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

Abstract

We have studied the kinetic and pharmacological properties of the Ca 2+-activated Cl − current (I 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 Ca 2+-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 Ca 2+ concentrations ([Ca 2+] i). The time constant of deactivation was Ca 2+-independent and decreased at more negative potentials. Steady-state currents showed strong outward rectification, but the instantaneous current-voltage relationship was almost ohmic. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit I Cl,Ca, Half maximal block for TFP occurred at 5.7 ± 2.1 μM ( n = 16). GTPγS did not activate I Cl,Ca, but activated a Cl −- current similar to the volume-activated Cl −- current (I Cl,vol). [Ca 2+] i for half maximal activation of I Cl,Ca was voltage-dependent, and suggests that the apparent binding constant for Ca 2+ 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 [Ca 2+] i did not depend on the membrane potential. RT-PCR experiments did not provide any evidence that the endothelial Ca 2+-activated Cl − channel might be identical with a recently cloned Ca 2+-sensitive Cl − channel (CaCC).