Pkg i[alpha] phosphorylates the [alpha] subunit and up regulates reconstituted gk ca channels from tracheal smooth muscle. Alioua, Abderrahmane, John P. Huggins, and Eric Rousseau. Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke QC, CANADA J1H 5N4, Marion Merrell Dow Research Institute, 16, rue d'Ankara, 67080 Strasbourg cedex, France.
APStracts 2:0053L, 1995.
Modulation of Ca2+-dependent K+ channels (GKCa) activities in airway smooth muscles (ASM) by cGMP-dependent protein kinase (PKG) is thought to play a central role in mediating the effect of some branchodilator agents that elevate cytoplasmic basal cGMP concentrations. However, no direct evidence supports this hypothesis in ASM. In the present work, we provide evidence that cGMP-dependent protein kinase I[alpha] up regulates GKCa channels, derived from bovine tracheal smooth muscle cells and reconstituted into planar lipid bilayers. In two different experimental approaches, PKG increased the open probability as well as the mean open time of GKCa channels, without any effect on unitary current amplitudes and unit conductance. Our results indicate that the kinetics of GKCa channels are controlled by a phosphorylation step mediated by PKG, and thus might be modulated by intracellular cGMP. Biochemical assays demonstrated that PKG phosphorylates several protein bands in the membrane fraction. Two of those proteins co-migrate with the same relative molecular mass as the 62kDa and 30kDa components of the purified channel complex identified as [alpha] and [beta] GKCa subunits, respectively. Our results also indicate that PKG phosphorylates the GKCa [alpha]-subunit with an apparent stroichiometry of 0.89, which would be consistent with the presence of a single-PKG sensitive phosphorylating site within its amino acid sequence. Furthermore, these results demonstrate for the first time that PKG directly phosphorylates GKCa from airway smooth muscle cells and thereby activates the channels at negative voltage or at low free Ca2+ concentrations. 

Received 4 November 1994; accepted in final form 6 April 1995.
APS Manuscript Number L318-4.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 19 April 1995.