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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
15:43

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Published on: January 7, 2013

Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5

Seung-Kuy Cha1, Tao Wu, Chou-Long Huang

  • 1Department of Medicine (Division of Nephrology), UT Southwestern Medical Center, Dallas, Texas 75390-8856, USA.

American Journal of Physiology. Renal Physiology
|February 29, 2008
PubMed

Insights

Protein kinase C activation increases cell surface abundance of TRPV5 channels by inhibiting endocytosis, thereby enhancing renal calcium reabsorption. This mechanism is crucial for acute hormonal regulation of calcium homeostasis.

Area of Science:

  • Nephrology
  • Molecular Biology
  • Cell Biology

Background:

  • Transient receptor potential vanilloid 5 (TRPV5) is the primary channel for renal calcium reabsorption.
  • Hormones regulate TRPV5, but the mechanism for phospholipase C-coupled hormones remains unclear.

Purpose of the Study:

  • To elucidate the mechanism by which phospholipase C-coupled hormones acutely regulate TRPV5 channel activity.
  • To investigate the role of protein kinase C (PKC) and caveolin-1 in TRPV5 regulation.

Main Methods:

  • Cell culture studies using HEK293 cells expressing TRPV5.
  • Pharmacological activation of PKC with OAG and inhibition with specific PKC inhibitors.
  • Gene silencing using small interfering RNA (siRNA) for caveolin-1 and clathrin heavy chain.
  • Analysis of TRPV5 surface abundance and current density.
  • Studies in caveolin-1 knockout cells and rescue experiments.
  • Site-directed mutagenesis of putative PKC phosphorylation sites on TRPV5.
  • Experiments involving parathyroid hormone (PTH) stimulation.

Main Results:

  • PKC activation by OAG increased TRPV5 current density and surface expression.
  • This effect was blocked by PKC inhibitors, dominant-negative dynamin, and caveolin-1 knockdown.
  • TRPV5 regulation by OAG was absent in caveolin-1 null cells but restored by caveolin-1 re-expression.
  • Mutations at serine-299 and/or serine-654 abolished OAG-mediated TRPV5 regulation.
  • PTH stimulation of TRPV5 was similarly dependent on PKC, caveolin-1, and TRPV5 phosphorylation sites.

Conclusions:

  • TRPV5 undergoes continuous caveolae-mediated endocytosis.
  • PKC activation inhibits TRPV5 endocytosis, increasing its surface abundance.
  • This PKC-dependent mechanism contributes to the acute stimulation of TRPV5 and renal calcium reabsorption by hormones like PTH.

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