UNC-8(G387E) currents are larger in divalent cation–free solution. (A) Example of currents recorded in an oocyte injected with UNC-8(G387E) and perfused with NaCl physiological solution, which contains 1 mM CaCl2 and 2 mM MgCl2. Currents were stimulated by voltage steps from −160 to +100 mV in 20-mV increments. The holding potential was −30 mV. (B) The same oocyte shown in A was perfused with a divalent cation–free NaCl solution containing 1 mM EGTA. Currents were recorded with the same voltage protocol used in A. (C) Currents were recorded from the same oocyte in divalent cation–free EGTA solution plus 1 mM amiloride. Dashed lines represent the zero current level. (D) Current–voltage relationships from noninjected oocytes and oocytes injected with UNC-8 or UNC-8(G387E), and perfused with either the NaCl physiological solution or the divalent cation–free or divalent cation–free plus EGTA NaCl solution, as indicated. The number of oocytes tested is shown in parentheses. (E) Shift in the oocyte resting potential upon switching from NaCl physiological solution (1 mM CaCl2 and 2 mM MgCl2) to divalent cation–free plus EGTA NaCl solution for noninjected oocytes (open squares; n = 9) and oocytes injected with UNC-8(G387E) (closed circles; n = 32). Small symbols and lines correspond to measurement from individual oocytes, and large symbols are means ± SE. (F) Amiloride dose–response curve obtained from oocytes injected with UNC-8(G387E) and perfused with divalent cation–free plus EGTA NaCl solution. Data points were fitted by Hill equation, which gave a Ki of 106 µM and an n value of 1. The inset shows the voltage dependence of amiloride blockade. Data points were fitted using the Woodhull model, which gave a δ of 0.08 (Woodhull, 1973). Data are expressed as mean ± SE (n = 8).
