Ca(2+)-induced down-regulation of epithelial amiloride-sensitive channels reconstituted into planar lipid bilayers

Multichannel experiments were carried out to investigate the Ca(2+)-induced down-regulation of epithelial Na+ channels reconstituted into planar lipid bilayer membranes. Reconstitution was achieved by fusion of vesiculated apical membrane fragments to solvent-free planar lipid bilayers. We found that the presence of micromolar concentrations of Ca2+ on the side to which the vesicles were added substantially lowered the channel-mediated current. The inhibition was strongly influenced by pH. At pH 8.0, all the current was blocked by 1 mM calcium, whereas at pH 7.1 the inhibition was about 80%. The blocking kinetics was clearly voltage-dependent. The mechanism of blocking cannot be explained either in terms of interactions with a single site, or by a model in which two blocking sites are assumed.