Parathyroid hormone activates TRPV5 via PKA-dependent phosphorylation.
Low extracellular calcium (Ca(2+)) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca(2+) balance. In the distal part of the nephron, PTH stimulates active Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) reabsorption in the distal nephron.
Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands.
Theun
T
de Groot
Kyupil
K
Lee
Michiel
M
Langeslag
Qi
Q
Xi
Kees
K
Jalink
René J M
RJ
Bindels
Joost G J
JG
Hoenderop
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D005576:Colforsin; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D010281:Parathyroid Hormone / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism; D050916:TRPV Cation Channels / Q000378:metabolism*; D013912:Threonine
C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D019269:Phosphatidylinositol 4,5-Diphosphate; D044168:Receptor, Parathyroid Hormone, Type 1; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D005576:Colforsin; D013912:Threonine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium