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.

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3␣1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, bettercharacterized TRP channels. We selected two splice variants, TRPM3␣1 and TRPM3␣2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3␣1 as well as TRPM3␣2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg 2؉ . However, these two variants are highly different in their ionic selectivity. Whereas TRPM3␣1-encoded channels are poorly permeable for divalent cations, TRPM3␣2-encoded channels are well permeated by Ca 2؉ and Mg 2؉ . Additionally, we found that currents through TRPM3␣2 are blocked by extracellular monovalent cations, whereas currents through TRPM3␣1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ionconducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channelencoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles. The transient receptor potential (TRP)1 gene family comprises at least 28 mammalian genes divided into seven subfamilies (1, 2). Most of the encoded proteins exhibit common structural features such as six predicted transmembrane (TM) domains with a putative pore loop between TM5 and TM6 and the so-called TRP box after TM6 (1, 2). Although all members of this group have been reported to form cationic channels, their mechanisms of activation, their regulation, and their biological functions are remarkably diverse. They also display a large variety of different cation selectivities (1, 2). For example, TRPM4 and TRPM5 have been described as impermeable for divalent cations (3-5), whereas TRPV5 and TRPV6 appear to be exclusively permeable for Ca 2ϩ (6, 7). The diversity of TRP channels is further increased by the fact that most members of the TRP gene family can give rise to several different transcripts due to alternative splicing (8).In a few cases, the functional consequences of these alternative splice events are now beginning to emerge. For example, missplicing of TRPM6 transcripts is associated with a hereditary disorder called hypomagnesemia with secondary hypocalcemia (9, 10), and an amino-terminal-truncated variant of TRPM4 appears to modulate Ca 2ϩ oscillations after receptor stimulation in T lymphocytes (11).However, up to now, the largest number of different splice variants ...

Anion channels are present in every mammalian cell and serve many different functions, including cell volume regulation, ion transport across epithelia, regulation of membrane potential and vesicular acidification. Here we characterize a proton-activated, outwardly rectifying current endogenously expressed in HEK293 cells. Binding of three to four protons activated the anion permeable channels at external pH below 5.5 (50% activation at pH 5.1). The proton-activated current is strongly outwardly rectifying, due to an outwardly rectifying single channel conductance and an additional voltage dependent facilitation at depolarized membrane potentials. The anion channel blocker 4,4 -diisothiocyanostilbene-2,2 -disulphonic acid (DIDS) rapidly and potently inhibited the channel (IC 50 : 2.9 µM). Flufenamic acid blocked this channel only slowly, while mibefradil and amiloride at high concentrations had no effect. As determined from reversal potential measurements under bi-ionic conditions, the relative permeability sequence of this channel was SCNNone of the previously characterized anion channel matches the properties of the proton-activated, outwardly rectifying channel. Specifically, the proton-activated and the volume-regulated anion channels are two distinct and separable populations of ion channels, each having its own set of biophysical and pharmacological properties. We also demonstrate endogenous proton-activated currents in primary cultured hippocampal astrocytes. The proton-activated current in astrocytes is also carried by anions, strongly outwardly rectifying, voltage dependent and inhibited by DIDS. Proton-activated, outwardly rectifying anion channels therefore may be a broadly expressed part of the anionic channel repertoire of mammalian cells.

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. Here, we report that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel.