The Roles Calmodulin and PI(4,5)P2 in Ca2+-Induced Inactivation of TRPV6 Channels

Abstract

The epithelial Ca2+ channel Transient Receptor Potential Vanilloid 6 (TRPV6) undergoes Ca2+-induced inactivation. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca2+, CaM and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in excised patches. The activity of TRPV6 decreased after excision, (run down) and channels could be re-activated with either PI(4,5)P2 or MgATP, which supplies substrate for endogenous lipid kinases to synthesize PI(4,5)P2. Ca2+-CaM inhibited the channel at physiologically relevant concentrations both when endogenous PI(4,5)P2 was used to maintain channel activity, and when exogenous PI(4,5)P2 was applied. Ca2+ alone also inhibited TRPV6 at high concentrations (IC50 ∼20 μM). Biochemical binding experiments showed that CaM binds to TRPV6 via a distal C-terminal binding site. A double mutation in this CaM binding site of TRPV6 (W695A-R699E) essentially eliminated inhibition by CaM in excised patches. In whole-cell patch clamp experiments, this mutation reduced, but did not eliminate Ca2+-induced inactivation. Providing excess PI(4,5)P2 reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P2 did not inhibit binding of the C-terminus of the channel to CaM. Overall, our data show a complex interplay between CaM and PI(4,5)P2, and that Ca2+, CaM and the depletion of PI(4,5)P2 all contribute to inactivation of TRPV6.