jessica H. Brann
similar scenario to that seen in photoreceptors, gustducin-mediated transduction may lead to a
decrease in cyclic nucleotides and consequently the activation of a cation channel in the
membrane that is normally suppressed by cyclic nucleotides. In other work, bitter compounds
such as caffeine and theophylline lead to the accumulation of cGMP as measured by quenchflow techniques. The increases in cGMP were blocked when inhibitors of guanylyl cyclase were
presented with taste stimuli; these experiments together implicate that bitter tastants may lead to
the activation of a cyclic nucleotide-gated channel in TRCs (Rosenzweig et al., 1999).
-gustducin knockouts have shown diminished responses to both bitter and sweet
compounds, both behaviorally and electrophysiologically (Wong et al., 1996). The
heterotrimeric G protein gustducin as been linked to two sets of transduction cascades in TRCs,
one in which -gustducin mediates a decrease in cyclic nucleotide monophosphates via
activation of phosphodiesterase (PDE). The  subunit of gustducin is thought to lead to
activation of PLC2 by G3/G13, thereby increasing of IP3 (Gilbertson et al., 2000; Perez et
al., 2002).
IP3 has been shown through multiple studies to be important to the transduction of bitter
taste. A study using Ca2+ imaging first showed that the bitter tastant denatonium induced
increases in the concentration of intracellular calcium (Akabas et al., 1988). The increase was
due to release from intracellular stores, which implied a second messenger system instead of
voltage-activated calcium entry from the extracellular milleu. Using a 45Ca2+ uptake assay,
Hwang et al. were able to demonstrate that the endoplasmic reticulum was the site of selective
calcium accumulation by the cell. IP3 was able to reduce 45Ca2+ accumulation; in addition,
heparin, an inhibitor of IP3 binding to its receptor on the ER, was able to inhibit this release of
calcium (Hwang et al., 1990). Futher studies were able to demonstrate by quench-flow analysis
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Jessica H. Brann
that IP3 was generated with the bitter tastant sucrose octaacetate (Spielman et al., 1994). A more
recent study returned to denatonium; this group replicated the first experiment, showing that
denatonium increased calcium levels, but they were also able to inhibit this intracellular calcium
accumulation by addition of a phospholipase C inhibitor. Thapsigargin treatment, a drug that
impairs the refilling of calcium stores in the endoplasmic reticulum, showed that the increases in
calcium in response to denatonium were not dependent on external calcium levels. No change in
the calcium elicited by denatonium was seen with the addition of inhibitors of PDE or adenylyl
cyclase (Ogura et al., 1997). A separate group of experiments showed IP3 accumulation upon
stimulation with denatonium and strychnine HCl in a rapid and transient manner via quench-flow
analysis. Interestingly, these experiments also showed a decrease in cGMP and cAMP levels.
While an antibody against -gustducin blocked the suppression of cyclic nucleotides, it had no
effect on IP3 accumulation. When an antibody against PLC2 was added, the denatoniuminduced increase in IP3 was blocked (Yan et al., 2001). Recently, the IP3 receptor type III
(IP3R3) has been localized to taste receptor cells, further emphasizing the role of IP3 in taste
transduction cascades. Both Asano-Miyoshi et al. and Clapp et al. independently showed that
IP3R3 is expressed in the same cells expressing PLC2 and G13 (Asano-Miyoshi et al., 2001;
Clapp et al., 2001). -gustducin was also expressed in a subset of those cells (Clapp et al.,
2001). In addition, two types of G protein-coupled taste receptors are found (in separate
populations) in those cells expressing PLC2/IP3R3 (Asano-Miyoshi et al., 2001).
In addition to IP3, DAG has been implicated in bitter taste transduction in the gerbil.
DAG in the presence of calcium can activate protein kinase C (PKC); PKC can then
phosphorylate proteins that regulate the cellular response to tastant stimulation, placing PKC in a
regulatory role, not a primary transduction role. However, DAG has also been shown to be a
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Jessica H. Brann
precursor of arachidonic acid, a twenty amino acid fatty acid. Fatty acids such as arachidonic
acid are precursors of a wide variety of lipid messengers collectively termed the eicosanoids,
which include prostaglandins, thromboxanes, and leukotrienes (Ganong 1991). Arachidonic acid
can be generated by phospholipase A2 from several lipid sources including phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, as well as
phosphatidylinositides. In addition, arachidonic acid can be generated from PIP2 via the PLC
pathway (Schiffman et al., 1995). In the gerbil, electrophysiological measurements have shown
that cell-permeant analogues of DAG, such as OAG and DiC8 (1-Oleoyl-2-acetyl-sn-glycerol
and 1,2-dioctanoyl-sn-glycerol, respectively) were able to decrease the TRCs response to bitter
stimuli (Schiffman et al., 1995). Analogues of DAG and eicosanoids have been demonstrated to
directly activate transient receptor potential (TRP) channels in other sensory systems and in
lymphocytes (Chyb et al., 1999; Hofmann et al., 1999; Gamberucci et al., 2002). Recently,
TRPM5, of the larger family of TRP channels, was cloned from TRCs. This channel is
selectively expressed in TRCs and is co-expressed with PLC2 and G13 (Perez et al., 2002). It
is possible that in bitter taste derivatives of DAG modulate the activity of the TRP channel
expressed in TRCs.
Sweet Taste. Like bitter taste, several mechanisms have been proposed to explain the
transduction of sweet tastants. In the fly, Boettcherisca peregrina, both ligand-gated and G
protein-coupled receptors have been shown to mediate sweet taste. Recent work has shown that
the response of the sugar receptor cell of Boettcherisca is inhibited in the presence of neomycin,
a drug that blocks PLC’s access to PIP2, therefore inhibiting IP3 and DAG production. Inhibition
was also seen with U 72133, a PLC inhibitor. Adenophostin A, an analog of IP3, was able to
increase the response of the sugar receptor cell in a dose-dependent manner. Ruthenium red, an
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Jessica H. Brann
IP3R channel antagonist, depressed the response of the sugar receptor cell, and 2-APB (2aminoethoxydiphenyl borate, another IP3R channel antagonist) blocked the response completely
(Koganezawa and Shimada, 2002). In dog, the response to sucrose appears to be via amiloridesensitive sodium channels (Lindemann 1996).
However, sweet taste seems to be a bit more complicated in vertebrates, as sucrose and
synthetic sweeteners stimulate separate transduction pathways. Sugars stimulate increases in
cAMP via the cyclic nucleotide pathway, as seen by calcium imaging studies demonstrating
calcium uptake from the extracellular space. In contrast, synthetic sweeteners and some amino
acids stimulate the phosphatidylinositol pathway and cause intracellular calcium release.
Interestingly, both pathways are present in the same TRCs (Lindemann, 2001). Membrane
permeant analogs of DAG (OAG and DiC8) were shown to increase responses to sweet stimuli
in TRCs of the gerbil (Schiffman et al., 1995). Varkevisser et al. used loose patch
electrophysiology to investigate the role of protein kinases in sweet transduction, as DAG in the
presence of calcium is able to activate PKC. TRCs were first tested for their sensitivity to a
synthetic sweetener, NC-01. PDBu, a membrane permeant PKC activator, was able to elicit
currents in those cells shown to be sensitive to NC-01 while it had no effect on the sweetinsensitive cells. Furthermore, Bis I, a membrane permeant PKC inhibitor, decreased the
response of the TRCs to NC-01. The authors therefore propose that PKC is able to selectively
phosphorylate and close sweet-senstive potassium channels, leading to membrane depolarization.
H-89, a membrane permeant inhibitor of protein kinase A (which would be stimulated via the
AC system), did not inhibit the TRC response to sucrose, suggesting that phosphorylation may
not have a role in the transduction of natural sugars such as sucrose (Varkevisser et al., 2000).
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Salt Taste. The transduction mechanism for salt taste is phosphoinositide-independent.
Instead, it is mediated by a group of amiloride-sensitive sodium channels (ASSCs) that are very
similar to the peripherially expressed epithelial sodium channels (ENaCs). ASSCs are
permeable to sodium, lithium, and protons, but not to potassium. Sodium is able to enter the cell
via these channels, triggering transmitter release at the basolateral membrane of the TRC onto
the nerve fibers (Lindemann, 1996; Herness and Gilbertson, 1999).
Sour Taste. Sour taste is also not mediated by the phosphoinositide system.
Alternatively, protons from acidic compounds mediate sour taste. ENaC is capable of carrying a
proton current if there is a significant gradient of protons in the extracellular space surrounding
the TRC in the taste bud. Other H+-gated channels, such as MDEG1 of the ENaC/Deg family
and HCN, a hyperpolarization-activated, cyclic nucleotide-gated cation channel, are able to
conduct protons and transduce sour tastants.
Nucleotide receptors. In addition to conventional tastants, cyclic nucleotides are able to
induce calcium accumulation in taste buds. Purinergic receptors are expressed on TRCs; P2X
receptors are ATP-gated calcium channels while P2Y receptors are G protein-coupled receptors
whose stimulation is coupled to calcium release within the cell. Using photometry and patchclamp electrophysiology techniques, Kim et al. were able to show that ATP presentation resulted
in an increase in intracellular calcium concentration. These responses were abolished by
application of suramine, a non-specific antagonist of nucleotide receptors. This demonstrates
that the increases in calcium were due to activation of the nucleotide receptor. Interestingly, a
P2Y agonist (2-methylthio-ATP) was able to induce responses in TRCs while a P2X agonist (methylene-D-ATP) was not. When an inhibitor of phospholipase C, U 73122, was added to the
TRC, the response elicited by ATP was dramatically reduced (Kim et al., 2000). Thus it appears
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that the purinergic receptor P2Y is present in TRCs and is able to utilize the phosphatidylinositol
system to transduce responses to ATP.
Mechanosensation
Although the original question asked that I discuss touch transduction, touch sensation –
as conveyed by free nerve endings, Ruffini endings, Pacinian corpuscles, and Merkel discs – is
currently undescribed. However, there is a fair amount of literature concerning the role of the PI
system in both pain and mechanosensation by hair cells of the cochlea. Therefore, I will instead
be describing those systems.
Pain and Itch Sensation. The molecular mechanisms of pain are relatively unknown.
Current investigations emphasize the role of small peptides and growth factors in initiating pain
cascades. Bradykinin is a nonapeptide produced in response to injury known to activate sensory
neurons transmiting pain information to the central nervous system. Bradykinin has been shown
to stimulate IP3 accumulation and mobilize IP3-sensitive calcium stores (Thayer et al., 1988).
More recently, bradykinin and nerve growth factor (NGF), both of which function as algesic
agents, were found to potentiate the activity of VR1, a heat-activated ion channel that is part of
the TRP superfamily. This group found that PIP2 likely inhibits VR1 directly; application of
sequestering PIP2 antibody or initiating hydrolysis of PIP2 via PLC had similar effects to
bradykinin or NGF. It is possible that derivatives of DAG may displace PIP2 from the VR1
channel and promotes inflammation as well (Chuang et al., 2001).
Itch sensation is detected when histamine is released by mast cells after degranulation
and can bind to H1 receptors. Several types of fibers express H1 receptors, including lamina I
spinothalamic tract neurons and peripheral C fibers. A recent study demonstrated the link
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between histamine release and the PI system. When histamine was applied to isolated spinal
ganglia, a two-phase increase in intracellular calcium was seen, consisting of an initial transient
followed by a sustained plateau. Application of histamine increased IP3 production in a dosedependent manner and was specific to the H1 receptor, as addition of mepyramine, an H1
receptor antagonist, inhibited IP3 accumulation. The initial calcium transient was shown to be
PLC-dependent, as pretreatment with the PLC inhibitor U 73122 suppressed the transient rise in
calcium. A combination of U 73122 and Ca2+-free media completely abolished the calcium
increase seen with histamine application, showing that the plateau phase was dependent on
extracelluar calcium influx (Nicolson et al., 2002).
Hearing and Vestibular Function. Hair cells are the sensory receptor cells of the hearing
and vestibular systems. The vestibular labyrinth has semicircular canals that detect motion in all
planes. Sensory cells line the maculae in the utricle and saccule of the otolith organ as well as
the cristae of the ampullae within each semicircular canal.
The hearing aparatus, the cochlea, is essentially a fluid-filled sac that developed as an
outcropping of this labyrinth. The basilar membrane within the cochlea transmits mechanical
force that is a representation of the sound waves encoutering the hearing apparatus in the
external ear. The organ of Corti along the basilar membrane is lined with rows of inner and
outer hair cells (IHC and OHC, respectively), all of which possess a ciliated apparatus that is
connected by tip-links. Tip-links are essentially small mechanical gates, that when stretched,
open unidentified ion channels that conduct potassium. The endolymph surrounding the hair cell
has a remarkably high concentration of K+, hence it is the charge carrier in this system. The
direction of deflection will either open (by tension) or relax (lack of tension) the transduction
channels and the cell will depolarize or hyperpolarize, respectively. The time period in between
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Jessica H. Brann
deflection of the hair cell to the point at which the cell depolarizes is extremely short, on the
order of 200 microseconds. The speed of this transduction mechanism indicates that a second
messenger does not mediate it (Gillespie and Walker, 2001; Jarman, 2002). However, it seems
likely that the PI system functions in hair cells in a regulatory fashion.
The mechanosensitive ion channels found in hair cells are nonselective for monovalent
cations and are permeable to calcium (Mammano et al., 1999). It is likely that these channels are
related to the TRP family, as another TRP family member, OSM-9, is known to be important to
other modes of mechanosensation in C. elegans (Colbert et al., 1997).
IHCs are the primary transducers of sound information, while OHCs appear to be
modulators of IHC activity. An interesting characteristic of OHCs is that they are motile cells.
This motility is thought to amplify the vibrations travelling down the basilar membrane, thereby
altering the amount of deflection that the tip-links on IHCs experience (Mammano et al., 1999).
Calcium is necessary for this motility. Aminoglycoside antibodies that preferentially bind to
PIP2 and prevent hydrolysis were shown to impair OHC motility, linking the PI system to this
important modulatory role (Schacht and Zenner, 1987).
Hair cells also express purinergic receptors, much like those seen on TRCs (see above).
Exposure to calcium, ATP, or IP3 elicits “slow” contractions of the OHC (Schacht and Zenner,
1987). Application of ATP to isolated hair cells increases intracellular calcium concentration.
This effect was blocked by heparin, a drug known to competitively inhibit IP3 binding to the
IP3R (Mammano et al., 1999). A more recent study using caged IP3 showed IP3 produced a
response analagous to that induced by ATP (Lagostena et al., 2001). These studies indicate that
ATP can activate calcium stores (localized to the basolateral membrane of the OHC) and alter
the activities of myosin and actin-binding proteins that underlie the motility of the OHC.
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Implications for Vomeronasal Signaling
The classic PI system is fairly straightforward, with a stimulus initiating the release of
two common second messengers, IP3 and DAG, from membrane-bound PIP2. However, the
ways in which we think about this system are slowly expanding. It is now apparent that the
second messengers may be acting in concert with their precursors to initiate cellular transduction,
or that the second messengers are acting as modulators of the cascade initiated by precursor
molecules.
My research concerns vomeronasal sensory transduction in the turtle, Sternotherus
odoratus. It is thought that the PI system is used for vomeronasal transduction in this model (see
Kashiwayanagi et al., 2000 for an example). Figure 3 (see below) shows the hypothetical
scheme with which our laboratory has been working. However, several ideas from the signaling
systems described above have implications for vomeronasal signaling that we must consider.
A common theme for transduction pathways appears to be a synergistic activation and
inhibition of two separate pathways; for example, in TRCs, denatonium and strychnine HCl
increase IP3 accumulation while simultaneously decreasing cAMP and cGMP levels. This has
been shown in the vomeronasal system in garter snakes with ESS stimulation as well (Wang et
al., 2002). It is not sufficient to think about vomeronasal signaling in the turtle as mediated
strictly by a single messenger, rather a complex of molecules are likely involved.
The TRP family of ion channels is expressed throughout sensory modalities; there is a
TRP channel in the VN system as well, namely TRPC2. There is also evidence that TRP
channels and IP3Rs interact (Tang et al., 2001; Brann et al., 2002). I think that IP3R3 is probably
expressed on the plasma membrane of the vomeronasal sensory neurons (VSNs) and is able to
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
modulate the opening of the TRPC2 channel. How this occurs is not clear, as current evidence
indicates that IP3R3 is not directly gating the channel.
I will be investigating the response of the VSN to a variety of PI system analogs,
activators and inhibitors, including ruthenium red, a blocker of the IP3R, 2aminoethoxydiphenylborate (2-APB, an antagonist of the IP3R). I will also be testing for inward
currents upon application of both IP3 and a well-known analog of IP3, adenophostin A. In
addition to artificially stimulating the IP3 portion of the PI system, I will need to investigate the
DAG portion as well. Analogs such as OAG and DiC8 would be interesting to use – I may see
currents elicited by these compounds as DAG and its derivatives has been found to directly
activate other TRP channels (see Chyb et al., 1999 for an example). Unfortunately, no known
blockers of the TRPC2 channel exist, so I cannot see if blocking the TRPC2 channel results in
suppression of odorant response.
I think that, particularly after discussing the transduction cascades used in invertbrate
phototransduction, it would be interesting to investigate the role of PIP2 on TRPC2
activation/deactivation. It may be that IP3 or DAG are modulators in this systems as well, and
that ligand activation of the VSN may cause movement of PIP2 through the membrane to cause
interaction with the TRPC2 channel. I would like to put the PIP2 antibody discussed above on
these cells to see if I can interrupt the odorant response. A recent paper by Spehr et al. showed
that presentation of arachidonic acid elicited currents from rat VSNs, but the kinetics and
duration of the response are not similar in time or amplitude to an odorant response, so the role
of AA and other derivatives of DAG in vomeronasal signaling is not clear at this time (Spehr et
al., 2002).
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Figure 1. Phototransduction cascades in vertebrate rods and Drosophila (Hardie and
Raghu, 2001)
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Figure 2: Proposed transduction mechanisms in vertebrate taste receptor cells (from Gilbertson et
al., 2000).
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Figure 3: Hypothetical scheme for VN signaling in the turtle, S. odoratus.
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Figure 4: TRP family of ion channels
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
Abbreviations List:
2-aminoethoxydiphenyl borate (2-APB)
Amiloride-sensitive sodium channels (ASSCs)
Arachidonic Acid (AA)
Cyclic nucleotide-gated channel (CNG channel)
Diacylglycerol (DAG)
1,2-dioctanoyl-sn-glycerol (DiC8)
Epithelial sodium channels (ENaCs)
Inner hair cell (IHC)
Inositol 1,4,5-trisphosphate (IP3)
Linolenic Acid (LA)
Nerve growth factor (NGF)
1-Oleoyl-2-acetyl-sn-glycerol (OAG)
Outer hair cell (OHC)
Phosphatidylinositol (PI)
Phosphatidylinositol 4-phosphate (PIP)
Phosphatidylinositol 4,5-bisphosphate (PIP2)
Phosphodiesterase (PDE)
Phospholipase C (PLC)
Polyunsaturated fatty acids (PUFAs)
Protein kinase C (PKC)
Taste receptor cell (TRC)
Transient receptor potential (TRP)
Vomeronasal sensory neurons (VSNs)
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Comprehensive Exam Question (Dr. Fadool)
Jessica H. Brann
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