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.