The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

doi:10.1074/jbc.273.49.32950

The Large Conductance, Voltage-dependent, and Calcium-sensitive K⁺ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation *in Vivo**

From the Departments of ^‡Anesthesiology and ^‡Molecular and Medical Pharmacology, and ^§§Brain Research Institute, UCLA, Los Angeles, CA 90095-1778 and ^**Institut für Pharmakologie und Toxikologie, Technische Universtät München, 80802 München, Germany

Abstract

Native large conductance, voltage-dependent, and Ca²⁺-sensitive K⁺ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming α-subunit of the Ca²⁺-sensitive K⁺ channel, Hslo, and the α-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ∼20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates γ-³³P into the immunopurified Hslo band of ∼125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the α-subunit of large conductance Ca²⁺-sensitive K⁺ channels is substrate for G-Iα kinase in vivo and support direct phosphorylation as a mechanism for PKG-Iα-induced activation of maxi-K channels.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL54970 (to L. T.) and American Heart Association National Center Grant-in-Aid 9750745N (P. M.).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.
↵§ These authors contributed equally to this work.
↵¶ Recipient of an AHA-GLA Research Fellowship 1169-FI1.
↵‖ Present address: Dept. of Pharmacology, Toho University School of Pharmaceutical Sciences, 2-2-1 Miyama, Funabashi-City, Chiba 274-8510, Japan.
↵¶¶ An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Anesthesiology, UCLA School of Medicine, BH-612 CHS, Box 951778, Los Angeles, CA 90095-1778. Tel.: 310-794-7809; Fax: 310-825-5379; E-mail:ltoro{at}ucla.edu.
Abbreviations:

maxi-K

large conductance, voltage-dependent, and Ca²⁺-sensitive K⁺ channel

Hslo

α-subunit of maxi-K channels

HF1

c-Myc-tagged Hslo

MOPS

3-(N-morpholino)propanesulfonic acid

TES

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid

PKG

cGMP-dependent protein kinase

PAGE

polyacrylamide gel electrophoresis

8-Br-cGMP

8-bromo-cyclic GMP

8-pCPT-cGMP

guanosine-3′,5′-cyclic monophosphate, 8-(4-chlorophenylthio)triethylammonium salt.
- Received March 27, 1998.
- Revision received September 21, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.