Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells

Abstract

The apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique. We defined the apical membrane as that membrane in contact with the growth medium. In about 50% of the patches, channels with single-unit conductances of 360 +/- 45 pS in symmetrical 105 mM NaCl solutions, and characteristic voltage-dependent inactivation were observed. Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl- over Na+ of about 9:1, calculated from the reversal potential using the constant-field equation. The channel was most active at membrane potentials between +/- 20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity. Reactivation from this inactivation was slow, sometimes requiring minutes. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels. The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers.