New ion channel may yield clues to hearing.
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TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase.
1. TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line. 2. In whole-cell patch-clamp recordings, intracellular adenine 5'-diphosphoribose (ADP-ribose) triggered an inward current in tetracycline-induced TRPM2-human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H(2)O(2) stimulated an increase in [Ca(2+)](i) (pEC(50) 4.54+/-0.02) in Fluo-4-loaded TRPM2-expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H(2)O(2). These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells. 3. PARP inhibitors SB750139-B (patent number DE10039610-A1 (Lubisch et al., 2001)), PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) and DPQ (3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone) inhibited H(2)O(2)-mediated increases in [Ca(2+)](i) (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively), increases in mitochondrial dysfunction (pIC(50) vs 300 microm H(2)O(2): 7.32+/-0.23; 6.69+/-0.22; 5.44+/-0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 microm H(2)O(2): 7.45+/-0.27; 6.35+/-0.18; 5.29+/-0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. 4. SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP-ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. 5. SB750139-B, PJ34 and DPQ inhibited increases in [Ca(2+)](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively). 6. These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.
Adenosine Diphosphate Ribose↗
Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.
TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.
Amino Acid Sequence↗
ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes.
The Mg(2+)-inhibited cation (MIC) current (I(MIC)) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg(2+) homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I(MIC), studied in the presence or absence of extracellular divalent cations, was sustained for >or=50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I(MIC) exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I(MIC) rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 microM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by prevention of its resynthesis (10 microM wortmannin or 15 microM phenylarsine oxide) induced rundown of I(MIC). Finally, loading ATP-depleted cells with exogenous PIP(2) (10 microM) prevented rundown. These results suggest that PIP(2), likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity.
Adenosine Triphosphate↗
Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells.
In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.
Animals↗
How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation.
Recognition of temperature is a critical element of sensory perception and allows us to evaluate both our external and internal environments. In vertebrates, the somatosensory system can discriminate discrete changes in ambient temperature, which activate nerve endings of primary afferent fibers. These thermosensitive nerves can be further segregated into those that detect either innocuous or noxious (painful) temperatures; the latter neurons being nociceptors. We now know that thermosensitive afferents express ion channels of the transient receptor potential (TRP) family that respond at distinct temperature thresholds, thus establishing the molecular basis for thermosensation. Much is known of those channels mediating the perception of noxious heat; however, those proposed to be involved in cool to noxious cold sensation, TRPM8 and TRPA1, have only recently been described. The former channel is a receptor for menthol, and links the sensations provided by this and other cooling compounds to temperature perception. While TRPM8 almost certainly performs a critical role in cold signaling, its part in nociception is still at issue. The latter channel, TRPA1, is activated by the pungent ingredients in mustard and cinnamon, but has also been postulated to mediate our perception of noxious cold temperatures. However, a number of conflicting reports have suggested that the role of this channel in cold sensation needs to be confirmed. Thus, the molecular logic for the perception of cold-evoked pain remains enigmatic. This review is intended to summarize our current understanding of these cold thermoreceptors, as well as address the current controversy regarding TRPA1 and cold signaling.
Animals↗
Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.
Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.
Aequorin↗
VEGF activates receptor-operated cation channels in human microvascular endothelial cells.
OBJECTIVE: Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs). METHODS AND RESULTS: Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone. CONCLUSIONS: VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.
Animals↗
Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro.
Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.
Acetylcholine↗
Effects of intracellular pH and Ca2+ on the activity of stretch-sensitive cation channels in leech neurons.
The effects of intracellular pH and calcium on the activity of the leech mechanosensitive cation channels have been studied. These channels exhibited two activity modes denoted as spike-like (SL) and multiconductance (MC). In the absence of mechanical stimulation, acidification of the intracellular side of membrane patches from 7.2 to 6.2 reversibly increased the mean channel open time as well as the opening frequency in the SL mode. Channels in MC mode were activated by a pH(i) reduction from 7.2 to 6.2, but were inhibited at pH(i) 5.5. Unlike MC mode, SL mode was strongly activated by intracellular Ca(2+). Fura-2 imaging experiments showed that intracellular calcium was induced to increase by hypotonic cell swelling. The major component of this response did not require extracellular calcium. A component of the swelling-induced calcium response was sensitive to blockers of stretch-sensitive cation channels. The results indicate that the two activity modes of mechanosensitive channels of leech neurons respond differently to changes of intracellular pH and calcium. The sensitivity of the channel to micromolar concentrations of internal free calcium, along with its permeability to this ion, is consistent with a role in the amplification of mechanically induced Ca(2+) signals in leech neurons.
Action Potentials↗
TRP gating is linked to the metabolic state and maintenance of the Drosophila photoreceptor cells.
The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.
Animals↗
Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms.
In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.
Animals↗
Coordinated gating of TRP-dependent channels in rhabdomeral membranes from Drosophila retinas.
Using a newly developed dissociation procedure, we isolated the specialized rhabdomeral membranes from Drosophila retinal photoreceptors. From these membranes, we have recorded spontaneous active currents in excised patch, voltage-clamp recordings. We observed rapid opening events that closely resembled those ascribed to one class of light-activated channels, TRP. All activity exhibited Ba(2+) permeability, little voltage dependence, and sensitivity to La(3+) block. Mutational analysis indicated that the spontaneous activity present in these membranes was TRP-dependent. Excised patches from wild-type rhabdomeral membranes exhibited a wide range of conductance amplitudes. In addition, large conductance events exhibited many conductance levels in the open state. Block of activity by La(3+) both developed and recovered in a stepwise manner. Our results indicate that TRP-dependent channels have a small unitary conductance and that many channels can be gated coordinately.
Animals↗
Direct activation of trpl cation channels by G alpha11 subunits.
G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.
Animals↗
TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels.
Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.
8,11,14-Eicosatrienoic Acid↗
Polycystin-2 is an intracellular calcium release channel.
Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.
Animals↗
NMDA enhances a depolarization-activated inward current in subthalamic neurons.
Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor stimulation evokes Ca2+- and Na+-dependent burst firing in subthalamic nucleus (STN) neurons. Using whole-cell patch pipettes to record currents under voltage-clamp, we identified a time-dependent depolarization-activated inward current (DIC) that may underlie NMDA-induced burst firing in STN neurons in rat brain slices. Continuous superfusion with NMDA (20 microM) elicited a marked TTX-insensitive inward current when the membrane was depolarized to the level of -70 or -50 mV, from a holding potential of -100 mV. This current had a long duration, and its peak amplitude occurred at a test potential of -60 mV. DIC could not be evoked using the non-NMDA receptor agonist D,L-alpha-amino-3-hydroxy-5-methylisoxalone-4-propionic acid (AMPA). DIC was blocked by either intracellular BAPTA or by removal of extracellular Ca2+, but selective blockers of T-type (mibefradil), L-type (nifedipine) and N-type (omega-conotoxin GVIA) Ca2+ channels did not. Perfusing slices with a low extracellular concentration of sodium abolished the NMDA-induced DIC, implying that both Ca2+ and Na+ are necessary for the expression of DIC. Transient receptor potential (TRP) channel blockers flufenamic acid and SKF96365 severely reduced DIC amplitude, whereas NMDA-gated currents were either increased or were unchanged. These results suggest that the activation of NMDA receptors enhances a Ca2+-activated non-selective cation current that may be mediated by a member of the TRP channel family in STN neurons.
Animals↗
Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila.
Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.
Animals↗