title: Single-molecule dynamics of gating in a neurotransmitter transporter homologue.
abstract: Neurotransmitter:Na(+) symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na(+) gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, New York 10032, USA.
authors.forename: Yongfang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Daniel
authors.initials: D
authors.lastname: Terry
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D000409:Alanine / Q000378:metabolism; D000494:Allosteric Regulation; D029063:Aquifoliaceae / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer; D007930:Leucine / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D012964:Sodium; D007930:Leucine; D003545:Cysteine; D000409:Alanine
references.citation: Nature. 2007 Aug 23;448(7156):952-6
references.pmid: 17687333
references.citation: Essays Biochem. 2000;35:3-18
references.pmid: 12471886
references.citation: Science. 2009 Aug 21;325(5943):1010-4
references.pmid: 19608859
references.citation: Science. 2007 Sep 7;317(5843):1390-3
references.pmid: 17690258
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3603-8
references.pmid: 17360689
references.citation: Anal Biochem. 1973 Dec;56(2):502-14
references.pmid: 4128882
references.citation: Bioconjug Chem. 1993 Mar-Apr;4(2):105-11
references.pmid: 7873641
references.citation: Protein Sci. 1999 Apr;8(4):921-9
references.pmid: 10211839
references.citation: Br J Pharmacol. 2006 Jan;147 Suppl 1:S82-8
references.pmid: 16402124
references.citation: Nature. 2008 Apr 3;452(7187):598-603
references.pmid: 18327250
references.citation: J Biol Chem. 2000 Nov 3;275(44):34106-13
references.pmid: 10926932
references.citation: Drug Alcohol Depend. 1998 Jun-Jul;51(1-2):87-96
references.pmid: 9716932
references.citation: Curr Opin Struct Biol. 2008 Feb;18(1):16-26
references.pmid: 18221865
references.citation: Biophys J. 2009 Mar 18;96(6):2371-81
references.pmid: 19289062
references.citation: Curr Protein Pept Sci. 2004 Apr;5(2):81-7
references.pmid: 15078219
references.citation: Annu Rev Phys Chem. 2004;55:79-96
references.pmid: 15117248
references.citation: Science. 2008 Dec 12;322(5908):1655-61
references.pmid: 19074341
references.citation: ACS Chem Biol. 2007 Jan 23;2(1):53-61
references.pmid: 17243783
references.citation: Nature. 2005 Sep 8;437(7056):215-23
references.pmid: 16041361
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5563-8
references.pmid: 19307590
references.citation: Biophys J. 1996 Jan;70(1):264-80
references.pmid: 8770203
references.citation: Nat Struct Mol Biol. 2009 Jun;16(6):652-7
references.pmid: 19430461
references.citation: Annu Rev Biophys. 2008;37:317-36
references.pmid: 18573084
references.citation: Curr Opin Struct Biol. 2009 Feb;19(1):103-9
references.pmid: 19223173
references.citation: Annu Rev Biophys Biomol Struct. 2005;34:399-414
references.pmid: 15869396
references.citation: J Biol Chem. 2008 Jun 20;283(25):17691-701
references.pmid: 18426798
references.citation: Biophys J. 2004 Mar;86(3):1488-501
references.pmid: 14990476
references.citation: Mol Pharmacol. 2005 Dec;68(6):1728-35
references.pmid: 16150932
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12893-8
references.pmid: 15317937
references.citation: J Biol Chem. 1994 Mar 11;269(10):7124-30
references.pmid: 8125921
references.citation: Cell. 2007 Jun 29;129(7):1249-50
references.pmid: 17604712
references.citation: J Mol Biol. 2008 Jun 13;379(4):695-703
references.pmid: 18485365
references.citation: Nat Rev Neurosci. 2003 Jan;4(1):13-25
references.pmid: 12511858
references.citation: Science. 1999 Oct 8;286(5438):295-9
references.pmid: 10514373
references.citation: Mol Pharmacol. 2006 Nov;70(5):1630-42
references.pmid: 16880288
references.citation: Annu Rev Biochem. 2008;77:149-76
references.pmid: 18410247
references.citation: J Cell Biol. 2003 Nov 10;163(3):445-50
references.pmid: 14610051
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12640-5
references.pmid: 17502603
references.citation: Nat Methods. 2008 Jun;5(6):507-16
references.pmid: 18511918
references.citation: Mol Cell. 2007 Feb 23;25(4):505-17
references.pmid: 17317624
references.citation: Q Rev Biophys. 2007 Feb;40(1):87-111
references.pmid: 17666122
references.citation: Biophys J. 2008 Nov 15;95(10):4613-21
references.pmid: 18708457
references.citation: J Mol Biol. 2008 Mar 28;377(3):804-18
references.pmid: 18280500
references.citation: Curr Opin Neurobiol. 2005 Jun;15(3):296-304
references.pmid: 15919190
references.citation: ChemMedChem. 2007 Jun;2(6):827-40
references.pmid: 17436258
references.citation: Mol Cell. 2008 Jun 20;30(6):667-77
references.pmid: 18570870
references.citation: Biophys J. 2008 Mar 1;94(5):1600-12
references.pmid: 18024499