G-protein-mediated regulation of a Ca(2+)-dependent K+ channel in cultured vascular endothelial cells.
The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca(2+)-activated K+ channel (KCa) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10 +/- 2 pS and at potentials negative to 0 mV 30 +/- 3 pS (n = 7) when examined in symmetrical K+ (150 mmol/l) solutions. The channel open probability (P(o)) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca2+ concentration from 100 nmol/l to 10 mumol/l at -60 mV produced a graded increase in channel P(o) from 0.15 to 0.96; the concentration required for half-maximum response (apparent K0.5) was 719 nmol/l. At a constant Ca2+ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P(o). This effect was dependent upon the simultaneous presence of both GTP and Mg2+, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The hydrolysis-resistant GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), induced a long-lasting increase in channel P(o). In the presence of Mg(2+)-GTP, the apparent K0.5 for Ca2+ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg(2+)-GTP further enhanced KCa activity, shifting the apparent K0.5 for Ca2+ from 228 nmol/l to 107 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Vaca
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Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030.
Schilling
W P
WP
Kunze
D L
DL
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods; D019204:GTP-Binding Proteins / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*
D015221:Potassium Channels; D019204:GTP-Binding Proteins; D001920:Bradykinin; D002118:Calcium