TRPM3 proteins that belong to the large and diverse transient receptor potential (TRP) family, form Ca²⁺ permeable cation channels that are directly activated by the naturally occurring steroid metabolite pregnenolone sulfate (PS). We recently demonstrated that TRPM3 channels are expressed in pancreatic ß-cells and that PS increases glucose-induced insulin release from pancreatic islets (Wagner et al., 2008, Nature Cell Biol. 10:1421–1430).

PS activates TRPM3 channels rapidly (< 100 ms) and only from the extracellular side, since it is inactive when applied to the cytosolic face of excised inside-out patches. However, PS partitions strongly into the plasma membrane and causes a marked rise of the membrane capacitance readily detectable with standard electrophysiological techniques (Mennerick et al., 2008, Biophys J 95:176–185). In the plasma membrane, the insertion of PS therefore is likely to cause deformations and mechanical stretch. This raises the question whether PS exerts its effect on TRPM3 channels via deformations of the plasma membrane or by directly binding to the ion channels at a specific binding site.

To address this question, we used the synthetic enantiomer of pregnenolone sulfate (ent-PS) that has the physical and chemical properties as the natural occurring enantiomer (nat-PS). Using Fura-2 based Ca²⁺-imaging and conventional whole-cell patch-clamping on HEK293 cells overexpressing TRPM3, we found that nat-PS elicits a response that was several fold higher than the response evoked by ent-PS. The dose-response curve for ent-PS was shifted to concentration values that were at least 10 times higher compared to those of the nat-PS curve. These data are incompatible with a model in which PS acts on TRPM3 channels exclusively by modifying the physical properties of the plasma membrane; they instead provide strong evidence for a specific steroid binding site, which likely is located on the TRPM3 protein itself.