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• W1995875445 abstract "The sodium- and chloride-dependent electrogenic γ-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuTAa. In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. Atvaryingextracellularsodium,lithiumstimulatedsodiumdependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport. The sodium- and chloride-dependent electrogenic γ-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuTAa. In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. Atvaryingextracellularsodium,lithiumstimulatedsodiumdependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport. Many neurotransmitters are removed from the synaptic cleft by transporters, which thereby enable efficient synaptic transmission. Many of these transporters are sodium- and chloride-dependent and form a family which, besides the transporters for γ-aminobutyric acid (GABA), 2The abbreviations used are: GABA, γ-aminobutyric acid; TM, transmembrane domain; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium) ethyl-]methanethiosulfonate; MTSES, (2-sulfonatoethyl)methanethiosulfonate.2The abbreviations used are: GABA, γ-aminobutyric acid; TM, transmembrane domain; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium) ethyl-]methanethiosulfonate; MTSES, (2-sulfonatoethyl)methanethiosulfonate. also includes those for serotonin, dopamine, norepinephrine, and glycine (for reviews, see Refs. 1Kanner B.I. J. Exp. Biol. 1994; 196: 237-249Crossref PubMed Google Scholar and 2Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (321) Google Scholar). The GABA transporter GAT-1 (3Radian R. Bendahan A. Kanner B.I. J. Biol. Chem. 1986; 261: 15437-15441Abstract Full Text PDF PubMed Google Scholar, 4Guastella J. Nelson N. Nelson H. Czyzyk L. Keynan S. Miedel M.C. Davidson N. Lester H.A. Kanner B.I. Science. 1990; 249: 1303-1306Crossref PubMed Scopus (717) Google Scholar) is the first identified member of this family, which catalyzes electrogenic sodium:chloride:GABA cotransport with a stoichiometry of 2:1:1 (5Keynan S. Kanner B.I. Biochemistry. 1988; 27: 12-17Crossref PubMed Scopus (132) Google Scholar, 6Kavanaugh M.P. Arriza J.L. North R.A. Amara S.G. J. Biol. Chem. 1992; 267: 22007-22009Abstract Full Text PDF PubMed Google Scholar, 7Mager S. Naeve J. Quick M. Labarca C. Davidson N. Lester H.A. Neuron. 1993; 10: 177-188Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 8Lu C.C. Hilgemann D.W. J. Gen. Physiol. 1999; 114: 429-444Crossref PubMed Scopus (101) Google Scholar). The role of chloride in this process is still under debate because it has been suggested that during sodium-coupled GABA transport, obligatory chlorideout/chloridein exchange takes place (9Loo D.D. Eskandari S. Boorer K.J. Sarkar H.K. Wright E.M. J. Biol. Chem. 2000; 275: 37414-37422Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In contrast to sodium, lithium cannot support transport of [3H]GABA (10Bennett E.R. Su H. Kanner B.I. J. Biol. Chem. 2000; 275: 34106-34113Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11MacAulay N. Zeuthen T. Gether U. J. Physiol. (Lond.). 2002; 544: 447-458Crossref Scopus (42) Google Scholar), but it can stimulate GABA-induced currents at reduced sodium concentrations, suggesting that during transport, one of the two sodium ions can be replaced by lithium (11MacAulay N. Zeuthen T. Gether U. J. Physiol. (Lond.). 2002; 544: 447-458Crossref Scopus (42) Google Scholar). In the absence of GABA, leak currents are observed when lithium replaces sodium. These leak currents have a much steeper voltage dependence than the GABA-induced transport currents observed in the presence of sodium (12Mager S. Kleinberger-Doron N. Keshet G.I. Davidson N. Kanner B.I. Lester H.A. J. Neurosci. 1996; 16: 5405-5414Crossref PubMed Google Scholar, 13Bismuth Y. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 1997; 272: 16096-16102Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These lithium leak currents are inhibited by low concentrations of sodium ions, and this inhibition is not competitive. These observations indicate that when sodium binds to GAT-1, the leak mode is converted into a conformation that enables coupled transport (11MacAulay N. Zeuthen T. Gether U. J. Physiol. (Lond.). 2002; 544: 447-458Crossref Scopus (42) Google Scholar, 14Kanner B.I. J. Biol. Chem. 2003; 278: 3705-3712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Recently, a high-resolution crystal structure from a bacterial family member of the Na+/Cl−-dependent neurotransmitter transporters, the leucine transporter LeuTAa, was determined (15Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1335) Google Scholar). The transporter consists of 12 transmembrane domains (TMs), with TMs 1-5 related to TMs 6-10 by a pseudo-2-fold axis in the membrane plane. Two sodium ions, Na1 and Na2, and a leucine molecule are occluded in this structure (15Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1335) Google Scholar). Although the overall sequence identity between the eukaryotic and prokaryotic counterparts is only 20-25%, there are clusters of high sequence conservation. These are distributed throughout the primary structure and include functionally important residues (reviewed in Ref. 15Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1335) Google Scholar). Therefore, it is possible that the published structure may be a useful model to study the neurotransmitter transporter members of this family.In this study, we have investigated the possibility that one or more amino acid residues, which correspond to those that coordinate Na1 and Na2 in LeuTAa, participate in the interaction of lithium with GAT-1. This would provide an important initial clue toward understanding the structural basis of cation selectivity in neurotransmitter transporters. As a parent construct, we have used GAT-1 with cysteine 74 mutated to alanine (C74A). Transport by this mutant, which has similar properties as wild type GAT-1, is not inhibited by membrane-impermeant sulfhydryl residues (14Kanner B.I. J. Biol. Chem. 2003; 278: 3705-3712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 16Zhou Y. Bennett E.R. Kanner B.I. J. Biol. Chem. 2004; 279: 13800-13808Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Our results provide evidence for the idea that the architecture of the sodium binding sites in GAT-1 is similar, but not identical, to that of LeuTAa. The sodium binding site of GAT-1 that corresponds to Na1 is highly specific, whereas the ability of lithium to substitute for sodium at the other sodium binding site and to mediate leak currents by the transporter is controlled by aspartate 395.EXPERIMENTAL PROCEDURESGeneration and Subcloning of Mutants—Mutations were made by site-directed mutagenesis of the wild type GAT-1 in the vector pBluescript SK(-) (Stratagene) according to the Kunkel method as described (17Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4544) Google Scholar, 18Kleinberger-Doron N. Kanner B.I. J. Biol. Chem. 1994; 269: 3063-3067Abstract Full Text PDF PubMed Google Scholar). Briefly, the parent DNA was used to transform Escherichia coli CJ236 (dut-, ung-). From one of the transformants, single-stranded uracil-containing DNA was isolated upon growth in uridine-containing medium according to the standard protocol from Stratagene using helper phage R408. This yields the sense strand, and consequently, mutagenic primers were designed to be antisense. Mutants were subcloned into a construct containing C74A-GAT-1 in the pOG1 vector using two unique restriction enzymes. The pOG1 vector is an oocyte expression vector that contains a 5′-untranslated Xenopus β-globin sequence, the T7 RNA promoter, and a 3′-poly(A) signal. The coding and non-coding strands were sequenced between the two restriction sites.Cell Growth and Expression—HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 200 units/ml penicillin, 200 μg/ml streptomycin, and 2 mm glutamine. Infection with recombinant vaccinia/T7 virus vTF7-3 (19Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1864) Google Scholar) and subsequent transfection with plasmid DNA, as well as [3H]GABA transport, was done as published previously (20Keynan S. Suh Y.J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (121) Google Scholar). Effects of sulfhydryl reagents on transport were studied as described (16Zhou Y. Bennett E.R. Kanner B.I. J. Biol. Chem. 2004; 279: 13800-13808Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).cRNA Transcription, Injection, and Oocyte Preparation— Capped run-off cRNA transcripts were made from transporter constructs in pOG1, linearized with SacII, using mMessage mMachine (Ambion). Oocytes were removed from anesthetized Xenopus laevis frogs and treated with collagenase (type 1A; Sigma number C-9891) until capillaries were absent and injected with 50 nl of undiluted cRNA on the same or the next day. Oocytes were maintained at 18 °C in modified Barth's saline containing the following (in mm): 88 NaCl, 1 KCl, 1 MgSO4, 2.4 NaHCO3, 1 CaCl2, 0.3 Ca(NO3)2, 10 HEPES, pH 7.5, with freshly added 2 mm sodium pyruvic acid and 0.5 mm theophylline, and supplemented with 10,000 units/liter penicillin, 10 mg/liter streptomycin, and 50 mg/liter gentamicin.Oocyte Electrophysiology—This was done as described (14Kanner B.I. J. Biol. Chem. 2003; 278: 3705-3712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Oocytes were placed in the recording chamber, penetrated with two micropipettes (backfilled with 2 m KCl, resistance varied between 0.5 and 2 megaohms), and voltage-clamped using a GeneClamp 500 amplifier (Axon Instruments) and digitized using a Digidata 1200A (Axon Instruments), both controlled with the pClamp6 suite (Axon Instruments). Currents were acquired with Clampex 6.03 at 10 kHz every 0.5 ms and low pass-filtered online. The membrane potential of the oocytes was jumped from −140 mV to +60 mV in 25-mV increments, using −25 mV as holding potential, unless stated otherwise in the figure legends. Each potential was held clamped for 500 ms. The membrane potential was measured relative to an extracellular Ag/AgCl electrode in the recording chamber. Recording solution contained 96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, and 5 mm Hepes, pH 7.4. In substitution experiments, sodium ions were replaced with equimolar choline or lithium (as indicated in the figure legends). Figures were prepared using Origin 6.1 (Microcal). The GABA-induced currents are defined as the currents in the absence of GABA subtracted from those in its presence (at the concentrations indicated in the figure legends). In those experiments, in which the effect of the impermeant sulfhydryl reagents MTSET or MTSES was analyzed, this was done exactly as described (21Borre L. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 2002; 277: 13501-13507Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).Homology Modeling—Figures were prepared using the Deep-View Swiss-PDB viewer downloaded from the ExPASy Proteomics Server. Homology modeling of the Na2 binding site of GAT-1 based on the LeuTAa structure (accession code 2A65) was performed using the Tripos Sybyl7.0 computational software. Initially, modeling of the Na2 binding pocket was carried out for amino acid residues located within 6 Å from the sodium ion for greater precision, and afterward, energy minimization of the wild type and D395T were carried out for amino acid residues within a distance of 4 Å from the Na2 ion and compared. Energy computation and minimization were performed using the Powell method with Simplex initial optimization, Tripos force field, Gasteiger-Huckel charges, and a constant dielectric function.RESULTSIon Dependence of GABA Transport by Putative Na2 Site Mutants—The GABA (1 mm)-induced transport currents by oocytes expressing C74A at various sodium concentrations are shown in Fig. 1A. When lithium was the substituting ion, the GABA-induced currents reached saturation at lower Na+ concentrations than when choline replaced sodium. In the presence of lithium, the transport currents at 2 mm sodium were approximately half of those at 96 mm, whereas this value increased to about 18 mm in its absence (Fig. 1A). Although the results shown are for currents recorded at −140 mV, similar results were also obtained at the other potentials analyzed, −40 and −90 mV, and similar results were also obtained with wild type GAT-1 (data not shown). In the LeuTAa structure, Na2 is coordinated by three main chain carbonyl oxygens and the hydroxyl oxygens from the TM8 residues threonine 354 and serine 355 (15Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1335) Google Scholar). The equivalents of these latter two residues in GAT-1 are aspartate 395 and serine 396, respectively. Significant transport currents were observed by the mutants in which aspartate 395 was replaced by serine or threonine, D395S or D395T, respectively. However, with the D395S/C74A and D395T/C74A mutants, no stimulation of the transport currents by lithium was observed (Fig. 1, B and C). Importantly, the sodium concentration dependence of the GABA-induced currents by these mutants in the presence of either choline or lithium was almost the same as that for C74A in the presence of choline (Fig. 1, A-C). Similar results were also observed when these mutants were in the background of wild type GAT-1 (data not shown). In the case of D395C, a very minor stimulation by lithium, much smaller than in the case of C74A, was seen (Fig. 1, A and D). For this mutant, GABA was used at 10 mm rather than at 1 mm because its Km at −140 mV and 96 mm sodium was ∼400 μm, which is ∼2.5-fold of that in D395S/C74A and D395T/C74A and ∼8-fold that of C74A (Table 1). However, the value of Km is also dependent on the sodium concentration (22Radian R. Kanner B.I. Biochemistry. 1983; 22: 1236-1241Crossref PubMed Scopus (102) Google Scholar). At 20 mm sodium, the Km for GABA in the D395S/C74A mutant was 478 ± 40 μm. Therefore, we also analyzed the currents induced by 10 mm GABA in the D395S/C74A and D395T/C74A mutants, and the results were similar to those depicted in Fig. 1, B and C (data not shown). Transport currents by the D395C/C74A mutant were not affected by preincubation of the oocytes with the membrane-impermeant sulfhydryl reagents MTSET or MTSES (data not shown). The D395E mutant was inactive (data not shown), but the D395N/C74A mutant exhibited GABA-induced transport currents, which were also not stimulated by lithium (Fig. 1E).TABLE 1Kinetic parameters for GABA transport in GAT-1 mutants Km and Vmax were calculated from linear regression using the Lineweaver-Burk plot.KmaThe Km values for GABA measuring the GABA-induced transport currents at −140 mV and 96 mm sodiumImaxbImax is given as a range because of variations in expression levels between various batches of oocytes and also sometimes within a given batchKmcThe Km values for GABA measuring initial rates of [3H]-GABA transport in HeLa cells at 150 mm sodium (3-min time points performed in triplicates) using 24-well platesVmaxμmnAμm(pmol·min−1 (mg of protein−1))C74A56 ± 4408 ± 645.5 ± 0.5343 ± 38N327A/C74A3.7 ± 1.018 ± 1N327C/C74A0.6 ± 0.14 ± 1D395S/C74A173 ± 13271 ± 64D395T/C74A153 ± 12328 ± 50D395C/C74A407 ± 54258 ± 27D395N/C74A1076 ± 180481 ± 82S396T/C74A6652 ± 1072331 ± 46S396C/C74A1277 ± 127167 ± 21S295A/C74A3693 ± 448154 ± 71S295T/C74A53 ± 3156 ± 22a The Km values for GABA measuring the GABA-induced transport currents at −140 mV and 96 mm sodiumb Imax is given as a range because of variations in expression levels between various batches of oocytes and also sometimes within a given batchc The Km values for GABA measuring initial rates of [3H]-GABA transport in HeLa cells at 150 mm sodium (3-min time points performed in triplicates) using 24-well plates Open table in a new tab In contrast to the transport currents induced by 1 mm GABA, when transport of [3H]GABA was measured in HeLa cells expressing C74A (Fig. 2A), the stimulation by lithium was barely visible. This is in agreement with previous studies (10Bennett E.R. Su H. Kanner B.I. J. Biol. Chem. 2000; 275: 34106-34113Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A major difference between the experiments depicted in Figs. 1A and 2A is that, besides the expression system, the actual GABA concentration in the latter is 50 nm, ∼2 orders of magnitude below the Km for radioactive uptake at 150 mm sodium (Table 1). Indeed, when the GABA concentration was increased to 20 μm, a clear stimulation by lithium was observed (Fig. 2B). When transport of [3H]GABA was measured under the same conditions in HeLa cells expressing D395S/C74A or D395T/C74A, the stimulation by lithium was not observed (Fig. 2, C and D). Again, similar results were obtained when wild type GAT-1 was used as a background (data not shown). In contrast to transport currents, uptake of [3H]GABA could not be observed by the D395C/C74A mutant.FIGURE 2Sodium dependence of [3H]GABA transport. HeLa cells expressing C74A, D395S/C74A, or D395T/C74A GAT-1 were assayed for [3H]GABA transport as described under “Experimental Procedures.” Transport was carried out for 3 min at the indicated sodium concentrations with either choline (•) or lithium (▴) as the substituting ion. Data shown at the indicated Na+ concentrations are normalized to those at 150 mm Na+ (no choline or lithium substitution). GABA concentrations used were either 50 nm, 87 Ci/mmol (A)or 20 μm, 0.44 Ci/mmol (B-D). Values are averages (± S.E.) of at least three experiments each performed in triplicate. [3H]GABA uptake at 150 mm Na+ by D395S/C74A and D395T/C74A was 41.5 ± 7.8% and 31.0 ± 8.9%, respectively, of that by C74A, (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)GABA-induced currents could be measured with oocytes expressing the S396T/C74A and S396C/C74A mutants (Fig. 3). In the case of S396T/C74A, the voltage dependence of these currents was similar to that of the GABA-induced currents by C74A shown in Fig. 4A (data not shown), indicating coupled transport. On the other hand, the voltage dependence of the GABA-induced currents by S396C/C74A was more similar to that of the lithium leak currents shown in Fig. 4B (data not shown), suggesting that perhaps GABA induces a sodium leak in this mutant rather than sodium-coupled GABA transport. The Km values for GABA in these two mutants were dramatically increased, ∼1.3 and 6.7 mm for S396C/C74A and S396T/C74A, respectively (Table 1). When the currents induced by 10 mm GABA were measured at varying sodium concentrations, a stimulation by lithium was observed (Fig. 3, A and B), although somewhat smaller than that observed in the case of C74A (Fig. 1A). The transport currents by S396C/C74A were not inhibited by preincubation of the oocytes with the membrane-impermeant sulfhydryl reagents MTSET or MTSES (data not shown). The S396T/C74A and S396C/C74A mutants did not exhibit any radioactive GABA transport (data not shown), apparently due to the high Km values of these mutants (Table 1). Radioactive uptake is measurable in the μm range (at higher concentrations, prohibitive amounts of radiolabel are needed), and due to the high Km, the transport rate at these concentrations would be an undetectably small fraction of Vmax.FIGURE 3Sodium dependence of GABA-induced currents by serine 396 mutants at −140 mV. Currents induced by 10 mm GABA were measured at −140 mV in oocytes expressing S396T/C74A (A) or S396C/C74A (B) at the indicated sodium concentrations as described under “Experimental Procedures.” Na+-substituting ions were either choline (•) or lithium (▴). Data are normalized to those obtained at 96 mm Na+ (no choline or lithium substitution). Data are averages of at least three oocytes ± S.E. The magnitudes of net GABA-induced currents (in nA) obtained in 96 mm Na+ at −140 mV by S396T/C74A and S396C/C74A were −330.8 ± 45.7 and −167.2 ± 20.7, respectively (n = 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Voltage dependence of GABA-induced currents and lithium leak currents. Oocytes expressing C74A (▪), D395S/C74A (▴), D395T/C74A (▾), or D395C/C74A (♦) were clamped at −25 mV before the membrane potential was jumped to the indicated values for 500 ms before returning to −25 mV again. A, GABA-induced currents in the presence of 96 mm sodium, normalized to those by oocytes expressing C74A measured at −140 mV. GABA concentration used was either 1 mm (C74A, D395S/C74A and D395T/C74A) or 10 mm (D395C/C74A). B, currents in 96 mm sodium were subtracted from those in 96 mm lithium, normalized to those by oocytes expressing C74A at −140 mV. Data are averages of three oocytes ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lithium Leak Currents by Asp-395 Mutants—The voltage dependence of the sodium-dependent GABA currents by the Asp-395 mutants was more linear than in C74A (Fig. 4A), perhaps due to an altered interaction between the two sodium sites. The similar voltage dependence of the GABA-induced currents in the presence of sodium in the Asp-395 mutants indicates that also in D395C/C74A, these currents reflect coupled transport, which is difficult to detect using the radioactive uptake assay due to the increased Km for GABA. Not only were these coupled currents by the Asp-395 mutants lacking the stimulation by lithium, but the leak currents were also not observed in the presence of lithium (Fig. 4B). The same result was also observed with D395N (data not shown). On the other hand, the S396T and S396C mutants exhibited lithium leak currents that had similar voltage dependence to those by wild type GAT-1 (data not shown).Ion Dependence of GABA Transport by Putative Na1 Mutants—In the LeuTAa structure, Na1 is coordinated by two main chain carbonyl oxygens, the carboxyl oxygen of the transported leucine, the side chain carbonyl oxygens of asparagine 27 and asparagine 286, and the hydroxyl oxygen from threonine 254 (15Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1335) Google Scholar). The equivalents of the latter three residues in GAT-1 are asparagine 66, asparagine 327, and serine 295. As described previously (14Kanner B.I. J. Biol. Chem. 2003; 278: 3705-3712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 16Zhou Y. Bennett E.R. Kanner B.I. J. Biol. Chem. 2004; 279: 13800-13808Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), mutation of asparagine 66 of GAT-1 to glutamine, aspartate, or cysteine caused defective transport currents as well as the lack of measurable [3H]GABA transport. No transport currents could be observed by the asparagine 327 to alanine or cysteine mutants (data not shown). However, [3H]GABA transport, albeit diminished, could be measured in both mutants. The background in this assay is very low, ∼0.3-0.4% of the signal, so that the sodium dependence of a mutant that has 5% of the activity of the wild type can still be measured. Analysis of the kinetic parameters of [3H]GABA transport by these mutants at 150 mm sodium revealed a 20- and 80-fold reduction of Vmax in N327A and N327C, respectively (Table 1). This is apparently the reason for the lack of detectable transport currents by these mutants in the oocyte system because in this assay, 5% of wild type activity can only be detected in the best expressing batches of oocytes. The size of these currents reflects Vmax. In contrast to the transport currents in which unlabeled GABA is used and its concentration can be increased at will, radioactive uptake is performed at concentrations below Km and therefore depends both on Vmax and on Km. The Km values for GABA were 3.7 ± 1.0 and 0.6 ± 0.1 μm for N327A/C74A and N327C/C74A, respectively, as compared with 5.5 ± 0.5 μm for C74A (n = 3) (Table 1). In both mutants, transport of [3H]GABA was stimulated by lithium (Fig. 5, A and B). Similar results were also obtained with the more conserved mutants N327D and N327Q (data not shown). Transport by N327C was not inhibited by preincubation of the HeLa cells with MTSET or MTSES (data not shown). Regarding the Km values for GABA, it is important to note that they can only be compared at similar sodium concentrations and membrane potentials. Lowering the sodium concentration results in an increased Km (22Radian R. Kanner B.I. Biochemistry. 1983; 22: 1236-1241Crossref PubMed Scopus (102) Google Scholar), and at less negative membrane potentials, the values for Km decrease (7Mager S. Naeve J. Quick M. Labarca C. Davidson N. Lester H.A. Neuron. 1993; 10: 177-188Abstract Full Text PDF PubMed Scopus (277) Google Scholar). These facts explain the relatively low Km value of ∼5 μm for radioactive uptake, measured at 150 mm sodium in HeLa cells expressing wild type GAT-1. The membrane potential in these cells infected with the recombinant Vaccinia virus is presumably not very negative. The Km in oocytes, expressing wild type GAT-1, at −140 mV and 96 mm sodium is ∼55 μm (Table 1).FIGURE 5Sodium dependence of [3H]GABA transport by asparagine 327 mutants. HeLa cells expressing N327A/C74A (A) or N327C/C74A (B) were assayed for [3H]GABA transport as described under “Experimental Procedures.” Transport was carried out for 3 min at the indicated sodium concentrations with either choline (•) or lithium (▴) as the substituting ion. Data shown at the indicated sodium concentrations are normalized to those at 150 mm Na+ (no choline or lithium substitution). Values are averages (± S.E.) of at least three experiments each performed in triplicate. [3H]GABA uptake at 150 mm Na+ by N327A/C74A and N327C/C74A was 5.61 ± 0.46% and 13.53 ± 0.84%, respectively, of that by C74A, (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the case of serine 295 mutants, transport currents induced by GABA could be measured with S295T/C74A and S295A/C74A (Fig. 6) but not with the cysteine replacement mutant (data not shown). The voltage dependence of the" @default.
• W1995875445 created "2016-06-24" @default.
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• W1995875445 creator A5043629759 @default.
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• W1995875445 date "2006-08-01" @default.
• W1995875445 modified "2023-09-30" @default.
• W1995875445 title "Identification of a Lithium Interaction Site in the γ-Aminobutyric Acid (GABA) Transporter GAT-1" @default.
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