title: Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone.
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.
authors.affiliation: Nijmegen Center for Molecular Life Sciences, 286 Physiology, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, Netherlands.
authors.forename: Theun
authors.initials: T
authors.lastname: de Groot
authors.forename: Nadezda V
authors.initials: NV
authors.lastname: Kovalevskaya
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Verkaart
authors.forename: Nathalie
authors.initials: N
authors.lastname: Schilderink
authors.forename: Marco
authors.initials: M
authors.lastname: Felici
authors.forename: Eline A E
authors.initials: EA
authors.lastname: van der Hagen
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
authors.forename: Geerten W
authors.initials: GW
authors.lastname: Vuister
authors.forename: Joost G
authors.initials: JG
authors.lastname: Hoenderop
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010281:Parathyroid Hormone / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D005456:Fluorescent Dyes; D010281:Parathyroid Hormone; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2