title: Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.
abstract: Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na(+), Cl(-), and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na(+):1 Cl(-):1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na(+), Cl(-), and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na(+), Cl(-), or GABA. The shifts in V rev for a tenfold change in the external Na(+), Cl(-), and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na(+), Cl(-), and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na(+):1 Cl(-):1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease.
authors.affiliation: Biological Sciences Department, California State Polytechnic University, 3801 West Temple Avenue, Pomona, CA, 91768-4032, USA.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Willford
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Anderson
authors.forename: Shelly R
authors.initials: SR
authors.lastname: Spencer
authors.forename: Sepehr
authors.initials: S
authors.lastname: Eskandari
mesh_terms: D000818:Animals; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials; D015398:Signal Transduction; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000235:genetics / Q000378:metabolism*
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; C493413:SLC6A1 protein, human; C493416:SLC6A11 protein, human; D005680:gamma-Aminobutyric Acid
references.citation: J Biol Chem. 1992 Nov 5;267(31):22007-9
references.pmid: 1429551
references.citation: J Neurophysiol. 2003 Sep;90(3):1363-74
references.pmid: 12966170
references.citation: J Neurophysiol. 2013 Feb;109(3):803-12
references.pmid: 23114210
references.citation: J Neurosci Res. 2001 Mar 15;63(6):461-8
references.pmid: 11241581
references.citation: Pflugers Arch. 2004 Nov;449(2):132-42
references.pmid: 15338308
references.citation: J Neurosci. 2004 Apr 14;24(15):3837-49
references.pmid: 15084665
references.citation: J Neurosci. 1989 Feb;9(2):648-56
references.pmid: 2918382
references.citation: BMC Biol. 2012 Mar 15;10:26
references.pmid: 22420899
references.citation: Nature. 2007 Oct 11;449(7163):726-30
references.pmid: 17704762
references.citation: J Physiol. 2006 Dec 1;577(Pt 2):591-9
references.pmid: 17008380
references.citation: J Neurochem. 1980 Dec;35(6):1394-9
references.pmid: 7441257
references.citation: Neuron. 2007 Dec 6;56(5):851-65
references.pmid: 18054861
references.citation: Neuron. 1992 Aug;9(2):337-48
references.pmid: 1497897
references.citation: Structure. 2013 May 7;21(5):694-705
references.pmid: 23664361
references.citation: Am J Physiol Cell Physiol. 2012 Apr 15;302(8):C1096-108
references.pmid: 22173867
references.citation: J Neurochem. 1973 Aug;21(2):345-56
references.pmid: 4724323
references.citation: Biochem Pharmacol. 1965 Mar;14:273-88
references.pmid: 14314324
references.citation: Nature. 2009 May 21;459(7245):347-55
references.pmid: 19458710
references.citation: J Biol Chem. 1993 Jan 25;268(3):2106-12
references.pmid: 8420981
references.citation: J Membr Biol. 2012 Dec;245(12):841-57
references.pmid: 22918627
references.citation: Can J Biochem. 1967 Dec;45(12 ):1795-807
references.pmid: 6082568
references.citation: J Gen Physiol. 1999 Sep;114(3):429-44
references.pmid: 10469733
references.citation: J Physiol. 1991 Nov;443:587-99
references.pmid: 1822540
references.citation: Neurochem Int. 1996 Oct;29(4):335-56
references.pmid: 8939442
references.citation: J Physiol. 2014 Mar 1;592(5):863-9
references.pmid: 23878376
references.citation: J Neurophysiol. 2006 Nov;96(5):2425-36
references.pmid: 16870837
references.citation: J Gen Physiol. 1999 Sep;114(3):445-57
references.pmid: 10469734
references.citation: Mol Pharmacol. 2005 Dec;68(6):1728-35
references.pmid: 16150932
references.citation: Neuroscience. 1988 Nov;27(2):563-70
references.pmid: 3217004
references.citation: J Neurosci Res. 2014 Mar;92(3):398-408
references.pmid: 24273061
references.citation: J Physiol. 1996 Feb 1;490 ( Pt 3):691-702
references.pmid: 8683468
references.citation: J Neurophysiol. 2003 Apr;89(4):2021-34
references.pmid: 12612025
references.citation: Front Cell Neurosci. 2011 Apr 05;5:2
references.pmid: 21503156
references.citation: J Neurochem. 1998 Nov;71(5):1785-803
references.pmid: 9798903
references.citation: Biophys J. 2005 Jul;89(1):211-31
references.pmid: 15849242
references.citation: Biochemistry. 1988 Jan 12;27(1):12-7
references.pmid: 3349023
references.citation: ChemMedChem. 2010 Jul 5;5(7):986-1000
references.pmid: 20491137
references.citation: Biochemistry. 1978 Apr 4;17(7):1207-11
references.pmid: 656383
references.citation: J Membr Biol. 2005 Jan;203(2):65-82
references.pmid: 15981712
references.citation: J Neurophysiol. 2002 Sep;88(3):1407-19
references.pmid: 12205162
references.citation: J Neurophysiol. 1998 Jul;80(1):270-81
references.pmid: 9658049
references.citation: J Neurochem. 1991 Jan;56(1):273-80
references.pmid: 1987321
references.citation: Neurochem Int. 2014 Jun;72:1-9
references.pmid: 24726769
references.citation: Neuron. 1993 Feb;10 (2):165-75
references.pmid: 7679913
references.citation: J Biol Chem. 1992 Oct 15;267(29):21098-104
references.pmid: 1400419
references.citation: J Neurochem. 1971 May;18(5):683-90
references.pmid: 5145146
references.citation: J Membr Biol. 2007 Dec;220(1-3):33-51
references.pmid: 17994179
references.citation: FEBS Lett. 1995 Oct 16;373(3):229-33
references.pmid: 7589472
references.citation: J Biol Chem. 2000 Dec 1;275(48):37414-22
references.pmid: 10973981
references.citation: J Membr Biol. 1976 Dec 28;30(2):153-73
references.pmid: 1011247
references.citation: J Biol Chem. 1999 Jun 11;274(24):16709-16
references.pmid: 10358010
references.citation: J Neurophysiol. 2005 Sep;94(3):2073-85
references.pmid: 15987761
references.citation: PLoS One. 2012;7(6):e39360
references.pmid: 22737235
references.citation: FEBS Lett. 1990 Aug 20;269(1):181-4
references.pmid: 2387399
references.citation: Brain Res. 1983 Mar 14;263(1):63-75
references.pmid: 6132667
references.citation: Science. 1987 Oct 16;238(4825):350-5
references.pmid: 2443977
references.citation: J Neurosci. 2000 Nov 1;20(21):7914-21
references.pmid: 11050111
references.citation: Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):723-7
references.pmid: 8570623
references.citation: FASEB J. 1997 Sep;11(11):835-41
references.pmid: 9285481
references.citation: J Neurosci. 2001 Apr 15;21(8):2630-9
references.pmid: 11306616
references.citation: Am J Physiol Cell Physiol. 2012 Jun 1;302(11):C1661-75
references.pmid: 22460712
references.citation: J Neurosci. 1996 Sep 1;16(17):5405-14
references.pmid: 8757253
references.citation: J Neurophysiol. 1992 Jun;67(6):1698-701
references.pmid: 1629773
references.citation: Neurosci Lett. 1992 Nov 23;147(1):16-20
references.pmid: 1336151
references.citation: Pflugers Arch. 2004 Feb;447(5):519-31
references.pmid: 12719981
references.citation: Neuron. 1993 Feb;10(2):177-88
references.pmid: 7679914
references.citation: J Neurosci. 2003 Apr 1;23(7):2618-26
references.pmid: 12684447
references.citation: Nature. 2005 Sep 8;437(7056):215-23
references.pmid: 16041361
references.citation: J Neurochem. 1978 Jul;31(1):251-6
references.pmid: 27588
references.citation: CRC Crit Rev Biochem. 1987;22(1):1-38
references.pmid: 2888595
references.citation: Eur J Pharmacol. 2003 Oct 31;479(1-3):127-37
references.pmid: 14612144
references.citation: Glia. 1991;4(3):245-55
references.pmid: 1680100
references.citation: Pharmacol Rev. 2011 Sep;63(3):585-640
references.pmid: 21752877
references.citation: Neuron. 2000 Feb;25(2):373-83
references.pmid: 10719892
references.citation: J Neurosci. 1992 Apr;12 (4):1394-408
references.pmid: 1556600
references.citation: Am J Physiol Cell Physiol. 2011 Nov;301(5):C1064-73
references.pmid: 21775701
references.citation: J Physiol. 1985 Sep;366:387-409
references.pmid: 2414435
references.citation: Neuroscience. 1993 Jun;54(4):1019-34
references.pmid: 8101980
references.citation: J Neurochem. 1972 Mar;19(3):841-55
references.pmid: 5030987
references.citation: J Membr Biol. 2002 Nov 1;190(1):57-73
references.pmid: 12422272
references.citation: Biochemistry. 1992 Feb 25;31(7):1974-9
references.pmid: 1536839
references.citation: Pflugers Arch. 2014 Jan;466(1):25-42
references.pmid: 24337881
references.citation: Brain Res Brain Res Rev. 2004 Jul;45(3):196-212
references.pmid: 15210304
references.citation: Neuron. 1994 Oct;13(4):949-60
references.pmid: 7524562
references.citation: Biochemistry. 1983 Mar 1;22(5):1236-41
references.pmid: 6838850
references.citation: J Biol Chem. 2003 May 16;278(20):17716-26
references.pmid: 12595533
references.citation: Neurochem Int. 2009 Dec;55(8):732-40
references.pmid: 19622377
references.citation: Methods Enzymol. 1998;296:233-47
references.pmid: 9779452
references.citation: Biophys J. 2007 Oct 1;93(7):2325-31
references.pmid: 17526579
references.citation: Mol Pharmacol. 2003 Oct;64(4):905-13
references.pmid: 14500747