Kinetic and pharmacological properties of the calcium-activated chloride-current in macrovascular endothelial cells.
We have studied the kinetic and pharmacological properties of the Ca(2+)-activated Cl- current (ICl,Ca) in cultured cell 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 Ca2+ concentrations ([Ca2+]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 ICl,Ca. Half maximal block for TFP occurred at 5.7 +/- 2.1 microM (n = 16). GTP gamma S did not activate ICl,Ca, but activated a Cl- current similar to the volume-activated Cl- current (ICl,vol). [Ca2+]i for half maximal activation of ICl,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, nH, 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 Ca(2+)-activated Cl- channel might be identical with a recently cloned Ca(2+)-sensitive Cl- channel (CaCC).
Nilius
B
B
Laboratorium voor Fysiologie, KU Leuven, Belgium. bernd.nilius@med.kuleuven.ac.be
Prenen
J
J
Voets
T
T
Van den Bremt
K
K
Eggermont
J
J
Droogmans
G
G
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002417:Cattle; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology / Q000379:methods; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D016133:Polymerase Chain Reaction; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D018118:Chloride Channels; D007093:Imidazoles; D014268:Trifluoperazine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C031938:calmidazolium; D019204:GTP-Binding Proteins; D002118:Calcium