Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Abstract

Chloride ions (Cl-) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl- channels. We have studied Cl- currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2-6 days old were characterized by an input resistance of 3.4 +/- 0.8 G omega (n = 11) and a capacitance of 63.8 +/- 10.8 pF (n = 26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3-4 days old held at -40 mV produced currents of 196 +/- 22 pA at 50 mV and -246 +/- 27 pA at -90 mV (n = 212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 microM) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1 mM) and DNDS (1-30 microM). Replacement of K+ with Cs+ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K+ dependent when the intrapipette solution was buffered to a Ca2+ concentration of 20 nM. The Cl-/Na+ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 1:2. Current equilibrium permeabilities, relative to Cl- were: I- (2.9) much greater than NO3- (1.1) greater than or equal to Br- (1.1) greater than or equal to Cl- (1.0) greater than or equal to F- (0.93) much greater than MeSO4- (0.19) greater than or equal to gluconate (0.18) greater than or equal to aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course.(ABSTRACT TRUNCATED AT 250 WORDS)