Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

doi:10.1074/jbc.274.16.10927

Cyclic GMP-dependent Protein Kinase Activates Cloned BK_Ca Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072*

From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

Abstract

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca²⁺-activated K⁺ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca²⁺-activated K⁺ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca²⁺-activated K⁺ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072.

Footnotes

↵* This work was supported by the Banyu Fellowships in Lipid Metabolism and Atherosclerosis, which are sponsored by Banyu Pharmaceutical Co., Ltd., The Merck Co. foundation (to M. F.), and National Institutes of Health Grants HL40399 (to K. K.) and DK41315 (to B. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, University of Nevada, Reno, NV 89557. Tel.: 775-784-4302; Fax: 775-784-6903; E-mail: kathy{at}physio.unr.edu.
Abbreviations:

BK_Cachannel

large conductance Ca²⁺-activated K⁺channel

PKG

cGMP-dependent protein kinase

K_Vchannel

voltage-gated K⁺ channel

NPo

channel open probability

IBTX

iberiotoxin

SNP

sodium nitroprusside

DETA

diethylenetriamine

PCR

polymerase chain reaction

pS

picosiemens
- Received December 1, 1998.
- Revision received February 2, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.