Mechanism of activation by cyclic gmp-dependent protein kinase (PKG) of large ca2+-acttvated k+ channels BK(Ca)J in human mesangial cells (MC)

James D. Stockand, Steven C. Sansnm

Research output: Contribution to journalArticle

Abstract

We previously showed that cGMP-elevating vasodilators modulate MC tone by regulating the feedback, hyperpolarizing signal carried by BK(Ca). Patch clamp and molecular methods were used to elucidate the cGMP-dependent mechanism for activating BK(Ca). In cell attached patches, activation by cGMP of BK(Ca) was prevented by staurosporine, implicating a protein kinase in the signal transduction pathway. In inside-out patches, BK(Ca) was activated by Mg-ATP plus cGMP but was not affected by the separate addition of either agent Cyclic AMP plus Mg-ATP failed to activate BK(Ca). Activation by cGMP plus Mg-ATP of BK(Ca) was blocked by KT5823, a specific inhibitor of PKG but not by KT5720, a specific inhibitor of PKA. Exogenous PKG but not PKA activated BK(Ca). PKG activated BK(Ca) by decreasing the long closed time constant from 192 to 22 ms, resulting in a decrease in the half activation potential, a measure of voltage threshold, by 60 mV. The gating charge and Hill coefficient, measurements of voltage and calcium sensitivity, respectively, were not affected. Using RT-PCR to amplify cDNA products of hsia, a consensus PKG phosphorylation site was established on BK(Ca). In conclusion, cGMP-dependent protein kinase phosphorylates directly and activates mesangial BK(Ca) by decreasing the activation threshold through a reduction of the duration of the long closed state.

Original languageEnglish (US)
Pages (from-to)A545
JournalFASEB Journal
Volume10
Issue number3
StatePublished - Dec 1 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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