Transport Coupling Stoichiometry Determination of Electrogenic Secondary Transporters

Abstract

Accurately determining stoichiometry is a vital step in the mechanistic characterization of secondary active transporters, but has been difficult for many bacterial transporters. To address this problem we have developed a new method to determine stoichiometry for electrogenic secondary transporters by measuring the reversal potential, the voltage at which there is no net flux of ions or substrate. Reversal potentials have been routinely measured for mammalian channels and transporters which are amenable to heterologous expression and electrophysiological characterization. However, such measurements are much more difficult for bacterial proteins, for which electrophysiology is not applicable. A recent flood of X-ray structures of bacterial transport proteins, with or without their coupling ions and substrates, and the accompanying efforts to understand their mechanisms using experimental and computational approaches increases the urgency of obtaining accurate transport stoichiometries. Our new method should be applicable to any electrogenic secondary transporter, as it is based on the measurement of radioactive substrate flux. We preload proteoliposomes containing purified reconstituted transporter with a set amount of coupling ion and radiolabeled substrate, incubate the vesicles in an external buffer also containing labeled substrate and coupling ion, then set the transmembrane voltage using K+/valinomycin. With labeled substrate at known concentrations, we can follow both substrate influx and efflux. By making measurements at different voltages inducing both influx and efflux of substrate, we can determine the voltage at which there is no net flux: the reversal potential. Knowing this voltage allows straightforward thermodynamic calculations that yield the coupling stoichiometry.We tested this method on VcINDY, an electrogenic Na+/Succinate2- symporter, and MelB; an electrogenic Na+/melibiose symporter, the latter has a known coupling stoichiometry of 1:1. Our results firmly establish that VcINDY has a 3:1 Na+:succinate2- coupling stoichiometry and confirm the 1:1 coupling stoichiometry of MelB.