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

Abstract

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+.

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

“…This conclusion is supported by previous data indicating that the aldosteroneinduced channels are accessible to impermeable reagents interacting with the apical surface prior to the application of the hormone (9)(10)(11). A possible mechanism for such a labile activation of transport could be release of channels from direct inhibition by cell Ca2" (22). Incubating the tissue with aldosterone for longer periods (e.g., 6-16 hr) produced an additional increase in ISC that was sustained by vesicles.…”

Effects of [Ca2+]i and pH on epithelial Na+ channel activity of cultured mouse cortical collecting ducts

“…The possible explanations are focused upon the elevation in [Ca 2+ ] i in the medium or alternation in interaction of Ca 2+ and ENaCs due to changes in pH o . The ability of Ca 2+ to inhibit Na + uptake in toad bladder was greatly reduced by decreasing pH i from 7.4 to 7.0 (Garty et al, 1987). In this case, it would be expected that a decrease of pH i may relieve the inhibition of the channel by Ca 2+ .…”

“…Trans epithelial Na + transport rate is increased by intracellular alkalization but decreased by intracellular acidosis (Blokkebak-Poulsen et al, 1991;Harvey et al, 1988;. Controversially, in toad bladder, acidification of the cytoplasm can stimulate Na + transport (Garty et al, 1987;Leaf et al, 1964). Electrophysiology experiments based on transfected oocytes and A6 cells demonstrated that the α subunit of ENaC is directly regulated by pH i (Chalfant et al, 1999) and a reduction of pH i decreased ENaC activity.…”

Effects of prostaglandin E2 on membrane voltage of the connecting tubule and cortical collecting duct from rabbits.

“…Taylor & Windhager (1979) suggested that luminal Na+ conductance can be regulated by an elevated cell Ca2+ concentration that might be involved in a feedback mechanism. Although the cellular mechanism whereby intracellular Ca2+ inhibits the conductance remains unclear, Ca2+ might directly block the Na+ channels by binding to a site on the cytoplasmic face of the channel protein (Chase & Al-Awquati, 1983;Garty, Asher & Yeger, 1987). There is also evidence that Ca2+ inhibits ap>al Na+ permeability indirectly by activating protein kinase C (Ling & Eaton,1°I t has been reported that in rabbit CCD, PGE2 increases celiJar Ca2+ by releasing Ca2+ from intracellular stores (Breyer, Jacobson & Hebert, 1990;Hebert et al, 1991).…”