cAMP-dependent phosphorylation modulates voltage gating in an endothelial Cl- channel.
Using the patch clamp technique in the cell-attached, inside-out, and outside-out configurations, we have identified a voltage-gated outwardly rectifying, large conductance (400 pS) Cl- channel in patches from the surface membrane of a cultured monolayer of bovine aortic endothelial cells. The channel is activated in cell-attached patches with 1 microM isoproterenol or 1 mM dibutyryladenosine 3',5'-cyclic monophosphate. In excised inside-out patches the voltage dependence of this channel could be fitted by a Boltzmann distribution with a half-activation voltage (V1/2) at 0 mV. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation induces a shift of -50 mV in V1/2. Alkaline phosphatases restores the voltage dependence of the channel to control values. The channel is reversibly blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The half-inhibitory concentration was approximately 70 microM. Our results suggest that beta-adrenergic stimulation (which increases intracellular cAMP levels in this endothelium) may increase Cl- permeability at the cell resting potential by shifting the voltage dependence of this channel.
Vaca
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Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Kunze
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D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D018118:Chloride Channels; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology*; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015640:Ion Channel Gating; D007545:Isoproterenol / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation
D018118:Chloride Channels; D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000242:Cyclic AMP; D007545:Isoproterenol