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Figure 1. Reaction cycle of gastric H,K-ATPase.(A) Reaction mechanism of gastric H,K-ATPase adapted from the Post-Albers scheme [1], [2], which had originally been postulated for the related Na,K-ATPase. Upon intracellular binding of protons to the E1 conformation (step 1), a phosphointermediate with occluded H+ ions (E1P(H+)) is formed (step 2), and after a conformational change to E2P (step 3), protons dissociate to the extracellular space (step 4). Subsequently, K+ ions bind from the extracellular side (step 5) and become occluded, a process which stimulates dephosphorylation (step 6), and after a conformational change from E2 to E1 (step 7) the K+ ions are intracellularly released (step 8). The gray box indicates the reaction sequence which can be studied by voltage pulses at [K+]ext = 0 in VCF experiments. (B) Pseudo three-state model for the reaction sequence including steps 1 to 4 in (A). A detailed description and analysis of this kinetic scheme is provided in Supporting Information (Appendix S2).
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Figure 2. Effects of extracellular pH on the E1P/E2P distribution of gastric H,K-ATPase.(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 (1-ΔF/F) from experiments as in (A,B) for pHex 5.5 (○), and for pHex 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 (pHex 7.4: V0.5 = −19.7±5.4 mV, zq = 0.26±0.02; pHex 5.5: V0.5 = −126.4±16.6 mV, zq = 0.27±0.04). The fluorescence amplitudes 1-ΔF/F were normalized to the difference between the saturation values at positive or negative potentials, respectively, as obtained from the fits. (D) Reciprocal time constants (τ−1) from fits of a single exponential function to voltage jump-induced fluorescence changes under K+-free conditions at pHex 5.5 (□) and pHex 7.4 (▪). Data are means ± S.E. from 15–17 oocytes. (E,F) Graphs showing the forward (kf) and reverse (kb) rate constants of the E1P↔E2P transition at pHex 7.4 (E) and pHex 5.5 (F), as calculated from the observed ktot = τ−1 in (D) and the voltage-dependent fluorescence amplitudes in (C) according to Supporting Information (Appendix S1). Superimposed in (E,F) are fits of a single exponential function to the calculated kf and kb values. The resulting fit parameters (rate constants at 0 mV: kb(0), kf(0), and zq values), as summarized in Table 1, were: pHex 7.4 (E): kf(0) = 2.61±0.05 s−1, zq,f = −0.06±0.01; kb(0) = 2.31±0.10 s−1, zq,b = 0.21±0.01; pHex 5.5 (F): kf(0) = 4.56±0.05 s−1, zq,f = −0.056±0.003; kb(0) = 1.22±0.10 s−1, zq,b = 0.230±0.004.
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Figure 3. Effects of intracellular acidification on the E1P/E2P distribution and Rb+ transport.(A–E) Fluorescence responses of TMRM-labeled HKαS806C/βWT under extracellular K+-free conditions (90 mM extracellular Na+) upon voltage jumps from −40 mV to potentials between −180 mV and +60 mV in −20 mV steps. Recordings in (A–D) originated from a single oocyte (A) at pHex 7.4, (B) after 1 min in presence of 40 mM Na-butyrate (pHex 7.4), and after 1 min (C) and 2 min (D) washout of butyrate (pHex 7.4 buffer). (E) Fluorescence responses from a different cell in pHex 5.5 buffer. (F) Voltage dependence of stationary fluorescence amplitudes 1-ΔF/F from the recordings in (A–E) at pHex 7.4 (▪), pHex 7.4+40 mM butyrate (□), and after 1 min (⋄) or 2 min (▵) washout of butyrate. Data at pHex 5.5 (•) are also shown. Fits of a Boltzmann-type function are superimposed to each data set, and the fluorescence amplitudes were normalized to saturation values from the fits. (G) Reciprocal time constants (τ−1) from fits of a single exponential function to fluorescence changes under K+-free conditions. Data obtained at pHex 7.4 in the presence of 40 mM butyrate (♦) are compared to those in butyrate-free solutions at pHex 5.5 (□) and at pHex 7.4 (▪). Data are means±S.E. from 12–17 oocytes. (H) H,K-ATPase-mediated Rb+ uptake (at 5 mM Rb+) measured on individual cells by atomic absorption spectroscopy in the absence (gray) or presence (black) of 10 µM SCH28080 at different pHex and ionic conditions. Results from non-injected and HKαS806C/βWT-expressing oocytes at pHex 5.5, pHex 7.4, and pHex 7.4+40 mM butyrate are shown. Data are means±S.E. from three experiments on different cell batches with 15–20 oocytes per condition, and normalized to the Rb+ uptake of HKαS806C/βWT at pHex 5.5 (mean specific activities of 15.4, 23.1 and 39.0 pmol/oocyte/min).
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Figure 4. Effects of extracellular K+ on voltage-dependent fluorescence changes.(A,B) Voltage step-induced fluorescence responses of TMRM-labeled oocytes expressing HKαS806C/βWT in K+-free (upper traces) or 5 mM K+-containing extracellular solution (lower traces) at pHex 5.5 (A), and pHex 7.4 (B), according to a voltage protocol as in Fig. 2A (inset). (C) Comparison of the time course of the fluorescence signals from panel (A) in response to voltage pulses to −180 mV and +60 mV in the absence of K+ and in the presence of 5 mM K+. Signals were normalized to the fluorescence amplitude reached at the end of the voltage pulse to −180 mV or +60 mV, respectively. (D) Comparison of normalized ΔF/F values (change in stationary fluorescence between −180 mV and +60 mV, divided by fluorescence at −40 mV) in the absence (gray bars) or presence (black bars) of 5 mM extracellular K+, both at pHex 5.5 and pHex 7.4. Data are means±S.D. from 3–5 oocytes, normalized to the mean ΔF/F at pHex 5.5 in K+-free solution.
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Figure 5. Effect of extracellular Na+ on the E1P↔E2P conformational transition.(A,B) Voltage dependence of fluorescence amplitudes 1-ΔF/F of TMRM-labeled HKαS806C/βWT at pHex 7.4 (A), and pHex 5.5 (B) in the presence of 90 mM extracellular Na+ (▪,□) compared to Na+-free conditions (•,○; Na+ replacement by 90 mM TMA+). Data are means±S.E. of 13–15 oocytes. Superimposed are curves resulting from a fits of a Boltzmann-type function to the data (pHex 7.4, 90 mM TMA+: V0.5 = −89.6±3.3 mV, zq = 0.48±0.04; pHex 7.4, 90 mM Na+: V0.5 = −19.7±5.4 mV, zq = 0.26±0.02; pHex 5.5, 90 mM TMA+: V0.5 = −125.2±11.4 mV, zq = 0.49±0.07; pHex 5.5, 90 mM Na+: V0.5 = −126.4±16.6 mV, zq = 0.26±0.03), parameters are listed in Table 1. The fluorescence amplitudes were normalized to the saturation values from the fits. (C,F) Reciprocal time constants (τ−1) from fits of a single exponential function to fluorescence signals in Na+-free and in 90 mM Na+-containing solutions for pHex 7.4 (C) and pHex 5.5 (F). Data are means±S.E. from 13–15 oocytes. (D,E) Calculated forward (kf) and reverse (kb) rate constants of the E1P↔E2P transition in the presence of 90 mM Na+ (D), and in Na+-free solution (E) at pHex 7.4, as calculated from the observed ktot = τ−1 values in (C) and the voltage-dependent fluorescence amplitudes in (A) according to Supporting Information (Appendix S1). (G,H) Calculated forward (kf) and reverse (kb) rate constants at pHex 5.5 in the presence of 90 mM Na+ (G) and in Na+-free solution (H), as calculated from the ktot = τ−1 values in (F) and fluorescence amplitudes in (B). Superimposed in (D,E,G,H) are fits of a single exponential function to the kf and kb values, the resulting fit parameters (kb(0), kf(0), and zq values) are summarized in Table 1.
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Figure 6. Temperature and voltage dependence of Rb+ uptake by gastric H,K-ATPase.(A) H,K-ATPase-mediated Rb+ uptake (in pmol/oocyte/min) at 5 mM Rb+ and a pHex of 7.4 (light gray bars) or 5.5 (gray bars) at temperatures between 18 and 34°C, as indicated. White bars represent Rb+ uptake of non-injected control oocytes at each temperature and pHex 5.5. The black bar at 34°C shows the residual Rb+ uptake at pHex 5.5 in the presence of 100 µM SCH28080. Data in each column are means of 20–25 oocytes from oocytes of one cell batch. (B) Arrhenius plot for temperature-dependent Rb+ uptakes from data as in (A) at pHex 7.4 (▪), and pHex 5.5 (○). Data represent means±S.E. of three independent experiments (similar to the one shown in A), after normalization to Rb+ uptake at 34°C for each experiment. Activation energies obtained from linear fits to the data (superimposed lines) are given for each pHex. (C) Rb+ uptake (in pmol/oocyte/min) at 5 mM Rb+ and pHex 7.4 or 5.5 for oocytes expressing HKαS806C/βWT, which had either been clamped to a membrane potential of −100 mV, or subjected to Rb+ uptake without voltage clamping (Vm∼−10 to −20 mV). Black bars represent Rb+ uptake of H,K-ATPase-expressing oocytes clamped at −100 mV in the presence of 100 µM SCH28080. Data are means±S.D. from several oocytes of a single batch (numbers stated on each column).
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