Transient Receptor Potential Channel Function Research Articles

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.

International Union of Basic and Clinical Pharmacology. LXXVI. Current Progress in the Mammalian TRP Ion Channel Family

Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca(2+) and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels.

Renal TRPathies

Many ion channels and transporters are involved in the filtration, secretion, and resorption of electrolytes by the kidney. In recent years, the superfamily of transient receptor potential (TRP) ion channels have received deserved attention because mutated TRP channels are linked to human kidney diseases. This review focuses on two TRP members--TRPC6 and TRPM6--and their functions in the kidney. Gain-of-function mutations in TRPC6 are the cause for progressive kidney failure with urinary protein loss such as FSGS. Thus, TRPC6 is an essential signaling component in a functional slit diaphragm formed by podocytes around the glomerular capillaries. Loss-of-function mutations in TRPM6 are a molecular cause of hypomagnesemia with secondary hypocalcemia, suggesting that TRPM6 is critically involved in transcellular Mg2+ transport in the kidney. Here, we highlight how recent studies analyzing function and expression of these channels in the kidney improve our mechanistic understanding of TRP channel function in general and pave the way to new, promising therapeutic strategies to target kidney diseases such as FSGS and hypomagnesemia with secondary hypocalcemia.

The Role of Transient Receptor Potential Channels in Metabolic Syndrome

Metabolic syndrome is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential (TRP) canonical (TRPC), vanilloid (TRPV), and melastatin (TRPM) channels, have been associated with the development of cardiovascular diseases. Thus, disruption of TRP channel expression or function may account for the observed increased cardiovascular risk in metabolic syndrome patients. TRPV1 regulates adipogenesis and inflammation in adipose tissues, whereas TRPC3, TRPC5, TRPC6, TRPV1, and TRPM7 are involved in vasoconstriction and regulation of blood pressure. Other members of the TRP family are involved in regulation of insulin secretion, lipid composition, and atherosclerosis. Although there is no evidence that a single TRP channelopathy may be the cause of all metabolic syndrome characteristics, further studies will help to clarify the role of specific TRP channels involved in the metabolic syndrome. (Hypertens Res 2008; 31: 1989-1995).

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca2+ and Mg2+ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.

Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.

TRP channels and mechanical stress

Transient receptor potential (TRP) channels respond to diverse external stimuli and convert them into intracellular calcium signals. Recent evidence shows that TRPV4, one of the vanilloid subfamily, plays a key role in mechanosensation in bone. Here I summarize the current knowledge on possible functions of TRP channels.

Determinants of 4α-Phorbol Sensitivity in Transmembrane Domains 3 and 4 of the Cation Channel TRPV4

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.

TRP channels and kidney disease: lessons from polycystic kidney disease

Important insights in to the function of members of the TRP (transient receptor potential) channel superfamily have been gained from the identification of disease-related mutations. In particular the identification of mutations in the PKD2 gene in autosomal dominant polycystic kidney disease has revealed a link between TRP channel function, mechanosensation and the role of the primary cilium in renal cyst formation. The PKD2 gene encodes TRPP2 (transient receptor potential polycystin 2) that has significant homology to voltage-activated calcium and sodium TRP channels. It interacts with polycystin-1 to form a large membrane-associated complex that is localized to the renal primary cilium. Functional characterization of this polycystin complex reveals that it can respond to mechanical stimuli such as flow, resulting in influx of extracellular calcium and release of calcium from intracellular stores. TRPP2 is expressed in the endoplasmic reticulum/sarcoplasmic reticulum where it also regulates intracellular calcium signalling. Therefore TRPP2 modulates many cellular processes via intracellular calcium-dependent signalling pathways.

Why Spider Bites Hurt

Peptide toxins produced by venomous creatures contain potent inhibitors of channel activity, and these inhibitors contribute to the shock, paralysis, and death that can accompany bites from one of these organisms. Siemens et al . show that, in addition to these potent channel inhibitors, spider venom has peptides of the inhibitor cysteine knot (ICK) family that activate transient receptor potential (TRPs) channels associated with pain perception. Venom from 22 spiders and scorpions was applied to human embryonic kidney (HEK) 293 cells transfected with TRPV1, TRPA1, or TRPM8 (all TRP channels found in primary afferent neurons, the activation of which is associated with pain and inflammation). Venom from one species of tarantula stimulated an increase in calcium signaling when applied to the TRPV1-expressing cells but not the TRPA1- or TRPM8-expressing cells. The active peptides were isolated and sequenced and named vanillotoxins 1, 2, and 3 (VaTx1, VaTx2, and VaTx3). VaTx1 and VaTx2 were most similar to an ICK that inhibits Kv2-type voltage-gated potassium channels, whereas VaTx3 resembles a toxin (huwentoxin 5) from another spider that has no known target. Huwentoxin 5 also triggered a calcium signal in TRPV1-expressing cells. VaTx1 and VaTx2 inhibited Kv2.1 activity, although VaTx2 was more potent at activating the TRPV1 channel than at inhibiting the Kv2.1 channel. VaTx3 did not inhibit Kv2.1 channels. Dose-response analysis showed that VaTx3 was most potent, followed by VaTx2, then VaTx1, and the abundance of these toxins in the venom was inversely correlated with their potency. To confirm the relevance of these results in pain transmission, the authors demonstrated that dissociated sensory neurons from mouse trigeminal ganglia responded to VaTx2 or VaTx3 with a robust calcium signal and that this response was abolished in neurons from mice deficient in TRPV1. Injection of VaTx3 into the paws of mice caused pain-related behaviors and swelling; these responses were absent in the TRPV1-deficient mice. These results provide mechanistic insight into the pain associated with bites from venomous animals and also new tools for studying TRP channel function. J. Siemens, S. Zhou, R. Piskorowski, T. Nikai, E. A. Lumpkin, A. I. Basbaum, D. King, D. Julius, Spider toxins activate the capsaicin receptor to produce inflammatory pain. Nature 444 , 208-212 (2006). [PubMed]

TRP proteins and cancer

Cancer is the second most common cause of death in western countries. It is therefore of fundamental importance to improve the treatment of patients with malignant tumors. This goal can only be achieved if we get closer insight in the various mechanisms leading to tumor formation. Significant progress in the understanding of carcinogenesis has been made during the last couple of years. Ion channels contribute to the regulation of cell proliferation which has initially been shown for K + channels. Meanwhile, other ion channels such as Cl −, Na + and Ca 2+ channels seem to influence cellular function like growth, migration and invasion. In addition, cation channels of the transient receptor potential (TRP) superfamily are implicated in cancer formation. Most recent data concerning TRP vanilloid (TRPV) type 6, TRP melastatin (TRPM) type 1 and 8 channels and their relevance for common human cancer types will be highlighted in this review. Furthermore, TRP channel structure and function will be discussed in the light of their possible importance as prognostic markers and targets for drug discovery.

Role of Src in C3 transient receptor potential channel function and evidence for a heterogeneous makeup of receptor- and store-operated Ca 2+ entry channels

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors and activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays we now report that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (i) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 had no effect; (ii) TRPC6 and TRPC7 were activated in src-, yes-, and fyn-deficient cells; and (iii) src, but not yes or fyn, rescued TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src was found to interact with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous.

AN INTRODUCTION TO TRP CHANNELS

The aim of this review is to provide a basic framework for understanding the function of mammalian transient receptor potential (TRP) channels, particularly as they have been elucidated in heterologous expression systems. Mammalian TRP channel proteins form six-transmembrane (6-TM) 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). Selected functional properties of TRP channels from each subfamily are summarized in this review. Although a single defining characteristic of TRP channel function has not yet emerged, TRP channels may be generally described as calcium-permeable cation channels with polymodal activation properties. By integrating multiple concomitant stimuli and coupling their activity to downstream cellular signal amplification via calcium permeation and membrane depolarization, TRP channels appear well adapted to function in cellular sensation. Our review of recent literature implicating TRP channels in neuronal growth cone steering suggests that TRPs may function more widely in cellular guidance and chemotaxis. The TRP channel gene family and its nomenclature, the encoded proteins and alternatively spliced variants, and the rapidly expanding pharmacology of TRP channels are summarized in online supplemental material.

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp 14 with an amino acid substitution juxtaposed to the TRP domain. The trp 14 flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp Δ 1272, had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na+/Ca2+ exchanger, CalX, indicating that the cell death was due primarily to deficient Ca2+ entry rather than disruption of the TRP-anchoring function.

TRP channels: novel gating properties and physiological functions

TRP channels: novel gating properties and physiological functions

Functional role of TRPC proteins in native systems: implications from knockout and knock‐down studies

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

A Functional Link between Store-operated and TRPC Channels Revealed by the 3,5-Bis(trifluoromethyl)pyrazole Derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.

Transient receptor potential and other ion channels as pharmaceutical targets in airway smooth muscle cells

Regardless of the triggering stimulus in asthma, contraction of the airway smooth muscle (ASM) is considered to be an important pathway leading to the manifestation of asthmatic symptoms. Therefore, the various ion channels that modulate ASM contraction and relaxation are particularly attractive targets for therapy. Although voltage-operated Ca2+ channels (VOCC) are the most extensively characterised Ca(2+)-permeable channels in ASM cells and are obvious pharmacological targets, blockers of VOCC have not been successful in alleviating ASM contraction in asthma. Similarly, although the Cl- and K+ channels also modulate ASM contraction and relaxation by regulating plasma membrane potential, pharmacological interventions directed against these channels have failed to abrogate ASM contraction in asthma. A large body of evidence suggests that store-operated Ca2+ channels (SOCC) and Ca(2+)-permeable second messenger-activated non-selective cation channels (NSCC) predominantly mediate ASM contraction. However, development of pharmacological interventions involving these channels has been hampered by the paucity of information regarding their molecular identity. Members of the mammalian transient receptor potential (TRP) protein family, which form voltage-independent channels with variable Ca2+ selectivity that are activated by store depletion and/or by intracellular messengers, are potential molecular candidates for SOCC and NSCC in ASM cells. While the function of TRP channels in ASM cells remains to be elucidated and there are, at present, essentially no good TRP channel antagonists, this group of proteins is a potentially valuable pharmaceutical target for the treatment of asthma.

Regulation of a Transient Receptor Potential (TRP) Channel by Tyrosine Phosphorylation: SRC FAMILY KINASE-DEPENDENT TYROSINE PHOSPHORYLATION OF TRPV4 ON TYR-253 MEDIATES ITS RESPONSE TO HYPOTONIC STRESS

The recently identified transient receptor potential (TRP) channel family member, TRPV4 (formerly known as OTRPC4, VR-OAC, TRP12, and VRL-2) is activated by hypotonicity. It is highly expressed in the kidney as well as blood-brain barrier-deficient hypothalamic nuclei responsible for systemic osmosensing. Apart from its gating by hypotonicity, little is known about TRPV4 regulation. We observed that hypotonic stress resulted in rapid tyrosine phosphorylation of TRPV4 in a heterologous expression model and in native murine distal convoluted tubule cells in culture. This tyrosine phosphorylation was sensitive to the inhibitor of Src family tyrosine kinases, PP1, in a dose-dependent fashion. TRPV4 associated with Src family kinases by co-immunoprecipitation studies and confocal immunofluorescence microscopy, and this interaction required an intact Src family kinase SH2 domain. One of these kinases, Lyn, was activated by hypotonic stress and phosphorylated TRPV4 in an immune complex kinase assay and an in vitro kinase assay using recombinant Lyn and TRPV4. Transfection of wild-type Lyn dramatically potentiated hypotonicity-dependent TRPV4 tyrosine phosphorylation whereas dominant negative-acting Lyn modestly inhibited it. Through mutagenesis studies, the site of tonicity-dependent tyrosine phosphorylation was mapped to Tyr-253, which is conserved across all species from which TRPV4 has been cloned. Importantly, point mutation of Tyr-253 abolished hypotonicity-dependent channel activity. In aggregate, these data indicate that hypotonic stress results in Src family tyrosine kinase-dependent tyrosine phosphorylation of the tonicity sensor TRPV4 at residue Tyr-253 and that this residue is essential for channel function in this context. This is the first example of direct regulation of TRP channel function through tyrosine phosphorylation.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca(2+)-permeable channel in the yeast vacuolar membrane.

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca(2+) activates the YVC1 current carried by cations including Ca(2+) across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.