Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.

TRPA1 functions as an excitatory ionotropic receptor in sensory neurons. It was originally described as a noxious cold-activated channel, but its cold sensitivity has been disputed in later studies, and the contribution of TRPA1 to thermosensing is currently a matter of strong debate. Here, we provide several lines of evidence to establish that TRPA1 acts as a cold sensor in vitro and in vivo. First, we demonstrate that heterologously expressed TRPA1 is activated by cold in a Ca 2؉ -independent and Ca 2؉ store-independent manner; temperature-dependent gating of TRPA1 is mechanistically analogous to that of other temperature-sensitive TRP channels, and it is preserved after treatment with the TRPA1 agonist mustard oil. Second, we identify and characterize a specific subset of cold-sensitive trigeminal ganglion neurons that is absent in TRPA1-deficient mice. Finally, cold plate and tail-flick experiments reveal TRPA1-dependent, cold-induced nociceptive behavior in mice. We conclude that TRPA1 acts as a major sensor for noxious cold.cold sensing ͉ pain ͉ sensory neurons ͉ TRP channels S ensing the environmental temperature is essential for animals to maintain thermal homeostasis and to avoid prolonged contact with harmfully hot or cold objects (1). Our understanding of the molecular basis of thermosensation has made great strides with the discovery that several members of the transient receptor potential (TRP) cation channel family exhibit highly temperature-sensitive gating and are expressed in cells of the sensory system (1). Mice lacking specific temperature-sensitive TRP channels illustrate how these channels serve as molecular thermometers in the peripheral sensory system (2). At least 3 heat-activated members of the TRPV subfamily (TRPV1, TRPV3, and TRPV4) are critically involved in sensing hot temperatures. TRPM8, a channel activated by cold temperatures and cooling compounds, such as menthol, plays a major role in cold sensing (1). Importantly, although TRPM8-deficient mice exhibit significant deficits in cold sensing in the temperature range between 28°C and 15°C, they retain a normal response to noxious cold temperatures, demonstrating the existence of TRPM8-independent mechanisms to detect noxious cold (3-5). TRPA1 has been put forward as a potential candidate to mediate detection of noxious cold, based on its expression in nociceptive neurons, and on the finding that heterologously expressed TRPA1 in CHO cells is activated by cold temperatures with a lower temperature threshold for activation than TRPM8 (6-8).At this point, however, the role of TRPA1 in (noxious) cold sensing is highly controversial. First, there is no consensus as to whether TRPA1 is directly gated by cold temperatures. Two groups have reported that they failed to detect cold-induced activation of heterologously expressed TRPA1 (9, 10), and a third report suggested that cold-induced activation of TRPA1 in overexpression systems is an indirect effect, caused by cold-induced Ca 2ϩ release from intracellular stores and subsequent Ca 2ϩ -d...

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...

Acute pain represents a crucial alarm signal to protect us from injury. Whereas the nociceptive neurons that convey pain signals were described more than a century ago, the molecular sensors that detect noxious thermal or mechanical insults have yet to be fully identified. Here we show that acute noxious heat sensing in mice depends on a triad of transient receptor potential (TRP) ion channels: TRPM3, TRPV1, and TRPA1. We found that robust somatosensory heat responsiveness at the cellular and behavioural levels is observed only if at least one of these TRP channels is functional. However, combined genetic or pharmacological elimination of all three channels largely and selectively prevents heat responses in both isolated sensory neurons and rapidly firing C and Aδ sensory nerve fibres that innervate the skin. Strikingly, Trpv1Trpm3Trpa1 triple knockout (TKO) mice lack the acute withdrawal response to noxious heat that is necessary to avoid burn injury, while showing normal nociceptive responses to cold or mechanical stimuli and a preserved preference for moderate temperatures. These findings indicate that the initiation of the acute heat-evoked pain response in sensory nerve endings relies on three functionally redundant TRP channels, representing a fault-tolerant mechanism to avoid burn injury.