title: Binding thermodynamics of a glutamate transporter homolog.
abstract: Glutamate transporters catalyze concentrative uptake of the neurotransmitter into glial cells and neurons. Their transport cycle involves binding and release of the substrate on the extra- and intracellular sides of the plasma membranes and translocation of the substrate-binding site across the lipid bilayers. The energy of the ionic gradients, mainly sodium, fuels the cycle. Here, we used a cross-linking approach to trap a glutamate transporter homolog from Pyrococcus horikoshii in key conformational states with the substrate-binding site facing either the extracellular or the intracellular side of the membrane to study binding thermodynamics. We show that the chemical potential of sodium ions in solution is exclusively coupled to substrate binding and release, not to substrate translocation. Despite the transporter's structural symmetry, the binding mechanisms are distinct on the opposite sides of the membrane and more complex than the current models suggest.
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA. nicolas.reyes@pasteur.fr
authors.forename: Nicolas
authors.initials: N
authors.lastname: Reyes
authors.forename: SeCheol
authors.initials: S
authors.lastname: Oh
authors.forename: Olga
authors.initials: O
authors.lastname: Boudker
mesh_terms: D001665:Binding Sites; D050491:Glutamate Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D043902:Pyrococcus horikoshii / Q000737:chemistry* / Q000378:metabolism*; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D050491:Glutamate Plasma Membrane Transport Proteins; D007477:Ions; D018377:Neurotransmitter Agents; D012964:Sodium
references.citation: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references.pmid: 15299374
references.citation: Protein Sci. 2001 Jul;10(7):1343-52
references.pmid: 11420436
references.citation: J Membr Biol. 2002 Feb 1;185(3):221-36
references.pmid: 11891580
references.citation: Annu Rev Phys Chem. 2005;56:521-48
references.pmid: 15796710
references.citation: Nature. 2007 Jan 25;445(7126):387-93
references.pmid: 17230192
references.citation: Biophys J. 1995 Apr;68(4):1406-15
references.pmid: 7787026
references.citation: Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):155-61
references.pmid: 18977733
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: J Mol Biol. 2008 Dec 26;384(4):1002-17
references.pmid: 18930735
references.citation: Nat Struct Mol Biol. 2012 Feb 12;19(3):355-7
references.pmid: 22343718
references.citation: J Neurosci. 1998 Dec 1;18(23):9620-8
references.pmid: 9822723
references.citation: Mol Pharmacol. 1998 Feb;53(2):195-201
references.pmid: 9463476
references.citation: Biochemistry. 2002 Jun 11;41(23):7501-7
references.pmid: 12044184
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12148-52
references.pmid: 15304639
references.citation: J Biol Chem. 2008 Oct 17;283(42):28680-90
references.pmid: 18678877
references.citation: J Mol Biol. 2004 Feb 27;336(4):829-42
references.pmid: 15095863
references.citation: Chem Rev. 1997 Aug 5;97(5):1251-1268
references.pmid: 11851450
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Nature. 2009 Dec 17;462(7275):880-5
references.pmid: 19924125
references.citation: J Biol Chem. 2006 Sep 22;281(38):27905-15
references.pmid: 16870620
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18025-30
references.pmid: 17991780
references.citation: Nature. 1996 Oct 17;383(6601):634-7
references.pmid: 8857541
references.citation: Acta Crystallogr D Biol Crystallogr. 2001 Jan;57(Pt 1):122-33
references.pmid: 11134934
references.citation: Nature. 2004 Oct 14;431(7010):811-8
references.pmid: 15483603
references.citation: Science. 2008 Oct 31;322(5902):709-13
references.pmid: 18927357
references.citation: Biochemistry. 2009 Sep 29;48(38):9147-55
references.pmid: 19708718
references.citation: Biochemistry. 2010 May 4;49(17):3511-3
references.pmid: 20349989
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 3;107(31):13912-7
references.pmid: 20634426
references.citation: Biochim Biophys Acta. 2011 Feb;1807(2):167-88
references.pmid: 21029721
references.citation: PLoS One. 2012;7(3):e33058
references.pmid: 22427946
references.citation: Bioorg Med Chem Lett. 2000 Nov 6;10(21):2407-10
references.pmid: 11078189
references.citation: Biophys Chem. 2011 Nov;159(1):6-13
references.pmid: 21536369
references.citation: Mol Biosyst. 2011 Mar;7(3):832-42
references.pmid: 21161089
references.citation: J Biol Chem. 2012 May 18;287(21):17198-17205
references.pmid: 22493292
references.citation: J Gen Physiol. 2001 Jun;117(6):547-62
references.pmid: 11382805
references.citation: Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9026-31
references.pmid: 10922058
references.citation: Biophys J. 2008 Sep;95(5):2292-300
references.pmid: 18515371
references.citation: Biophys J. 2010 Sep 8;99(5):1416-25
references.pmid: 20816053
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Nat Struct Mol Biol. 2004 Oct;11(10):945-9
references.pmid: 15334070
references.citation: J Biol Chem. 2010 Jun 4;285(23):17725-33
references.pmid: 20378543
references.citation: J Biol Chem. 2009 Jun 26;284(26):17540-8
references.pmid: 19380583
references.citation: Proteins. 1995 Aug;22(4):404-12
references.pmid: 7479713
references.citation: J Neurosci. 2011 Apr 20;31(16):6255-62
references.pmid: 21508248
references.citation: J Phys Condens Matter. 2010 Aug 18;22(32):323101
references.pmid: 21386475
references.citation: J Physiol. 2006 Dec 1;577(Pt 2):591-9
references.pmid: 17008380
references.citation: Bioorg Med Chem. 2008 Aug 15;16(16):7740-8
references.pmid: 18650095