Kinetic and pharmacological properties of the calcium-activated chloride-current in macrovascular endothelial cells

We have studied the kinetic and pharmacological properties of the Ca2+-activated Cl- current (l(Cl,Ca)) in cultured calf pulmonary artery endothelial (CPAE) cells by means of combined patch clamp and Fura-2 micro fluorescence measurements. The current was activated by loading the cells via the patch pipettes with Ca2+-buffered solutions. Currents activated slowly at positive potentials, and decayed rapidly at negative potentials. The time constant of activation decreased at more positive membrane potentials and more elevated intracellular Ca2+ concentrations ([Ca2+](i)). The time constant of deactivation was Ca2+-independent and decreased at more negative potentials. Steadystate currents showed strong outward rectification, but the instantaneous current-voltage relationship was almost ohmic. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit l(Cl,Ca). Half maximal block for TFP occurred at 5.7 +/- 2.1 mu M (n = 16). GTP gamma S did not activate l(Cl,Ca), but activated a Cl- current similar to the volume-activated Cl- current (l(Cl),(vol)). [Ca2+](i) for half maximal activation of l(Cl,Ca) was voltage-dependent, and suggests that the apparent binding constant for Ca2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the binding site is 12% within the electrical field from the cytoplasmic side. The Hill-coefficient, n(H), of the binding was larger than 1 and increased with depolarization. The maximal Cl- conductance at saturating [Ca2+](i) did not depend on the membrane potential. RT-PCR experiments did not provide any evidence that the endothelial Ca2+-activated Cl- channel might be identical with a recently cloned Ca2+-sensitive Cl- channel (CaCC).