Substrate-dependent proton antiport in neurotransmitter:sodium symporters

Neurotransmitter:sodium symporters (NSS), targets for psychostimulants and therapeutic drugs, play a critical role in neurotransmission. Whereas eukaryotic NSS exhibit Cl(−)-dependent transport, bacterial NSS feature Cl(−)-independent substrate transport. Recently we showed in LeuT and Tyt1 that mutation of an acidic side chain near one of the Na(+)-binding sites renders substrate binding and/or transport Cl(−) dependent. We reasoned that the negative charge - provided either by Cl(−) or by the transporter itself - is required for substrate translocation. Here we show that Tyt1 reconstituted in proteoliposomes is strictly dependent on the Na(+) gradient and is stimulated by an inside negative membrane potential and by an inversely-oriented H(+) gradient. Remarkably, Na(+)/substrate symport elicited H(+) efflux, indicative of Na(+)/substrate symport-coupled H(+) antiport. Mutations that render the transport phenotype Cl(−)-dependent essentially abolish the pH dependence. We propose unifying features of charge balance by all NSS members with similar mechanistic features but with different molecular solutions.