Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells

Abstract

Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphothericin Bperforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of −20 mV to +20 mV. Addition of 1 mM Zn²⁺ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 μmol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 μmol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 μmol/l). Both agents increase the cytoplasmic Ca²⁺ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 μmol/l), which spontaneously releases NO, did not increase Cl⁻ channel activity in intact cells. Polymyxin B (100 μmol/l), an inhibitor of protein kinase C, clearly enhanced Cl⁻ channel activity in intact cells, resulting in the observation of Cl⁻ channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl⁻ channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca²⁺-dependent process which is different from the NO/cGMP pathway.