Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions

Direct inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable 22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati, J. Gen. Physiol. 81:643-666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive 22Na+ uptake could not be blocked by Ca2+ (up to 1 mM). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 microM at pH 7.8 to greater than 10 microM at pH 7.0. Strong pH effects were also demonstrated by measuring the pH dependence of 22Na+ uptake in vesicles that contained 0.5 microM Ca2+. This Ca2+ activity produced a maximal inhibition of 22Na+ uptake at pH greater than or equal to 7.4 but had no effect at pH less than or equal to 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.