pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers

Members of the degenerin/epithelial Na+ channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechan oelectrical transduction. We studied brain Na+ channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca2+. Upon incorporation of vesicles made from membranes of oocytes express ing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) poi nt mutation (G433F), functional channels with different properties were obt ained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans comp artment of the bilayer activated WT BNaC-2 and decreased its permeability f or Na+ over K+. Moreover, these maneuvers made the WT channel more resistan t to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na+ over K+ were unaffec ted by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca2+] to < 10 nm increased WT single channel open probability 10- fold, but not that of GF BNaC-2. Ca2+ blocked both WT and GF BNaC-2 in a do se- and voltage-dependent fashion; single channel conductances were unchang ed. A drop in pH reduced the ability of Ca2+ to inhibit these channels. The se results show that BNaC-2 is an amiloride-sensitive sodium channel and su ggest that pH activation of these channels could be, in part, a consequence of H+ "interference" with channel regulation by Ca2+.