Intracellular Long-Chain Acyl CoAs Activate TRPV1 Channels

TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs), another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP₂-binding residues in TRPV1. In contrast to PIP₂, LC-CoA modulation of TRPV1 is independent of Ca²⁺ _i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca²⁺ _i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.     

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

Although PI(4,5)P₂ is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P₂ and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC₅₀ for activation by PI(4,5)P₂, it decreased the EC₅₀ for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P₂ from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P₂-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.     

Ethanol inhibits cold-menthol receptor TRPM8 by modulating its interaction with membrane phosphatidylinositol 4,5-bisphosphate

Ethanol has opposite effects on two members of the transient receptor potential (TRP) family of ion channels: it inhibits the cold-menthol receptor TRPM8, whereas it potentiates the activity of the heat- and capsaicin-gated vanilloid receptor TRPV1. Both thermosensitive cation channels are critically regulated by the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)). The effects of this phospholipid on TRPM8 and TRPV1 are also functionally opposite: PIP(2) is necessary for the activation of TRPM8 but it constitutively inhibits TRPV1. This parallel led us to investigate the possible role of PIP(2) in the ethanol-induced modulation of rat TRPM8, heterologously expressed in HEK293T cells. In this study, we characterize the effects of ethanol (0.1-10%) on whole-cell currents produced by menthol and by low temperature (< 17 degrees C). We show that the inclusion of PIP(2) in the intracellular solution results in a strong reduction in the ethanol-induced inhibition of menthol-evoked responses. Conversely, intracellular dialysis with anti-PIP(2) antibody or with the PIP(2) scavenger, poly L-lysine, enhanced the ethanol-induced inhibition of TRPM8. A 20 min pre-incubation with wortmannin caused a modest decrease in inhibition produced by 1% ethanol, indicating that the ethanol-induced inhibition is not mediated by lipid kinases. These findings suggest that ethanol inhibits TRPM8 by weakening the PIP(2)-TRPM8 channel interaction; a similar mechanism may contribute to the ethanol-mediated modulation of some other PIP(2)-sensitive TRP channels.     

Integration of phosphoinositide- and calmodulin-mediated regulation of TRPC6

Multiple TRP channels are regulated by phosphoinositides (PIs). However, it is not known whether PIs bind directly to TRP channels. Furthermore, the mechanisms through which PIs regulate TRP channels are obscure. To analyze the role of PI/TRP interactions, we used a biochemical approach, focusing on TRPC6. TRPC6 bound directly to PIs, and with highest potency to phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). We found that PIP(3) binding disrupted the association of calmodulin (CaM) with TRPC6. We identified the PIP(3)-binding site and found that mutations that increased or decreased the affinity of the PIP(3)/TRPC6 interaction enhanced or reduced the TRPC6-dependent current, respectively. PI-mediated disruption of CaM binding appears to be a theme that applies to other TRP channels, such as TRPV1, as well as to the voltage-gated channels KCNQ1 and Ca(v)1.2. We propose that regulation of CaM binding by PIs provides a mode for integration of channel regulation by Ca(2+) and PIs.     

Sub-Ciliary Segregation of Two Drosophila Transient Receptor Potential Channels Begins at the Initial Stage of Their Pre-Ciliary Trafficking

Cilia are important eukaryotic cellular compartments required for diverse biological functions. Recent studies have revealed that protein targeting into the proper ciliary subcompartments is essential for ciliary function. In Drosophila chordotonal cilium, where mechano-electric transduction occurs, two transient receptor potential (TRP) superfamily ion channels, TRPV and TRPN, are restricted to the proximal and distal subcompartments, respectively. To understand the mechanisms underlying the sub-ciliary segregation of the two TRPs, we analyzed their localization under various conditions. In developing chordotonal cilia, TRPN was directly targeted to the ciliary tip from the beginning of its appearance and was retained in the distal subcompartment throughout development, whereas the ciliary localization of TRPV was considerably delayed. Lack of intraflagella transport-related proteins affected TRPV from the initial stage of its pre-ciliary trafficking, whereas it affected TRPN from the ciliary entry stage. The ectopic expression of the two TRP channels in both ciliated and non-ciliated cells revealed their intrinsic properties related to their localization. Taken together, our results suggest that sub-ciliary segregation of the two TRP channels relies on their distinct intrinsic properties, and begins at the initial stage of their pre-ciliary trafficking.     

Biophysical characterization of the isolated C-terminal region of the transient receptor potential vanilloid 1

Transient receptor potential (TRP) proteins are sensory-related cation channels. TRPV subfamily responds to vanilloids, generating a Ca(2+) current. TRPV1, a thermal-sensitive non-selective ion channel, possesses six transmembrane helices and the intracellular N- and C-terminal domains. The latter contains the PIP(2) and calmodulin binding sites, the TRP domain and a temperature-responding flexible region. Although the function of C-TRPV1 is known, there are no experimental reports on its structural features. Here, we describe the conformational features of C-TRVP1, by using spectroscopic and biophysical approaches. Our results show that C-TRVP1 is an oligomeric protein, which shows features of natively unfolded proteins.     

dTULP,the Drosophila melanogaster Homolog of Tubby,Regulates Transient Receptor Potential Channel Localization in Cilia

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.     

Phospholipase C Mediated Modulation of TRPV1 Channels

The transient receptor potential vanilloid type 1 (TRPV1) channels are involved in both thermosensation and nociception. They are activated by heat, protons, and capsaicin and modulated by a plethora of other agents. This review will focus on the consequences of phospholipase C (PLC) activation, with special emphasis on the effects of phosphatidylinositol 4,5-bisphosphate (PIP2) on these channels. Two opposing effects of PIP2 have been reported on TRPV1. PIP2 has been proposed to inhibit TRPV1, and relief from this inhibition was suggested to be involved in sensitization of these channels by pro-inflammatory agents. In excised patches, however, PIP2 was shown to activate TRPV1. Calcium flowing through TRPV1 activates PLC and the resulting depletion of PIP2 was proposed to play a role in capsaicin-induced desensitization of these channels. We will describe the data indicating involvement of PLC and PIP2 in sensitization and desensitization of TRPV1 and will also discuss other pathways potentially contributing to these two phenomena. We attempt to resolve the seemingly contradictory data by proposing that PIP2 can both activate and inhibit TRPV1 depending on the experimental conditions, more specifically on the level of stimulation of these channels. Finally, we also discuss data in the literature indicating that other TRP channels, TRPA1 and some members of the TRPC subfamily, may also be under a similar dual control by PIP2.     

Phosphoinositide regulation of non-canonical transient receptor potential channels

Transient receptor potential (TRP) channels are involved in a wide range of physiological processes, and characterized by diverse activation mechanisms. Phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP₂, or PtdIns(4,5)P₂] recently emerged as regulators of many TRP channels. Several TRP channels require PIP₂ for activity, and depletion of the lipid inhibits them. For some TRP channels, however, phosphoinositide regulation seems more complex, both activating and inhibitory effects have been reported. This review will discuss phosphoinositide regulation of members of the TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin) and TRPP (Polycystin) families. Lipid regulation of TRPC (Canonical) channels is discussed elsewhere in this volume.     

Regulation of the Temperature-dependent Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) by Phospholipids in Planar Lipid Bilayers

TRPV1 (transient receptor potential vanilloid 1) proteins are heat-activated nonselective cation channels. TRPV1 channels are polymodal in their function and exhibit multifaceted regulation with various molecular compounds. In this regard, phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, are important channel regulators. However, their effects on TRPV1 channel activity have not been conclusively determined. To characterize temperature-induced activation of TRPV1 in the presence of different phospholipids, we purified the TRPV1 protein from HEK-293 cells and incorporated it into planar lipid bilayers. In the presence of 2.5 μm phosphatidylinositol 4,5-bisphosphate, TRPV1 channels demonstrated rapid activation at 33–39 °C and achieved full channel opening at 42 °C. At this temperature range, TRPV1 heat activation exhibited steep temperature dependence (temperature coefficient (Q₁₀) of 18), and the channel openings were accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. At a similar temperature range, another phosphoinositide, phosphatidylinositol 4-phosphate, also potentiated heat activation of TRPV1, but with much lower efficiency. Negatively charged phosphatidylglycerol could also induce heat activation of TRPV1 channels, although with a small-conductance state. Our data demonstrate that phospholipids, specifically phosphoinositides, are important regulators of TRPV1 and are required for heat-induced channel activity.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

Modulation of TRPV2 by endogenous and exogenous ligands: A computational study

Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.     

TRP channels in kidney disease

Mammalian TRP channel proteins form six-transmembrane cation-permeable channels that may be grouped into six subfamilies on the basis of amino acid sequence homology (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML). Recent studies of TRP channels indicate that they are involved in numerous fundamental cell functions and are considered to play an important role in the pathophysiology of many diseases. Many TRPs are expressed in kidney along different parts of the nephron and growing evidence suggest that these channels are involved in hereditary, as well as acquired kidney disorders. TRPC6, TRPM6, and TRPP2 have been implicated in hereditary focal segmental glomerulosclerosis (FSGS), hypomagnesemia with secondary hypocalcemia (HSH), and polycystic kidney disease (PKD), respectively. In addition, the highly Ca(2+)-selective channel, TRPV5, contributes to several acquired mineral (dys)regulation, such as diabetes mellitus (DM), acid-base disorders, diuretics, immunosuppressant agents, and vitamin D analogues-associated Ca(2+) imbalance whereas TRPV4 may function as an osmoreceptor in kidney and participate in the regulation of sodium and water balance. This review presents an overview of the current knowledge concerning the distribution of TRP channels in kidney and their possible roles in renal physiology and kidney diseases.     

Promiscuous Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) Channels by Negatively Charged Intracellular Lipids: THE KEY ROLE OF ENDOGENOUS PHOSPHOINOSITIDES IN MAINTAINING CHANNEL ACTIVITY*

The regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channels by phosphoinositides is controversial. Data in cellular systems support the dependence of TRPV1 activity on phosphoinositides. The purified TRPV1, however, was recently shown to be fully functional in artificial liposomes in the absence of phosphoinositides. Here, we show that several other negatively charged phospholipids, including phosphatidylglycerol, can also support TRPV1 activity in excised patches at high concentrations. When we incorporated TRPV1 into planar lipid bilayers consisting of neutral lipids, capsaicin-induced activity depended on phosphatidylinositol 4,5-bisphosphate. We also found that TRPV1 activity in excised patches ran down and that MgATP reactivated the channel. Inhibition of phosphatidylinositol 4-kinases or enzymatic removal of phosphatidylinositol abolished this effect of MgATP, suggesting that it activated TRPV1 by generating endogenous phosphoinositides. We conclude that endogenous phosphoinositides are positive cofactors for TRPV1 activity. Our data highlight the importance of specificity in lipid regulation of ion channels and may reconcile discordant data obtained in various experimental settings.     

PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2+

The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C-activating hormones via alteration of the sensitivity to intracellular Mg2+.     

Linoleic acid inhibits TRP channels with intrinsic voltage sensitivity: Implications on the mechanism of linoleic acid action

Open channel block (OCB) is a process by which ions bind to the inside of a channel pore and block the flow of ions through that channel. Repulsion of the blocking ions by membrane depolarization is a known mechanism for open channel block removal. For the N-methyl-D-aspartate (NMDA) channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of Transient Receptor Potential (TRP) channels, including the Drosophila TRP and TRP-Like (TRPL) channels, also exhibit open channel block. For the Drosophila TRP and TRPL channels, removal of open channel block is necessary for the production of the physiological response to light. Recently, we have shown that lipids such as polyunsaturated fatty acids (PUFAs), represented by linoleic acid (LA), alleviate OCB under physiological conditions, from the Drosophila TRP and TRPL channels and from the mammalian NMDA channel. Here we show that OCB removal by LA is not confined to the Drosophila TRPs but also applies to mammalian TRPs such as the heat activated TRPV3 channel. TRPV3 shows OCB alleviation by LA, although it shares little amino acid sequence homology with the Drosophila TRPs. Strikingly, LA inhibits the heat-activated TRPV1 and the cold temperature-activated TRPM8 channels, which are intrinsic voltage sensitive channels and do not show OCB. Together, our findings further support the notion that lipids do not act as second messengers by direct binding to a specific site of the channels but rather act indirectly by affecting the channel-plasma membrane interface.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

Mechanotransduction by TRP Channels: General Concepts and Specific Role in the Vasculature

Transient receptor potential (TRP) ion channel superfamily is involved in sensing and transmission of a broad variety of external or internal stimuli, including but not limited to mechanical stress. Based on homology analysis, genetic and molecular studies have recently identified TRP channels in different tissues, comprising blood vessels. In invertebrates, many TRP channels including five TRPV channels identified in Caenorhabditis elegans and two in Drosophila have been implicated in mechanosensory behaviors as molecular basis of volume regulation, hearing and touch sensitivity. Consistently, in mammals many TRP family members such as TRPC1, TRPC3, TRPC6, TRPM4, TRPM7, TRPN1, TRPA1, TRPY1, TRPP1, TRPP2, and notably, TRPV1, TPRV2 as well as TRPV4 have been reported to be involved in mechanotransduction. This review summarizes recent and at times controversial findings on the role and regulation of TRP channels in mechanotransduction. Specifically, we highlight the relevance of TRPV channels in vascular regulation and focus on TRPV4 in the vascular system of the lung, which is constantly exposed to a unique combination of circumferential and longitudinal strains. In light of our observation in intact pulmonary microvessels that mechanical stress induced Ca²⁺ signaling in endothelial cells is closely related to TRPV4 activity, we postulate that TRPV4 plays a critical role in lung vascular mechanotransduction. The progress in this rapidly expanding field may allow for the identification of new molecular targets and the development of new therapeutic approaches in a number of intractable diseases related to mechanical stress.     

TRP channels as candidates for hearing and balance abnormalities in vertebrates

In this review, we summarize the potential functional roles of transient receptor potential (TRP) channels in the vertebrate inner ear. The history of TRP channels in hearing and balance is characterized at great length by the hunt for the elusive transduction channel of sensory hair cells. Such pursuit has not resulted in unequivocal identification of the transduction channel, but nevertheless revealed a number of candidates, such as TRPV4, TRPN1, TRPA1, and TRPML3. Much of the circumstantial evidence indicates that these TRP channels potentially play significant roles in inner ear physiology. Based on mutations in the corresponding mouse genes, TRPV4 and TRPML3 are possible candidates for human hearing, and potentially also balance disorders. We further discuss the role of the invertebrate TRP channels Nanchung, Inactive, and TRPN1 and how the functional analysis of these channels provides a link to vertebrate hearing and balance. In summary, only a few TRP channels have been analyzed thus far for a prospective role in the inner ear, and this makes the search for additional TRPs associated with inner ear function quite a tantalizing endeavor.