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Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation

Theun de Groot^*, Kyupil Lee^*, Michiel Langeslag, Qi Xi^*, Kees Jalink, René J.M. Bindels^* and Joost G.J. Hoenderop^*

*Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, and
Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands

Correspondence: Dr. Joost Hoenderop, 286 Physiology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, Netherlands. Phone: +31-24-3610580; Fax: +31-24-3616413; E-mail: j.hoenderop{at}ncmls.ru.nl

Received for publication August 20, 2008. Accepted for publication March 9, 2009.

Low extracellular calcium (Ca²⁺) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca²⁺ balance. In the distal part of the nephron, PTH stimulates active Ca²⁺ reabsorption via the adenylyl cyclase–cAMP–protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca²⁺ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca²⁺ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca²⁺ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca²⁺ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca²⁺ reabsorption in the distal nephron.

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Copyright © 2009 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673