Calcium-activated chloride channels in vascular endothelial cells

Abstract Various procedures that increase [Ca 2+ ] i , such as stimulation with vasoactive agonists (acetylcholine, histamine, bradykinin, ATP, UT?), but also ionomycin or loading the cells with Ca 2+ via the patch pipette, activate Ca 2+ -activated Cl − currents ( I CI(Ca) ) in a variety of vascular endothelial cells. This current is strongly outwardly rectifying and has a reversal potential close to the Cl − equilibrium potential. Current kinetics at positive potentials is characterized by a slowly activating component and a rapid deactivation at negative potentials. Activation is faster at more positive membrane potentials and higher intracellular Ca 2+ concentrations ([Ca 2+ ] i ). Deactivation is Ca 2+ independent and faster at more negative potentials. Outward tail currents are slowly decaying, whereas inward tail currents decay much faster. Steady-state currents show strong outward rectification, but the instantaneous current-voltage relationship is nearly ohmic. The halide permeability sequence of the Ca 2+ -activated conductance is Eisenmann I with P I > P Cl > P F > P gluconate . The single channel conductance is approximately 7 pS at 300 m M extracellular Cl − and less than 3 pS at 140 m M Cl − The open probability of the channel is high at positive potentials, but very small at negative potentials. DIDS and niflumic acid inhibit I Cl(Ca) in a voltage-dependent manner, i.e., they exert a more potent block at positive potentials. The block by NPA, NPPB, and tamoxifen is voltage independent. Niflumic acid and tamoxifen are the most potent blockers. The calmodulin antagonists trifluoperazine (TIP) and calmidazolium inhibit I Cl(Ca) . The current is inhibited by intracellularly applied Ins(1,4,5,6)P 4 and Ins(3,4,5,6)P 4 with a concentration for half-maximal inhibition of approximately 10 μ M . Inhibition by tetrakisphosphates occurred without significant changes in kinetic properties. Gating can be described by a two-step binding of Ca 2+ on a high-affinity site inside the channel. [Ca 2+ ] i for half-maximal activation of I Cl(Ca) is voltage dependent, and suggests that the apparent binding constant for Ca 2+ decreases with depolarization. Its value at 0 mV is 430 n M , and the putative binding site is 12% within the electrical field from the cytoplasmic side. The Hill coefficient, n H , of binding is larger than 1 and increases with depolarization. The maximal Cl − conductance at saturating [Ca 2+ ] i does not depend on membrane potential. Ca 2+ -activated Cl − currents coexist in vascular endothelial cells with at least two other Cl − channels, i.e., the volume-regulated anion channels VRAC and CFIR. Their molecular identity as well as their functional role are still uncertain. Likely, coactivation with agonist-induced Ca 2+ release and Ca 2+ entry points to a possible role in regulation of Ca 2+ homeostasis in endothelial cells. In addition, they might be involved in the regulation of more complex cell functions in endothelial cells, such as transcellular transport, exocytosis, and cell proliferation.