Properties of the Steroid Binding Site of TRPM3 Channels

TRPM3 channels are non-selective cation channels that have been implicated in a variety of functions, including the detection of noxious heat and the secretion of insulin. These channels can be activated by the endogenous steroid pregnenolone sulfate and the voltage-gated calcium channel blocker nifedipine, but it is unclear how these compounds interact with the channels.In electrophysiological and calcium imaging experiments we provide evidence that pregnenolone sulfate and nifedipine bind to distinct binding sites. Furthermore, we show that pregnenolone sulfate needs to bind to a chiral, and thus proteinaceous, binding site in order to activate TRPM3 by utilizing the enantiomer of pregnenolone sulfate. Employing further structural analogs of pregnenolone sulfate, we additionally determined that the binding site of pregnenolone sulfate needs to accommodate a large, negatively charged substituent at the C3 position of the steroid backbone.By combining these data we devised a strategy to find candidate amino acid residues of TRPM3 important for channel activation, possibly by being part of the pregnenolone sulfate binding site. We systematically mutated positively charged amino acids accessible from the extracellular side (from which pregnenolone sulfate is capable of activating TRPM3). We then evaluated whether mutated channels that displayed a reduced response to pregnenolone sulfate still responded to nifedipine comparably to wild-type channels. We identified one amino acid with these properties, which is predicted to be located at the transmembrane-extracellular domain interface.These data will lead to the characterization of the steroid binding site responsible for activating TRPM3 channels. Likely, this will help to identify more potent and specific pharmacological tools, with the ultimate goal to manipulate these channels for experimental and therapeutic purposes.