Figure 4.

pH_o dependence of I_H without Na⁺_o and K⁺_o in Xenopus oocytes. (A) Continuous recording from an oocyte expressing RD-α1β3 held at −50 mV. Maximum I_P was measured at 3 mM K⁺_o without Na⁺_o at pH_o 7.6. pH_o was then lowered, inducing an inward holding current that was inhibited by subsequent application of ouabain. After an 8-min ouabain washout with 120 mM Na⁺ (to maintain internal Na loading), the maneuver was repeated again, first applying K⁺, then lowering pH_o, then adding ouabain. Note that I_P is the same in each K⁺ application, indicating that all Na/K pumps recovered from inhibition before testing a different pH_o. Ramp-shaped vertical deflections are 100-ms-long pulses to different voltages. The current at the end of those pulses was used to obtain I/V curves. (B) Ouabain-sensitive current (normalized to I_H at −160 mV, pH 7.6, before lowering pH_o) as a function of voltage. The data points are an average of eight oocytes where at least four different pH_o values were tested. The currents at pH 5.6 and 5.0 were measured at the peak of the activation response, before significant inhibition had occurred. (C) pH dependence from the curves in B at the indicated voltages. The solid lines are global fits of Michaelis-Menten equations to the data from individual cells with K_0.5 (in μM) ± fitting errors of 0.20 ± 0.04 (−180 mV, black), 0.34 ± 0.10 (−140 mV, red), 0.79 ± 0.23 (−100 mV, blue), and 1.36 ± 0.39 (−60 mV, green). The K_0.5-versus-voltage curve is plotted in the inset; the red line is a fit of the equation K_0.5= K⁰_0.5exp (λF/RT) + C to the data, with parameters K⁰_0.5 = 4.4 μM, λ = 0.51, and C = 0.07. R is the gas constant, F the Faraday constant, and T the temperature.