Proteoliposome Reconstitution and Membrane Potential Measurement

Proteoliposomes were reconstituted with an internal buffer of 25 mM HEPES, pH 7.53, 100 mM NaCl, 100 mM KCl, and preloaded with 0.4 mM substrate (Gdm⁺ or guanylurea) and 1 mM pyranine (trisodium 8-hydroxypyrene-1, 3, 6-trisulfonate; Sigma-Aldrich) using three freeze/thaw cycles. Unilamellar liposomes were formed by extrusion through a 400-nm membrane filter and the external pyranine was removed by passing liposomes through a Sephadex G-50 column spin column equilibrated in an internal buffer with substrate. The external assay buffers contained 25 mM HEPES, pH 7.53, 0.4 mM substrate, and varying KCl concentration (3–46 mM) to establish the membrane potential, with NaCl to bring the total salt concentration to 200 mM. Proteoliposomes were diluted 200-fold into the external buffer, and after ∼30 s to establish a baseline, valinomycin (final concentration 0.2 ng/ml) was added together with the substrate (final concentration 4 mM). Fluorescence spectra were monitored (λ_ex = 455 nm; λ_em = 515 nm) for ∼300 s. The membrane potential was calculated using the Nernst potential for K⁺: ${\psi }_{calc}=\frac{RT}{F}\ln \frac{{\left[K^{+}\right]}_{out}}{{\left[K^{+}\right]}_{in}}\text{.}$
Fluorescence emission time courses were corrected for baseline drift measured prior to substrate and valinomycin addition. The stoichiometry was determined from the voltage at which electrochemical equilibrium occurred (no change in fluorescence over time) using the following equation: ${\mathrm{E}}_{rev}=\left(\frac{n}{m-n}*\frac{RT}{F}\mathit{ln}\frac{{\left[substrat{e}^{+}\right]}_{in}}{{\left[substrat{e}^{+}\right]}_{out}}\right)\text{,}$ where n and m represent the stoichiometric coefficients of substrate and protons, respectively.