Substrate-dependent proton antiport in neurotransmitter:sodium symporters

Neurotransmitter-sodium symporters (NSS), targets for psychostimulants and therapeutic drugs, have a critical role in neurotransmission. Whereas eukaryotic NSS show chloride-dependent transport, bacterial NSS feature Cl(-)-independent substrate transport. Recently we showed that mutation of an acidic residue near one of the sodium ion-binding sites in LeuT of Aquifex aeolicus or Tyt1 of Fusobacterium nucleatum 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 proton gradient. Notably, 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 different molecular solutions.