Effects of extracellular pH on the E₁P/E₂P distribution of gastric H,K-ATPase.

<p>(A,B) Fluorescence responses of site-specifically labeled gastric H,K-ATPase under K⁺-free conditions (90 mM Na⁺ in the extracellular solution) upon voltage jumps from −40 mV to voltages between −180 mV and +60 mV in (−20 mV steps, see inset in A) at an extracellular pH of 7.4 (A) or 5.5 (B). (C) Voltage dependence of normalized fluorescence amplitudes <i>(1-ΔF/F)</i> from experiments as in (A,B) for pH_ex 5.5 (○), and for pH_ex 7.4 (•). Data are means±S.E. of 11–14 oocytes. Superimposed as dashed lines are curves resulting from fits of a Boltzmann-type function to the data sets (pH_ex 7.4: <i>V_0.5</i> = −19.7±5.4 mV, <i>z_q</i> = 0.26±0.02; pH_ex 5.5: <i>V_0.5</i> = −126.4±16.6 mV, <i>z_q</i> = 0.27±0.04). The fluorescence amplitudes <i>1-ΔF/F</i> were normalized to the difference between the saturation values at positive or negative potentials, respectively, as obtained from the fits. (D) Reciprocal time constants (<i>τ⁻¹</i>) from fits of a single exponential function to voltage jump-induced fluorescence changes under K⁺-free conditions at pH_ex 5.5 (□) and pH_ex 7.4 (▪). Data are means ± S.E. from 15–17 oocytes. (E,F) Graphs showing the forward (<i>k_f</i>) and reverse (<i>k_b</i>) rate constants of the E₁P↔E₂P transition at pH_ex 7.4 (E) and pH_ex 5.5 (F), as calculated from the observed <i>k_tot = τ⁻¹</i> in (D) and the voltage-dependent fluorescence amplitudes in (C) according to Supporting Information (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone.0033645.s003" target="_blank">Appendix S1</a></b>). Superimposed in (E,F) are fits of a single exponential function to the calculated <i>k_f</i> and <i>k_b</i> values. The resulting fit parameters (rate constants at 0 mV: <i>k_b</i>(0), <i>k_f</i>(0), and <i>z_q</i> values), as summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone-0033645-t001" target="_blank">Table 1</a>, were: pH_ex 7.4 (E): <i>k_f(0)</i> = 2.61±0.05 s⁻¹, <i>z_q,f</i> = −0.06±0.01; <i>k_b(0)</i> = 2.31±0.10 s⁻¹, <i>z_q,b</i> = 0.21±0.01; pH_ex 5.5 (F): <i>k_f(0)</i> = 4.56±0.05 s⁻¹, <i>z_q,f</i> = −0.056±0.003; <i>k_b(0)</i> = 1.22±0.10 s⁻¹, <i>z_q,b</i> = 0.230±0.004.