Correlating Charge Movements with Local Conformational Changes of a Na-Coupled Cotransporter

Abstract

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (presteady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at externally accessible sites in the Na+-coupled inorganic cotransporter (SLC34A2) and the mutants expressed in Xenopus oocytes. Fluorophore labeling at one site resulted in complete suppression of transport activity, whereas Cys-substitution and labeling at 6 other sites had marginal effect on kinetics. For these 6 mutants, the properties of the presteady-state charge relaxations (mid-point potential, apparent valence) were similar, whereas fluorescence intensity changes (delta-F) differed significantly depending on the labeling site. By using a 5-state kinetic model, we simulated the measured delta-F and determined the contributions from each state as a function of membrane voltage to obtain a unique set of apparent quantum yields for each mutant. At one site, delta-F originated from the fluorophore sensing inward and outward conformations, whereas for the other sites delta-F was associated principally with one or the other orientation. In response to step changes in voltage, the presteady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant, the time constants matched well with and without external Na+, providing direct evidence that the fluorophore at this site reported conformational changes accompanying intrinsic charge movement and cation interactions in response to voltage steps. For other mutants, correlations were found only in the presence of Na+ and V>0. Additional evidence for the movement of parts of the protein into a more aqueous environment was obtained using iodide as a collisional quencher.