Abstract
TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel’s periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel’s selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.
Highlights
TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake
TRPV6 inhibitors can be used for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including TRPV6-rich tumors[36,37,38,39]
By studying structural bases of TRPV6 inhibition by ruthenium red (RR) and econazole we identified two novel TRPV6 inhibitory binding sites: in the ion channel selectivity filter (Fig. 3) and at the interface between transmembrane helices S4 and S5 (Fig. 4)
Summary
TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. We combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). RR inhibits TRPV6 by binding in the middle of the ion channel’s selectivity filter and plugging its pore like a bottle cork. Structures of TRPV6 identified a 2-APBbinding site at the cytoplasmic end of the S1–S4 transmembrane helical bundle and suggested an allosteric mechanism of channel closure through modulation of protein–lipid interactions[48]. Several structures of TRPV6 in complex with PCHPDs identified their main binding site at the intracellular entrance to the ion channel pore, which overlaps with the binding site of CaM49. PCHPDs act as TRPV6 open-channel blockers that convert the channel into a non-conducting state, mimicking inactivation by CaM14,49
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