Ca2+-dependent regulation and binding of calmodulin to multiple sites of Transient Receptor Potential Melastatin 3 (TRPM3) ion channels.

TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.

Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo.

Transient receptor potential melastatin 3 (TRPM3) is a calcium-permeable nonselective cation channel that is expressed in a subset of dorsal root (DRG) and trigeminal ganglia sensory neurons. TRPM3 can be activated by the neurosteroid pregnenolone sulfate (PregS) and heat. TRPM3⁻/⁻ mice display an impaired sensation of noxious heat and thermal hyperalgesia. We have previously shown that TRPM3 is blocked by the citrus fruit flavanones hesperetin, naringenin, and eriodictyol as well as by ononetin, a deoxybenzoin from Ononis spinosa. To further improve the tolerability, potency, and selectivity of TRPM3 blockers, we conducted a hit optimization procedure by rescreening a focused library that was composed of chemically related compounds. Within newly identified TRPM3 blockers, isosakuranetin and liquiritigenin displayed favorable properties with respect to their inhibitory potency and a selective mode of action. Isosakuranetin, a flavanone whose glycoside is contained in blood oranges and grapefruits, displayed an IC50 of 50 nM and is to our knowledge the most potent inhibitor of TRPM3 identified so far. Both compounds exhibited a marked specificity for TRPM3 compared with other sensory TRP channels, and blocked PregS-induced intracellular free Ca²âº concentration signals and ionic currents in freshly isolated DRG neurons. Furthermore, isosakuranetin and previously identified hesperetin significantly reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Because the physiologic functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo.

Transient receptor potential melastatin 1 (TRPM1) is an ion-conducting plasma membrane channel inhibited by zinc ions.

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic ß cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.