TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.
We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. ] i inhibits the channel with an IC 50 of 406 nM. Ruthenium Red at a concentration of 1 M completely blocks inward currents at ؊80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.
TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities.
Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca 2 þ -activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca 2 þ influx through Ca 2 þ -permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca 2 þ ([Ca 2 þ ] i ). Here we show that phosphatidylinositol 4,5-biphosphate (PIP 2 ) counteracts desensitization to [Ca 2 þ ] i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP 2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca 2 þ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP 2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP 2 , whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP 2 sensitivity. Our data demonstrate that PIP 2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP 2 action. PLC-mediated PIP 2 breakdown may constitute a physiologically important brake on TRPM4 activity.
TRPM4 is a Ca 2؉ -activated but Ca 2؉ -impermeable cation channel. An increase of [Ca 2؉ ] i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. Cation channels of the transient receptor potential (TRP) 1 superfamily have received much attention during the last years because of their pivotal role in various cell functions linked to the modulation of intracellular Ca 2ϩ signals, mostly in nonexcitable cells (1-3). More than 20 mammalian TRP members are known, which are classified into the TRPC (C for "canonical"), TRPV (V for "vanilloid"), and TRPM (M for "melastatin") subfamilies (1, 2). However, the functional properties of most members of this novel and fast growing channel family are not yet analyzed in detail. The predicted transmembrane topology of TRPs is similar to that of voltage-gated and cyclic nucleotide gated channels; they consist of six transmembrane-spanning helices (TM1 to -6), cytoplasmic N and C termini, and a pore region between TM5 and TM6 (2, 3). Since the fourth transmembrane helix is not positively charged, TRP channels were considered as voltage-independent. The voltage-sensing features of some members of the TRPV subfamily could be attributed to voltage-dependent block of the channel pore by intra-or extracellular divalent cations (4, 5).Members of the TRPM subfamily are much less studied at the functional level than those of the TRPV and TRPC family. They are characterized by relatively long N and C termini, and some of them have entire enzyme domains linked to their C termini: an ADP-ribose pyrophosphatase in TRPM2 (6) and an atypical ␣-kinase domain in TRPM6 and TRPM7 (7-11). Surprisingly, TRPM4b, which is a Ca 2ϩ -impermeable monovalent cation channel of 25-picosiemens unitary conductance belonging to the TRPM subfamily, is in contrast to other TRP channels not inactivated but activated by intracellular Ca 2ϩ , [Ca 2ϩ ] i (12). A short form of TRPM4, TRPM4a, is characterized in less detail and displays completely different properties with regard to Ca 2ϩ permeability and activation (13). In this study, we report cloning of the human and mouse TRPM4 cDNAs. Transcripts of these genes are expressed in heart, kidney, and endothelial cells, indicating that this channel plays a role in the cardiovascular system. We demonstrate that TRPM4 is a Ca 2ϩ -and voltage-dependent channel. It could therefore modulate the electrical activity of cells that generate action potentials. This is, to our knowledge, the first description of voltage-dependent properties of a TRP channel, suggesting a special role for this channel in excitable cells. MATERIALS AND METHODS Cloning of Human and Mouse TRPM4 cDNAs-The human expressed sequence tag 885075 (GenBank TM ), homologous to the human TRPM1 cDNA, was identified and sequenced on both strands; it contained a ϳ1500-bp DNA fragment, which represented part of the...
Nilius, Bernd, Joris Vriens, Jean Prenen, Guy Droogmans, and Thomas Voets. TRPV4 calcium entry channel: a paradigm for gating diversity. Am J Physiol Cell Physiol 286: C195-C205, 2004;10.1152/ajpcell.00365.2003.-The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca 2ϩ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4␣-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca 2ϩ entry channels. from intracellular stores and entry of extracellular Ca 2ϩ via diverse Ca 2ϩ entry channels. In the last 10 years, several novel Ca 2ϩ entry channels belonging to the still expanding family of TRP cation channels have been discovered. More than 20 mammalian TRP genes have been identified, encoding membrane proteins with six transmembrane segments (TM1-TM6) and a putative pore region formed by a short hydrophobic stretch between TM5 and TM6 (for detailed reviews, see Refs. 11,48,49). On the basis of their homology, mammalian TRP proteins are classified into three subfamilies (50): TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin). The core transmembrane channel structure of TRP channels resembles that of the pore-forming subunits of voltage-gated and cyclic nucleotide-gated channels and consists of a coassembly of four subunits (32). THE TRPV SUBFAMILYTRPV1 (VR-1), the founding member of the TRPV family, was identified by expression cloning as a capsaicin-and heatgated channel (9). A similar expression cloning strategy for proteins responsible for reabsorption of Ca 2ϩ in the kidney (31) and the gut (63) led to the discovery of TRPV5 (ECaC1) and TRPV6 (CaT1). The remaining three members (TRPV2-4) were identified by using electronic search strategies designed to recognize proteins related to TRPV1 or the related OSM-9 protein from Caenorhabditis elegans (for a detailed review, see Refs. 4,27). Functionally, the six mammalian members of the TRPV subfamily can be subdivided in two groups: TRPV1 to TRPV4 are Ca 2ϩ -permeable, nonselective cation channels with steep temperature dependence; TRPV5 and TRPV6 are highly Ca 2ϩ -selective channels with low temperature sensitivity. TRPV channels are also present in invertebrates: C. elegans genome encodes five TRPVs, OCR-1 to OCR-4 and the abovementioned OSM-9; Drosophila melanogaster expresses two TRPVs.TRPV1 is an outwardly rectifying cation-selective ion channel with a preference for calcium (P Ca /P Na ϳ 10) and magnesium (P Mg /P Na ϳ 5) (9), which depends on a single aspartic acid residue in the pore region of the prot...
TRPM4, a Ca2؉ -activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca 2؉ . The mechanisms underlying the alterations in Ca 2؉ sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca 2؉ sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca 2؉ sensitivity of wildtype TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca 2؉ dramatically reduced TRPM4 activation. We identified five Ca 2؉ -calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca 2؉ sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca 2؉ sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus. TRPM41 is a Ca 2ϩ -activated and voltage-dependent Ca 2ϩ -impermeable cation channel with a unitary conductance of 25 picosiemens that belongs to the melastatin subfamily of transient receptor potential membrane proteins (1-4). Ca 2ϩ -activated, Ca 2ϩ -impermeable nonselective cation channels that share functional properties with expressed TRPM4 (or the closest homologue, TRPM5) have been found in many excitable and non-excitable cells (Refs. 5-8; for reviews, see Refs. 9 and 10). These nonselective channels may regulate important processes including cardiac rhythmicity and neural bursting activity, and their Ca 2ϩ -dependent activation has been suggested to form a general feedback control mechanism for Ca 2ϩ influx in nonexcitable cells.The functional analysis of TRPM4 and TRPM5 and their comparison with native nonselective cation channels are complicated by a peculiar property of these channels: when activated by an increase in free intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), the currents decay rapidly due to a decrease in the sensitivity of the channels to Ca 2ϩ (1,3,4,11). Moreover, recent studies on TRPM4 exhibit an unusually large variability in reported values for Ca 2ϩ sensitivity and activation time courses (1-4). Most likely, these discrepancies reflect a highly regulated Ca 2ϩ affinity of TRPM4, which may be of physiological relevance.Another puzzling property of TRPM4 is its sensitivity to ATP. We have shown recently that cytosolic ATP 4Ϫ acts as a potent inhibitor of TRPM4 currents in inside-out patches, with half-maximal inhibition at ϳ2 M (3). However, robust TRPM4 currents can be measured in the whole-cell mode, even under conditions in which the free cytosolic ATP 4Ϫ concentration exceeds 100 M. One possible explanation of this apparent paradox could be that ATP has both an inhibitory and a stimulatory effect on TRPM4, but experimental data to support this idea are currently lacking.In the present study, we investigated potential cellular...
Abbreviations used in this paper: FRET, fl uorescence resonance energy transfer; hpf, hours postfertilization; MO, morpholino oligonucleotide; RR, ruthenium red; TRP, transient receptor potential.The online version of this paper contains supplemental material.
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