PKG-I alpha phosphorylates the alpha-subunit and upregulates reconstituted GKCa channels from tracheal smooth muscle.
Modulation of Ca(2+)-dependent K+ channel (GKCa) activities in airway smooth muscles (ASM) by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) is thought to play a central role in mediating the effect of some bronchodilator agents that elevate cytoplasmic basal cGMP concentrations. However, no direct evidence supports this hypothesis in ASM. In the present work, we provide evidence that PKG-I alpha upregulates 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 62- and 30-kDa components of the purified channel complex, identified as GKCa-alpha and -beta subunits, respectively. Our results also indicate that PKG phosphorylates the GKCa-alpha subunit with an apparent stoichiometry 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.
Alioua
A
A
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Huggins
J P
JP
Rousseau
E
E
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015640:Ion Channel Gating; D007527:Isoenzymes / Q000378:metabolism; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008564:Membrane Potentials; D009130:Muscle, Smooth / Q000502:physiology*; D010766:Phosphorylation; D015221:Potassium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D014132:Trachea / Q000502:physiology*
D007527:Isoenzymes; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D015221:Potassium Channels; D017869:Cyclic GMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D006152:Cyclic GMP; D002118:Calcium