Abstract
The epithelial Ca(2+) channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca(2+)-induced inactivation that protects the cell from toxic Ca(2+) overload and may also limit intestinal Ca(2+) transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca(2+), calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P(2), and Ca(2+)-CaM inhibited the channel at physiologically relevant concentrations. Ca(2+) alone also inhibited TRPV6 at high concentrations (IC(50) = ∼20 μM). A double mutation in the distal C-terminal 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 Ca(2+)-induced inactivation. Providing excess PI(4,5)P(2) reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P(2) did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P(2) and show that Ca(2+), CaM, and the depletion of PI(4,5)P(2) all contribute to inactivation of TRPV6.
Highlights
A large number of calmodulin-binding sites have been proposed in transient receptor potential vanilloid 6 (TRPV6)
We have identified the site that is responsible for inhibition of TRPV6 by calmodulin in excised inside-out patch clamp experiments
We have studied the effects of Ca2ϩ and CaM on the activity of the human TRPV6 in large excised inside-out patches from Xenopus oocytes expressing this channel (Fig. 1)
Summary
A large number of calmodulin-binding sites have been proposed in TRPV6. Results: We have identified the site that is responsible for inhibition of TRPV6 by calmodulin in excised inside-out patch clamp experiments. TRPV5, a close homologue of TRPV6, is expressed in the kidney, where it plays an important role in Ca2ϩ reabsorption in the distal convoluted tubule [1] Both these channels are constitutively active and undergo Ca2ϩ-induced inactivation [3], which is thought to protect cells from toxic Ca2ϩ levels, and it was proposed to limit intestinal Ca2ϩ transport [4]. CaM binding to the full-length protein was eliminated by a combined mutation of Trp-695 and Arg-699 residues in the distal C-terminal CaM-binding site of TRPV6 In excised patches, this mutant was essentially not inhibited by CaM; in whole cell patch clamp experiments, Ca2ϩ-induced inactivation was reduced but not eliminated. Our data show complex regulation of TRPV6 activity by the interplay between PI[4,5]P2, Ca2ϩ, and CaM
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
