Molecular Mechanisms of Calmodulin Action on TRPV5 and Modulation by Parathyroid Hormone

Abstract

ABSTRACT The epithelial Ca 2+ channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca 2+ reabsorption in the kidney. Ca 2+ influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca 2+ influx. This inactivation is mediated by the last ∼30 residues of the carboxy (C) terminus of the channel. Since the Ca 2+ -sensing protein calmodulin has been implicated in Ca 2+ -dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca 2+ -dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca 2+ -dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.