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Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL 33101.
1. Isolated native endothelial cells, obtained by treatment of rabbit aortic endothelium with papain and dithiothreitol, were voltage clamped, and single channel (unitary) and spontaneous transient outward currents (STOCs) were recorded from both whole cells and excised membrane patches. 2. In inside-out patches, the reversal potential of unitary currents was dependent on the extracellular K+ concentration and had a single-channel slope conductance of 220 pS in symmetrical 140 mM-K+ solutions. The open-state probability (Po) of the unitary K+ currents was sensitive to the intracellular Ca2+ concentration with half-maximal activation at approximately 1 microM at +20 mV. The ionic selectivity and Ca2+ sensitivity indicate that a large conductance, Ca(2+)-activated K+ channel is present in freshly dissociated rabbit aortic endothelial cells. 3. The frequency and amplitude of whole-cell unitary currents and amplitude of spontaneous transient outward currents were voltage-dependent. Whole-cell outward K+ currents evoked by depolarizing voltage ramps had amplitudes often corresponding to the simultaneous opening of more than five single Ca(2+)-activated K+ channels. Lowering the intracellular EGTA concentration tenfold, and hence the Ca2+ buffering capacity of the cell, increased unitary K+ current activity and shifted the relationship between Po and membrane potential by approximately -20 mV. 4. Bradykinin (1 microM), adenosine 5'-triphosphate (3 microM) and acetylcholine (3 microM) applied extracellularly evoked a biphasic increase in N Po (where N is number of channels activated) of the Ca(2+)-activated K+ channel studied in the whole-cell recording configuration. The development of a biphasic response to agonist stimulation requires a source of extracellular Ca2+. The sustained increase in N Po of the Ca(2+)-activated K+ channel was attenuated upon the removal of external Ca2+ (Mg2+ replacement) or in the presence of the Ca2+ entry blocker, Ni2+, and the potassium channel blockers tetrabutylammonium (TBA) or tetraethylammonium (TEA). 5. Unitary and spontaneous transient outward currents were inhibited by extracellularly applied TEA (0.5 mM), TBA (0.5-5 mM) and charybdotoxin (100 nM). Ca(2+)-activated K+ currents were blocked completely by 5 mM-TEA, whereas 3,4-diaminopyridine (1 mM), Ba2+ (10 mM) and apamin (0.1-1 microM) did not abolish these K+ currents. 6. The K+ channel opener cromakalim (10 microM) evoked a sustained increase in N Po of the Ca(2+)-activated K+ channels which was not potentiated by the addition of bradykinin. Glibenclamide (10 microM) alone increased N Po and partially inhibited the cromakalim-induced increase in N Po with respect to control.(ABSTRACT TRUNCATED AT 400 WORDS)
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