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.