Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone
Theun de Groot1, Nadezda V Kovalevskaya, Sjoerd Verkaart
1Nijmegen Center for Molecular Life Sciences, 286 Physiology, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, Netherlands.
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.
Insights
Calmodulin binding to the TRPV5 channel
Area of Science:
- Nephrology
- Molecular Biology
- Ion Channel Physiology
Background:
- The epithelial calcium channel TRPV5 is crucial for active calcium reabsorption in the kidney.
- TRPV5 undergoes rapid, calcium-dependent inactivation mediated by its C-terminus to prevent excessive calcium influx.
- Calmodulin, a calcium-sensing protein, is known to regulate other TRP channels.
Purpose of the Study:
- To investigate the role of calmodulin in the function and regulation of the TRPV5 channel.
- To elucidate the interaction between calmodulin and the C-terminus of TRPV5.
- To understand how parathyroid hormone (PTH) affects TRPV5 activity via calmodulin.
Main Methods:
- High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy to study calmodulin-TRPV5 C-terminal fragment interactions.
- Site-directed mutagenesis of TRPV5 C-terminal residues to disrupt calmodulin binding.
- Patch clamp analysis to assess channel inactivation and open probability in response to mutations and hormonal stimulation.
Main Results:
- NMR revealed a Ca(2+)-dependent interaction where one calmodulin binds two TRPV5 C-termini (residues 696-729).
- TRPV5 mutants (W702A, R706E) lacking calmodulin binding showed significantly reduced Ca(2+)-dependent inactivation.
- Parathyroid hormone (PTH) stimulation led to T709 phosphorylation, reducing calmodulin binding and increasing TRPV5 open probability.
Conclusions:
- Calmodulin negatively modulates TRPV5 channel activity by binding to its C-terminus.
- PTH-induced phosphorylation of T709 reverses this inhibition, enhancing channel activity.
- Understanding this regulatory mechanism is key to comprehending renal calcium handling.
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