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Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G

Hiu-Yee Kwan1, Yu Huang, Xiaoqiang Yao

  • 1Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China.

Proceedings of the National Academy of Sciences of the United States of America
|February 26, 2004
PubMed

Insights

Canonical transient receptor potential (TRPC) channels, specifically TRPC3, are directly phosphorylated by protein kinase G (PKG). This phosphorylation by PKG inhibits store-operated calcium influx in HEK293 cells.

Area of Science:

  • Cellular physiology
  • Molecular biology
  • Ion channel function

Background:

  • Canonical transient receptor potential (TRPC) channels are crucial Ca2+-permeable cation channels involved in various cellular processes.
  • TRPC channel activity is known to be modulated by intracellular Ca2+ stores, diacylglycerol, and Ca2+/calmodulin.
  • The direct regulation of TRPC channels by phosphorylation remains largely uninvestigated.

Purpose of the Study:

  • To investigate whether TRPC channels are directly regulated by phosphorylation.
  • To determine the role of protein kinase G (PKG) in the phosphorylation and regulation of TRPC isoform 3 (TRPC3) channels.

Main Methods:

  • Overexpression of TRPC3 in HEK293 cells stably expressing PKG.
  • Assessment of store-operated Ca2+ influx and its modulation by cGMP.
  • Site-directed mutagenesis of potential PKG phosphorylation sites (T11 and S263) on TRPC3.
  • In vitro PKG phosphorylation assays using purified wild-type and mutant TRPC3 proteins.

Main Results:

  • cGMP inhibited TRPC3-mediated store-operated Ca2+ influx, an effect abolished by PKG inhibitors KT5823 or H8.
  • Mutations at TRPC3 phosphorylation sites T11A and S263Q significantly reduced the inhibitory effect of cGMP.
  • Wild-type TRPC3 underwent direct phosphorylation by PKG in vitro, which was abolished by KT5823 and significantly reduced in T11A and S263Q mutants.

Conclusions:

  • TRPC3 channels are directly phosphorylated by PKG at specific sites (T11 and S263).
  • PKG-mediated phosphorylation of TRPC3 channels inhibits their activity, specifically abolishing store-operated Ca2+ influx.
  • This study reveals a novel mechanism of TRPC channel regulation through direct phosphorylation by PKG.

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