Amiloride-sensitive epithelial Na+ channels reconstituted into planar lipid bilayer membranes

Abstract

High resistance epithelia actively transport sodium from the luminal side to the blood. Aldosterone and vasopressin stimulate this sodium transport system; the diuretic drug amiloride inhibits it in a reversible fashion. The first step in the transepithelial transport of Na+ is the facilitated diffusion of Na+ across the apical membrane via Na+-specific, amiloride-sensitive channels. We report here the first direct measurements of single, amiloride-sensitive Na+ channel activity. The channel was isolated after incorporation of purified apical membrane vesicles from A6 cells into planar lipid bilayers. The channel had the following characteristics: single-channel conductance ranged from 4 to 80 pS at 200 mM NaCl; it was perfectly cation-selective; amiloride reduced the open-state conductance in a dose-dependent fashion when present in the cis compartment, and induced flickering when present in the trans chamber; channel conductance and gating were voltage-independent; and the Na+/K+ selectivity ratio of the channel was 2:1.