TRPV3 mutants causing Olmsted Syndrome induce impaired cell adhesion and nonfunctional lysosomes

TRPV3 is a non-selective cationic channel and is important for several physiological functions. It can be activated by physiological temperature and selective endogenous and exogenous compounds. TRPV3 is one of the key ion channel involved in Ca²⁺-signaling in keratinocyte and thus involved in skin-related functions. Recently, naturally occurring mutations in TRPV3, namely G573A, G573S, G573C and W692G have been detected which are linked with the development of pathophysiological conditions such as Olmsted Syndrome (OS) and other skin disorders. Our qualitative and quantitative data suggests that these naturally occurring TRPV3 mutants are mainly restricted in the ER. Expression of OS-mutants cause impaired vesicular trafficking resulting reduced surface localization of these mutants and other membrane proteins too. OS-mutants also cause reduced cell adhesion, altered distribution and less number of lysosomes. Our data confirms that TRPV3 is a lysosomal protein suggesting that Olmsted Syndrome is a lysosomal disorder. These findings may have a broad implication in the context of keratinocyte functions, skin-degeneration and in skin-cancer.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (delta384-444) variants and the new TRPV4-C (delta237-284), TRPV4-D (delta27-61), and TRPV4-E (delta237-284 and delta384-444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.     

Cross-talk between the calcium channel TRPV4 and reactive oxygen species interlocks adhesive and degradative functions of invadosomes

Invadosomes support cell invasion by coupling both acto-adhesive and extracellular matrix degradative functions, which are apparently antagonistic. β1-integrin dynamics regulate this coupling, but the actual sensing mechanism and effectors involved have not yet been elucidated. Using genetic and reverse genetic approaches combined with biochemical and imaging techniques, we now show that the calcium channel TRPV4 colocalizes with β1-integrins at the invadosome periphery and regulates its activation and the coupling of acto-adhesive and degradative functions. TRPV4-mediated regulation of podosome function depends on its ability to sense reactive oxygen species (ROS) in invadosomes’ microenvironment and involves activation of the ROS/calcium-sensitive kinase Ask1 and binding of the motor MYO1C. Furthermore, disease-associated TRPV4 gain-of-function mutations that modulate ECM degradation are also implicated in the ROS response, which provides new perspectives in our understanding of the pathophysiology of TRPV4 channelopathies.     

Progesterone interacts with the mutational hot-spot of TRPV4 and acts as a ligand relevant for fast Ca2+-signalling

Steroids are also known to induce immediate physiological and cellular response which occurs within minutes to seconds, or even faster. Such non-genomic actions of steroids are rapid and are proposed to be mediated by different ion channels. Transient receptor potential vanilloid sub-type 4 (TRPV4), is a non-specific polymodal ion channel which is involved in several physiological and cellular processes. In this work, we explored the possibilities of Progesterone (P4) as an endogenous ligand for TRPV4. We demonstrate that P4 docks as well as physically interacts with the TM4-loop-TM5 region of TRPV4, a region which is a mutational hotspot for different diseases. Live cell imaging experiments with a genetically encoded Ca²⁺-sensor suggests that P4 causes quick influx of Ca²⁺ specifically in the TRPV4 expressing cells, which can be partially blocked by TRPV4-specific inhibitor, suggesting that P4 can act as a ligand for TRPV4. Such P4-mediated Ca²⁺-influx is altered in cells expressing disease causing TRPV4 mutants, namely in L596P, R616Q, and also in embryonic lethal mutant L618P. P4 dampens, both in terms of “extent” as well as the “pattern” of the Ca²⁺-influx by other stimulus too in cells expressing TRPV4-Wt, suggesting that P4 crosstalk with the TRPV4-mediated Ca²⁺-signalling, both in quick and long-term manner. We propose that P4 crosstalk with TRPV4 might be relevant for both acute and chronic pain as well as for other health-related functions.     

The vanilloid transient receptor potential channel TRPV4: From structure to disease

The Transient Receptor Potential Vanilloid 4 channel, TRPV4, is a Ca²⁺ and Mg²⁺ permeable non-selective cation channel involved in many different cellular functions. It is activated by a variety of physical and chemical stimuli, including heat, mechano-stimuli, endogenous substances such as arachidonic acid and its cytochrome P450-derived metabolites (epoxyeicosatrienoic acids), endocannabinoids (anandamide and 2-arachidonoylglycerol), as well as synthetic α-phorbol derivatives. Recently, TRPV4 has been characterized as an important player modulating osteoclast differentiation in bone remodelling and as a urothelial mechanosensor that controls normal voiding. Several TRPV4 gain-of-function mutations are shown to cause autosomal-dominant bone dysplasias such as brachyolmia and Koszlowski disease. In this review we comprehensively describe the structural, biophysical and (patho)physiological properties of the TRPV4 channel and we summarize the current knowledge about the role of TRPV4 in the pathogenesis of several diseases.     

Multiubiquitination of TRPV4 reduces channel activity independent of surface localization

Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.     

Why individual thermo sensation and pain perception varies? Clue of disruptive mutations in TRPVs from 2504 human genome data

Every individual varies in character and so do their sensory functions and perceptions. The molecular mechanism and the molecular candidates involved in these processes are assumed to be similar if not same. So far several molecular factors have been identified which are fairly conserved across the phylogenetic tree and are involved in these complex sensory functions. Among all, members belonging to Transient Receptor Potential (TRP) channels have been widely characterized for their involvement in thermo-sensation. These include TRPV1 to TRPV4 channels which reveal complex thermo-gating behavior in response to changes in temperature. The molecular evolution of these channels is highly correlative with the thermal response of different species. However, recent 2504 human genome data suggest that these thermo-sensitive TRPV channels are highly variable and carry possible deleterious mutations in human population. These unexpected findings may explain the individual differences in terms of complex sensory functions.     

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.     

A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.     

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.     

An inhibitor of TRPV1 channels isolated from funnel Web spider venom

Capsaicin receptor channels (TRPV1) are nonselective cation channels that integrate multiple noxious stimuli in sensory neurons. In an effort to identify new inhibitors of these channels we screened a venom library for activity against TRPV1 channels and found robust inhibitory activity in venom from Agelenopsis aperta, a north American funnel web spider. Fractionation of the venom using reversed-phase HPLC resulted in the purification of two acylpolyamine toxins, AG489 and AG505, which inhibit TRPV1 channels from the extracellular side of the membrane. The activity of AG489 was characterized further, and the toxin was found to inhibit TRPV1 channels with a K(i) of 0.3 microM at -40 mV. Inhibition of TRPV1 channels by AG489 is strongly voltage-dependent, with relief of inhibition at positive voltages, consistent with the toxin inhibiting the channel through a pore-blocking mechanism. We used scanning mutagenesis throughout the TM5-TM6 linker, a region thought to form the outer pore of TRPV1 channels, to identify pore mutations that alter toxin affinity. Four mutants dramatically decrease toxin affinity and several mutants increase toxin affinity, consistent with the notion that the TM5-TM6 linker forms the outer vestibule of TRPV1 channels and that AG489 is a pore blocker.     

Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3

TRPV4, a member of the vanilloid subfamily of the transient receptor potential (TRP) channels, is activated by a variety of stimuli, including cell swelling, moderate heat, and chemical compounds such as synthetic 4alpha-phorbol esters. TRPV4 displays a widespread expression in various cells and tissues and has been implicated in diverse physiological processes, including osmotic homeostasis, thermo- and mechanosensation, vasorelaxation, tuning of neuronal excitability, and bladder voiding. The mechanisms that regulate TRPV4 in these different physiological settings are currently poorly understood. We have recently shown that the relative amount of TRPV4 in the plasma membrane is enhanced by interaction with the SH3 domain of PACSIN 3, a member of the PACSIN family of proteins involved in synaptic vesicular membrane trafficking and endocytosis. Here we demonstrate that PACSIN 3 strongly inhibits the basal activity of TRPV4 and its activation by cell swelling and heat, while leaving channel gating induced by the synthetic ligand 4alpha-phorbol 12,13-didecanoate unaffected. A single proline mutation in the SH3 domain of PACSIN 3 abolishes its inhibitory effect on TRPV4, indicating that PACSIN 3 must bind to the channel to modulate its function. In line herewith, mutations at specific proline residues in the N terminus of TRPV4 abolish binding of PACSIN 3 and render the channel insensitive to PACSIN 3-induced inhibition. Taken together, these data suggest that PACSIN 3 acts as an auxiliary protein of TRPV4 channel that not only affects the channel's subcellular localization but also modulates its function in a stimulus-specific manner.     

Distinct sites on G protein beta gamma subunits regulate different effector functions

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents. Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K(+) channels. With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors. These data show that distinct domains within Gbetagamma subserve specific functional roles.     

Single point mutations of aromatic residues in transmembrane helices 5 and -6 differentially affect TRPV4 activation by 4α-PDD and hypotonicity: Implications for the role of the pore region in regulating TRPV4 activity

The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure–function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5–6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants – F617L and Y621L – showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.     

Modulation of TRPV4 by diverse mechanisms

Transient receptor potential ion channels (TRP) are a superfamily of non-selective ion channels which are opened in response to a diverse range of stimuli. The TRP vanilloid 4 (TRPV4) ion channel is opened in response to heat, mechanical stimuli, hypo-osmolarity and arachidonic acid metabolites. However, recently TRPV4 has been identified as an ion channel that is modulated by, and opened by intracellular signalling cascades from other receptors and signalling pathways. Although TRPV4 knockout mice show relatively mild phenotypes, some mutations in TRPV4 cause severe developmental abnormalities, such as the skeletal dyplasia and arthropathy. Regulated TRPV4 function is also essential for healthy cardiovascular system function as a potent agonist compromises endothelial cell function, leading to vascular collapse. A better understanding of the signalling mechanisms that modulate TRPV4 function is necessary to understand its physiological roles. Post translational modification of TRPV4 by kinases and other signalling molecules can modulate TRPV4 opening in response to stimuli such as mechanical and hyposmolarity and there is an emerging area of research implicating TRPV4 as a transducer of these signals as opposed to a direct sensor of the stimuli. Due to its wide expression profile, TRPV4 is implicated in multiple pathophysiological states. TRPV4 contributes to the sensation of pain due to hypo-osmotic stimuli and inflammatory mechanical hyperalsgesia, where TRPV4 sensitizaton by intracellular signalling leads to pain behaviors in mice. In the vasculature, TRPV4 is a regulator of vessel tone and is implicated in hypertension and diabetes due to endothelial dysfunction. TRPV4 is a key regulator of epithelial and endothelial barrier function and signalling to and opening of TRPV4 can disrupt these critical protective barriers. In respiratory function, TRPV4 is involved in cystic fibrosis, cilary beat frequency, bronchoconstriction, chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury, acute respiratory distress syndrome and cough.In this review we highlight how modulation of TRPV4 opening is a vital signalling component in a range of tissues and why understanding of TRPV4 regulation in the body may lead to novel therapeutic approaches to treating a range of disease states.     

DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor

Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in?vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also coexpress DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.     

The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active

TRPV3 is a non-selective cation channel activated by warm to hot temperatures. In rodents, TRPV3 is highly expressed in basal keratinocytes of skin and oral/nasal epithelia. TRPV3 knockout mice showed impaired responses to innocuous and noxious heat but otherwise normal appearance and reactions to many sensory modalities. However, point mutations of TRPV3 at Gly573 to Ser and Cys have recently been linked to autosomal dominant hairless phenotypes and spontaneous dermatitis in mice and rats, implicating an important role for TRPV3 in alopecia and skin diseases. Exactly, how the mutations affect TRPV3 function was unexplained. Here, we show that both G573S and G573C mutations of murine TRPV3 are constitutively active in heterologous systems. In HEK 293 cells, expression of the TRPV3 mutants causes cell death. In Xenopus oocytes, the constitutively active mutant channel is irresponsive to thermal and chemical stimuli but it reduces the temperature threshold and enhances the responses to heat and TRPV3 agonists of the wild type channel when they are co-expressed. We conclude that the G573S and G573C substitutions render the TRPV3 channel spontaneously active under normal physiological conditions, which in turn alters ion homeostasis and membrane potentials of skin keratinocytes, leading to hair loss and dermatitis-like skin diseases.