title: Substrate-dependent proton antiport in neurotransmitter:sodium symporters.
abstract: 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.
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.
authors.forename: Yongfang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Ernest L
authors.initials: EL
authors.lastname: Mehler
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D001407:Bacillus / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D016967:Fusobacterium nucleatum / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008958:Models, Molecular; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D050480:Plasma Membrane Neurotransmitter Transport Proteins