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• W2079382471 abstract "The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na+/Cl−-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na+ coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). InXenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na+-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ ↔ CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na+, Cl−, and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation. The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na+/Cl−-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na+ coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). InXenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na+-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ ↔ CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na+, Cl−, and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation. extracellular loop 1 methanethiosulfonate (2-sulfonatoethyl) methanethiosulfonate [2-(trimethylammonium)-ethyl]-methanethiosulfonate dithioerythritol Na+/Cl−-coupled transporters control the extracellular and intracellular concentrations of neurotransmitters like bioamines, γ-aminobutyric acid, and glycine. Through the thermodynamically coupled uptake of Na+/Cl−, and the transmitter, transporters help terminate synaptic transmission, keeping a low extracellular concentration of neurotransmitter, and limiting the spill-over of neurotransmitter between neighboring synapses. At some presynaptic boutons, their concentrative power may also be required for an efficient supply of neurotransmitter to the low affinity vesicular transporters. Within this superfamily, the two glycine transporters (GlyT1 and GlyT2) have distinctive properties. A well established difference concerns their substrate specificity, because sarcosine is a substrate of GlyT1b (1Supplisson S. Bergman C. J. Neurosci. 1997; 17: 4580-4590Crossref PubMed Google Scholar) but not of GlyT2a (2López-Corcuera B. Martinez-Maza R. Núñez E. Roux M. Supplisson S. Aragón C. J. Neurochem. 1998; 71: 2211-2219Crossref PubMed Scopus (85) Google Scholar). Recently, other pharmacological differences have been identified; the tricyclic antidepressant amoxapine and ethanol both selectively inhibit GlyT2a (3Núñez E. López-Corcuera B. Vazquez J. Gimenez C. Aragón C. Br. J. Pharmacol. 2000; 129: 200-206Crossref PubMed Scopus (46) Google Scholar, 4Núñez E. López-Corcuera B. Martinez-Maza R. Aragón C. Br. J. Pharmacol. 2000; 129: 802-810Crossref PubMed Scopus (23) Google Scholar). Electrophysiological characterization of the two transporters has revealed additional features differentiating GlyT2a from GlyT1b: (i) a stoichiometry of 3 Na+/1 Cl−/glycine compared with only 2 Na+/1 Cl−/glycine for GlyT1b (resulting in a charge to glycine flux ratio of ∼2 for GlyT2a versus 1 for GlyT1b) (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar); (ii) a reduced capacity for reverse uptake (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar); (iii) an uncoupled, outwardly rectifying Cl−conductance; and (iv) a biexponential transient current recorded in the absence of glycine. 1M. J. Roux and S. Supplisson, unpublished observations.1M. J. Roux and S. Supplisson, unpublished observations.The amino acids involved in these differences between GlyT1b and GlyT2a have not been identified yet. Sequence comparison of EL12in the Na+/Cl−-coupled transporter superfamily shows that GlyT2a together with PROT (6Fremeau Jr., R.T. Caron M.G. Blakely R.D. Neuron. 1992; 8: 915-926Abstract Full Text PDF PubMed Scopus (203) Google Scholar) and ATB0,+ (7Sloan J.L. Mager S. J. Biol. Chem. 1999; 274: 23740-23745Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) are the only members that lack a conserved cysteine in EL1 that has been identified in GAT-1 (Cys74; Ref. 8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar) and SERT (Cys109; Ref.9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar) as a primary target for the membrane-impermeant sulfhydryl reagent MTSET. Mutation in GlyT2a EL1 induced an increase in the outwardly rectifying leak current characteristic of this transporter,1 which further suggested that the conformation of this loop may differ between GlyT1b and GlyT2a. To assess this issue, we tested the reactivity to sulfhydryl reagents of GlyT1b and GlyT2a. Transporter inhibition by MTSET required prolonged application and high concentrations of this reagent, which suggests a low accessibility of the reactive cysteine either because it is partially buried inside the protein or because it is infrequently exposed to the surface (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar). In fact, the orientation of EL1 has been a matter of debate (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar, 10Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Olivares L. Aragón C. Giménez C. Zafra F. J. Biol. Chem. 1997; 272: 1211-1217Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The original topological model proposed for the γ-aminobutyric acid transporter GAT-1 (13Guastella 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 (718) Google Scholar) predicted cytoplasmic N and C termini and 12 transmembrane segments linked by six extracellular loops and five intracellular loops. In accordance to this model, most positions in EL1 were found to be accessible to extracellularly applied MTSET when mutated to cysteine in GAT-1 (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar). In addition, MTSET dramatically increased the oocyte leak current (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar). However, inhibition by MTSET was not detected in uptake experiments in GAT-1 transfected mammalian cells (10Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas the inactivation produced by the membrane permeant (2-aminoethyl)methane thiosulfonate was attributed to cysteine 399 located in intracellular loop 4 (14Golovanevsky V. Kanner B.I. J. Biol. Chem. 1999; 274: 23020-23026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We characterized under voltage clamp the effects of MTSET and (2-sulfonatoethyl) methanethiosulfonate (MTSES) on the glycine uptake, the glycine-evoked current, and the charge movement of the two wild type glycine transporters, as well as on those of two point mutants of the first amino acid of EL1, GlyT1b-C62A and GlyT2a-A223C, which exchanged the MTSET reactivity. The difference in residue number for an equivalent position in EL1 sequence is explained by the much longer N terminus of GlyT2a (15Liu Q.R. López-Corcuera B. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1993; 268: 22802-22808Abstract Full Text PDF PubMed Google Scholar). In addition, the GlyT2a-EL1 chimera resulting from the mutation of the first three amino acids of GlyT2a-EL1 (AFQ) to the GlyT1b sequence (CYR) was studied in detail because it influenced MTSET reactivity and had a dramatic effect on the outward Cl− current specific of GlyT2a.1 Rat GlyT1b (gift from K. Smith, Synaptic Corporation (16Smith K.E. Borden L.A. Hartig P.R. Branchek T. Weinshank R.L. Neuron. 1992; 8: 927-935Abstract Full Text PDF PubMed Scopus (379) Google Scholar)) and rat GlyT2a (15Liu Q.R. López-Corcuera B. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1993; 268: 22802-22808Abstract Full Text PDF PubMed Google Scholar) cDNAs were subcloned as indicated by Supplisson and Bergman (1Supplisson S. Bergman C. J. Neurosci. 1997; 17: 4580-4590Crossref PubMed Google Scholar). Point mutations were performed using the method of Kunkel (17Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4894) Google Scholar). cRNAs were transcribed in vitro using the T7 mMessage-mMachine kit (Ambion, Austin, TX), and 10–100 ng were injected into oocytes using a nanoliter injector (World Precision Instruments, Sarasota, FL). Xenopus laevis oocytes were prepared and incubated as indicated by Roux and Supplisson (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Two-electrode voltage clamp was performed using a O-725A amplifier (Warner Instrument, Hamden, CT), a Digidata 1200 interface, and the pClamp7 software (Axon Instruments, Foster City, CA). Glass electrodes were filled with a 3 m KCl solution and had a resistance of 0.5–2 MΩ. The typical extracellular recording Ringer solution contained 100 mm NaCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.2 adjusted with KOH. In some experiments, choline and gluconate were substituted for Na+ and Cl−, respectively. Agar bridges (equilibrated in a 3m potassium gluconate solution) were used when required. Salts and glycine were purchased from Sigma. Aliquots of dithioerythritol (300 mm, Sigma), MTSET, and MTSES (100 mm, Toronto Research Chemical Co., Toronto, Canada) were prepared in ice-cold water and kept at −20 °C. They were thawed and diluted in the appropriate solution just prior to perfusion (<10 s). Data are given as the means ± S.E. with the number of experiments. Transient currents specific to GlyT1b or GlyT2a were evoked in the absence of glycine by stepping the voltage at various potentials. These currents are Na+-dependent and capacitative in nature because their time integrals estimated following the onset and return from the voltage step are equal in amplitude but opposite in sign. For GlyT1b, the charge movement was fitted with a single exponential function of the current decay upon repolarization. For GlyT2a and GlyT2a-EL1, the charge movement could not be fitted with a single exponential and was quantified by integration of the transient currents recorded during the membrane repolarization to the holding potential (off current) after subtraction of the current recorded in absence of Na+. The charge distribution as a function of membrane potential was fitted with a Boltzmann distribution as shown Equation 1. Q(Vm)=Qmax1+expzδ(V½−Vm)FRT+CteEquation 1 where V 12is the half-distribution potential, zδ is the equivalent charge per transporter moving through the membrane electric field, and R,T, and F have their usual meanings.Qmax is the total charge movement and is defined as Qmax = zδ F NT, where NT is the number of transporters/oocyte. Oocytes (n = 5–15) expressing wild type glycine transporters or various EL1 constructs were incubated for 10 min in 5 ml of choline chloride Ringer solution in the presence or the absence of 2 mm MTSET. Then oocytes were incubated for 10 min in control NaCl Ringer solution or in choline chloride Ringer solution containing 5 μm[U-14C]glycine (specific activity, 103 μCi/μmol; Amersham Pharmacia Biotech). Oocytes were washed three times in ice-cold NaCl Ringer solution and placed individually in vials containing 500 μl of SDS (2%). The radioactivity was measured on a Kontron liquid scintillation counter. Oocytes were voltage clamped at −40 mV and perfused for 60–90 s with a solution containing 25 μm[U-14C] glycine and 75 μm cold glycine. After complete wash-out and after the current returned to the base-line level, oocytes were transferred in 5 ml of ice-cold Ringer solution, washed twice, then lysed in vials containing 500 μl of SDS (2%), and measured for radioactivity. The charge coupling was determined as the ratio between the glycine-evoked current integral and the glycine uptake as described by Roux and Supplisson (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The glycine transporters GlyT1b and GlyT2a, expressed inXenopus oocytes, showed differential sensitivities to MTSET. The glycine-evoked current (IT) (Fig.1 a) and the transient currents (Fig. 1 b) recorded in the absence of glycine in GlyT1b+ oocytes were strongly and irreversibly reduced following a 5-min superfusion with choline chloride Ringer solution containing 1 mm MTSET. In contrast, similar applications of MTSET affected neither IT (Fig. 1 c) nor the transient currents nor the outward rectifying leak conductance (Fig. 1 d) recorded in GlyT2a+ oocytes. To test whether these differences could result from the presence (GlyT1b) or the absence (GlyT2a) of a cysteine in the first extracellular loop (EL1), as suggested by results obtained on GAT-1 (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar) and SERT (9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar), we applied MTSET both on the point mutant GlyT2a-A223C (Fig. 1,e and f) and on the GlyT2a-EL1 chimera (Fig. 1,g and h) in which the AFQ sequence of GlyT2a was replaced by the GlyT1b sequence CYR (TableI for sequence comparison). With GlyT2a-A223C and GlyT2a-EL1, application of MTSET reducedIT (Fig. 1, e and g), the transient currents, and the outwardly rectifying leak conductance (Fig.1, f and h). However, the inhibition of GlyT2a-EL1 was partial, as for GlyT1b, whereas GlyT2a-A223C was fully inhibited under the same conditions.Table ISequence alignment of the TM1–TM2 region of wild type and mutant glycine transporters.All amino acids in the variable region of EL1 (box) are shown. The putative TM regions are shown in gray. Open table in a new tab All amino acids in the variable region of EL1 (box) are shown. The putative TM regions are shown in gray. MTSET did not change the leak current in oocytes expressing either GlyT1b (Fig. 1 b) or GlyT2a (Fig. 1 d), but it affected the leak current of GlyT2a-A223C and GlyT2a-EL1. Like GlyT2a, both of these constructs exhibit an outwardly rectifying “leak” conductance at depolarized potentials that is particularly large in the case of GlyT2a-EL1. Although this outward leak conductance was not changed by MTSET in GlyT2a (Fig.1 d), it was suppressed in both GlyT2a-A223C and GlyT2a-EL1 (Fig. 1, f and h). In contrast, we observed after MTSET treatment a limited increase in the inward steady state leak current at hyperpolarized potential for these two constructs (Fig. 1,f and h). A 5-min application of MTSET reduced the charge movement of GlyT1b (Fig. 2 a), GlyT2a-EL1 (Fig. 2 e), and GlyT2a-A223C (Fig. 2 g) but not that of GlyT2a (Fig. 2 c). The reduction in Qmax (Table II), which is proportional to the number of functional transporters present in the plasma membrane (see “Experimental Procedures”), indicates that the loss of transport activity observed after cysteine modification by MTSET results from the hindrance of a kinetic step, which precedes substrate binding and translocation because the charge movement of glycine transporters corresponds to Na+- and voltage-dependent transitions that occur in the absence of glycine (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The Na+ dependence of the transient currents for oocytes expressing GlyT1b, GlyT2a, GlyT2a-El1, and GlyT2a-A223C is shown in Fig. 3 with the unsubstracted off currents recorded during the repolarization step to the holding potential in NaCl Ringer solution, in choline chloride Ringer solution, and after MTSET application.Table IIPercentage of reduction of Qmax and Imaxinduced by 1 mM MTSET for 5 min in oocytes expressing wild type and EL1 mutant of glycine transporterGlyT1bGlyT2GlyT2a-EL1GlyT2a-A223CCharge movementV½(mV)−19.7 ± 5.2−35.7 ± 1.5−41.3 ± 0.8−22.5 ± 1.4zδ0.55 ± 0.041.48 ± 0.021.46 ± 0.081.6 ± 0.0Percentage off reduction of Omax by MTSET64.0 ± 2.99.0 ± 1.060.6 ± 2.498.0 ± 0.7Uptake currentPercentage off reduction of Imax by MTSET70.8 ± 2.08.5 ± 1.267.7 ± 1.097.6 ± 0.1n4444 Open table in a new tab Figure 3Na+-dependent charge movement. Unsubtracted off currents for GlyT1b, GlyT2a, GlyT2a-EL1, and GlyT2a-A223C recorded during the repolarization step to the holding potential (−40 mV) from +50 to −130 mV, in the presence of NaCl and choline chloride and after exposure to MTSET (1 mm).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the potential range from −140 to +50 mV, theIT-voltage relationships of GlyT1b, GlyT2a, and GlyT2a-A223C (Fig. 2, b, d, and g) were quasi-linear, whereas that of GlyT2a-EL1 was inwardly rectifying (Fig. 2 f). For comparison, IT values were normalized to the Qmax determined before MTSET treatment. The transport currents were reduced in the same proportion at all potentials by MTSET for GlyT1b, GlyT2a-EL1, and GlyT2a-A223C transporters, as shown in Fig. 2 (b, f, andh). For each oocyte, the percentage of the remaining transport current was subsequently calculated as the mean of the experimental values between −140 and 0 mV (every 10 mV). Values obtained at voltages above 0 mV were ignored because of their small amplitudes (especially for GlyT2a-EL1). The reduction inIT after MTSET treatment was comparable with the reduction observed in Qmax as indicated in Table II. As expected for an inactivation process, the glycine EC50was not changed by MTSET application for the two transporters tested, GlyT1b (22.5 ± 1.10 μm (n = 7) and 19.5 ± 1.4 μm (n = 3) before and after MTSET application, respectively) and GlyT2a-EL1 (24.1 ± 3.1 μm (n = 3) and 23.2 ± 1.8 μm (n = 3) before and after MTSET application, respectively). To compare the accessibility of the EL1 cysteine for the various sensitive constructs, we studied the time course of the MTSET inhibition. The progressive decrease in uptake current plotted as a function of the cumulative exposure to MTSET is shown in Fig.4. The decay in current had time constants of 3.8 and 3.9 min for GlyT1b and GlyT2a-EL1, respectively, but was 5-fold faster with GlyT2a-A223C, with a time constant of 0.67 min. The difference in the time constants indicates that in glycine transporters, the accessibility of the EL1 cysteine to the extracellular solution and its reactivity to MTSET are strongly influenced by the next two residues that are modified in GlyT2a (FQversus YR in GlyT1b). To confirm that EL1 was fully responsible for GlyT1b sensitivity to MTSET, we constructed the reverse chimera GlyT1b-EL1. For this transporter, no change in the amplitude of glycine-evoked current was observed for period of incubation up to 20 min (Fig. 4). To verify that the reduction of the glycine-evoked current by MTSET was reflecting a comparable change in the influx rate of glycine in oocytes expressing sensitive glycine transporters, we repeated the MTSET inactivation protocol using tracer flux of glycine. A summary of these uptake experiments is shown in Fig.5 A that confirms the large reduction of the Na+-dependent glycine uptake with oocytes expressing GlyT1b, GlyT2a-EL1, and GlyT2a-A223C. No significant inhibition by MTSET is observed with GlyT2a, GlyT1b-EL1, and the point mutant GlyT1b-C62A. Finally, we checked the charge coupling of each transporter by applying radiolabeled glycine under voltage clamp conditions as described by Roux and Supplisson (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The results shown in Fig. 5 Bindicate that the specific charge coupling of the wild type transporters (1 and ∼2 for GlyT1b and GlyT2a, respectively) are not affected by EL1 sequence modification and that glycine-evoked current is proportional to glycine uptake. MTSET applications (1 mm for 5 min at −40 mV) were performed on GlyT1b, GlyT2a-EL1, and GlyT2-A223C in the presence or the absence of the transporter substrates to see whether these substrates protected against MTSET. As described for Fig.6, inhibition by MTSET was reduced by the joint presence of Na+ and Cl− and was further reduced by glycine. Sarcosine, a specific substrate of GlyT1b, protected this transporter to a similar extent as glycine but did not protect GlyT2a-EL1. In all transporters tested, Na+, Cl−, or glycine alone did not induce any protection (Fig.6). This is in good agreement with the fact that neither the uptake current nor the charge movement can be observed when one of the co-transported ions is absent (Ref. 5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar).1 The best protection was observed for GlyT2a-EL1, for which a quasi-complete protection by NaCl and glycine was found. GlyT1b oocytes were perfused with 1 mm MTSET for 5 min in choline chloride Ringer solution at holding potentials between −120 and 0 mV. Inhibition increased with hyperpolarization, from 62.3 ± 5.2% (n = 4) at 0 mV to 84.4 ± 0.9% (n = 4) at −120 mV (Fig.7 A). To test whether this voltage dependence was due to a voltage-dependent transition of the transporter in the absence of Na+ or to the positive charge of MTSET, we repeated the experiments using the negatively charged MTSES. Fig. 7 A shows that MTSES inhibition of GlyT1b had the same sign of voltage dependence as MTSET, with the inhibition increasing from 51.5 ± 1.3% at 0 mV to 79.0 ± 0.9% at −120 mV. The inhibition of GlyT2a-EL1 by MTSET or MTSES was not voltage-dependent (Fig. 7 B). In addition, the potency of MTSES on GlyT2a-EL1 and GlyT2a-A223C (Fig. 7 C) was much lower than that of MTSET, as expected from their reported relative reactivity (18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). This contrasts with the fact that the two reagents inhibited GlyT1b with comparable potencies (Fig.7 A). Covalent modification of cysteine by MTSET produced an inactivation of sensitive glycine transporters that each contain a cysteine in their EL1 sequence. This inactivation persisted after complete wash-out of MTSET (Fig.8 B). We tested whether the application of a reducing agent like DTE may reverse the alkylation by MTS reagents, as reported for the glucose transporter SGLT1 (19Loo D.D. Hirayama B.A. Gallardo E.M. Lam J.T. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (140) Google Scholar). The data shown in Fig. 8 indicate that superfusion with DTE (5 min, 10 mm in choline chloride Ringer solution) restored partially or totally the glycine-evoked current, with a more dramatic effect with GlyT2a-A223C. In addition, the small increase in leak current observed with GlyT2a-EL1 (Fig. 1 h) and GlyT2a-A223C (Figs.1 f and 8, A and B) was reversed by DTE. The topological organization of the Na+/Cl− neurotransmitter transporter family has now received good experimental support in favor of the original model of 12 putative transmembrane segments proposed for GAT-1 (13Guastella 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 (718) Google Scholar). Nevertheless the precise organization of the first third of the protein, which contains amino acids critical for function, is still debated (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar, 9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar, 10Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Olivares L. Aragón C. Giménez C. Zafra F. J. Biol. Chem. 1997; 272: 1211-1217Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Golovanevsky V. Kanner B.I. J. Biol. Chem. 1999; 274: 23020-23026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Clark J.A. J. Biol. Chem. 1997; 272: 14695-14704Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 21Chen J.G. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1044-1049Crossref PubMed Scopus (115) Google Scholar). Recent insight into the localization of the loops and the accessibility of membrane segments has been obtained (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar, 9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar, 12Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Golovanevsky V. Kanner B.I. J. Biol. Chem. 1999; 274: 23020-23026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) through the identification of solvent-accessible residues using the substituted cysteine accessibility method (for review, see Ref. 18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). This method has been extensively applied to determine the residues lining the pore of ionic channels and receptors (22Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 23Kuner T. Wollmuth L.P. Karlin A. Seeburg P.H. Sakmann B. Neuron. 1996; 17: 343-352Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 24Sun Z.P. Akabas M.H. Goulding E.H. Karlin A. Siegelbaum S.A. Neuron. 1996; 16: 141-149Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 25Akabas M.H. Cheung M. Guinamard R. J. Bioenerg. Biomembr. 1997; 29: 453-463Crossref PubMed Scopus (13) Google Scholar) and to map the ligand-binding crevice in G-protein-coupled receptors (26Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (167) Google Scholar). Glycine transporters are good candidates for the substituted cysteine accessibility method because one of them (GlyT2a) is totally insensitive to MTS reagents in uptake assays and voltage clamp studies, thus allowing further structure/function characterization without the need for a cysteine-less mutant (18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). Sequence comparison reveals that GlyT2a lacks the well conserved cysteine in EL1 that is present in GlyT1b and to which MTSET sensitivity was attributed in GAT-1 (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar) and SERT (9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar). This cysteine substitution (A223C) is the first of three consecutive differences between GlyT1b (CYR) and GlyT2a (AFQ) within a stretch of 25 otherwise conserved amino acids in the transmembrane segment 1 and 2 region (Table I), which is known to be critical for function (27Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, 28Pantanowitz S. Bendahan A. Kanner B.I. J. Biol. Chem. 1993; 268: 3222-3225Abstract Full Text PDF PubMed Google Scholar, 29Mager S. Kleinberger-Doron N. Keshet G.I. Davidson N. Kanner B.I. Lester H.A. J. Neurosci. 1996; 16: 5405-5414Crossref PubMed Google Scholar, 30Barker E.L. Moore K.R. Rakhshan F. Blakely R.D. J. Neurosci. 1999; 19: 4705-4717Crossref PubMed Google Scholar). We decided to probe EL1 accessibility and function in glycine transporters combining glycine uptake assay and transport current recordings. We have shown previously that charge and glycine uptake are precisely correlated in oocytes expressing GlyT1b (+1 elementary charge (e)/glycine) and GlyT2a (+∼2 e/glycine) (5Roux M.J. Supplisson S. Neuron. 2000; 25: 373-383Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and that the two transporters differ notably in their pre-steady state kinetics and leak current. The interest of a dual approach was reinforced by the fact that conflicting results have been reported on GAT-1 concerning MTSET accessibility (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar, 10Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 14Golovanevsky V. Kanner B.I. J. Biol. Chem. 1999; 274: 23020-23026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We report here that the first amino acid of EL1 (Cys62) in GlyT1b, which is also found in GAT-1 (Cys74), SERT (Cys109), and DAT (Cys90), is accessible to extracellularly applied MTSET and MTSES. All transporter-associated currents (pre-steady state and uptake) were blocked similarly after cysteine modification by MTS reagents. In contrast, we found that none of the electrophysiological properties (glycine-evoked current, charge movement, and leak current) of the wild type GlyT2a were affected by MTSET. Introduction of a cysteine (A223C), either by point mutation or by replacing EL1 with the GlyT1b sequence, conferred MTS reagent sensitivity. Previous results on the cysteines of Na+/Cl−-coupled transporters involved high concentrations of MTS reagents (≥1 mm) and prolonged applications (5–10 min in most cases) to achieve only partial inhibition (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar, 9Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (149) Google Scholar, 10Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Golovanevsky V. Kanner B.I. J. Biol. Chem. 1999; 274: 23020-23026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Similarly, the reduction of uptake current of the wild type GlyT1b was only partial after a 5-min exposure to MTSET (1 mm). This partial inhibition resulted from a loss of function of the fraction of transporters that had a modified cysteine. Complete inhibition of GlyT1b and GlyT2a-EL1 required ≥20 min of incubation with MTSET, whereas GlyT2a-A223C was fully inhibited in about 4 min. Rate constants estimated from the time constants of the inhibition (18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar) were in the order of 4.4 m−1s−1 for GlyT1b and GlyT2a-EL1 and of 25m−1 s−1 for GlyT2a-A223C. These rate constants are many orders of magnitude slower than for the reaction of MTSET with 2-mercaptoethanol in solution (18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar) or for exposed cysteines near the entrance of the pore in acetylcholine receptor (31Pascual J.M. Karlin A. J. Gen. Physiol. 1998; 111: 717-739Crossref PubMed Scopus (104) Google Scholar). Nevertheless, these rate constants are in the range reported for cysteines located near the cytoplasmic end of the acetylcholine receptor pore in the absence of ligand (31Pascual J.M. Karlin A. J. Gen. Physiol. 1998; 111: 717-739Crossref PubMed Scopus (104) Google Scholar). This indicates that even in the more reactive GlyT2a-A223C the EL1 cysteine is exposed only transiently to the extracellular solution or is located in a diffusion-limiting crevice. The rate of inactivation was influenced by the two other nonconserved residues of EL1. Differences in reactivity may be attributed to the presence (GlyT1b, GlyT2a-EL1) or the absence (GlyT2a-A223C) of the positive charge of an arginine in position n + 2. GAT-1, which has a lysine in the equivalent position, is inhibited by MTSET with a time course (8Yu N. Cao Y. Mager S. Lester H.A. FEBS Lett. 1998; 426: 174-178Crossref PubMed Scopus (30) Google Scholar) comparable with GlyT1b and GlyT2a-EL1. This positive charge may also be responsible for the similar rate of inhibition by MTSET and MTSES on GlyT1b, which contrasts with the >10-fold difference in reactivity in solution between these two compounds (18Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). Comparable changes in reactivity ratio have been observed in the case of the CFTR channel (32Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar), for which residues located deep inside the pore (Thr351 and Gln353) show a higher reactivity for MTSES than MTSET. In this channel, the arginine at position 352 is considered to be a key residue for anion selectivity (32Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar). In the wild type GlyT1b, Arg64 may facilitate the access to Cys62 for the negatively charged but less reactive MTSES. The orientation of the arginine, in addition to its charge, plays a role in controlling the access to EL1 cysteine, because GlyT2a-EL1 was much less sensitive to MTSES than to MTSET (by a factor of ∼5, as estimated from the inhibition after 5 min). In addition, position 225 of GlyT2a (an arginine in GlyT2a-EL1) is not accessible to extracellularly applied MTSET. 3B. López-Corcuera, R. Martı́nez-Maza, E. Núñez, A. Geerlings, and C. Aragón, submitted for publication. Together, these results suggest that EL1 has a different conformation in GlyT1b and GlyT2a. Another difference between the two transporters was that membrane potential influenced the MTS inhibition of GlyT1b but not of GlyT2a-EL1 in the absence of external Na+. Because the voltage dependence of MTSET and MTSES inhibition of GlyT1b were similar, a role of the reagent charge could be ruled out. Some voltage-dependent transition in the transporter must favor EL1 cysteine exposure at negative potentials. This transition is unlikely to correspond to extracellular Cl− binding to the transporter, because similar inhibition levels were achieved in choline chloride and choline-gluconate solutions at −40 mV. Although glycine transporters capacitative currents are Na+-dependent as are those of other Na+-coupled transporters, around 20% of GlyT1b charge movement persists after Na+ removal (choline substitution) from the extracellular solution.1 Such residual charge movement is not observed in GlyT2a or GlyT2a-EL1, consistent with the fact that no voltage dependence was observed for GlyT2a-EL1 inhibition by MTSET or MTSES. GlyT1b probably has either endogenous charges or ions bound to intracellular sites and can undergo voltage-dependent transitions even in the absence of ions bound to the extracellular sites. The presence of both Na+ and Cl− in the extracellular medium, which are required for the charge movement observed in response to voltage jumps in the absence of glycine, partially protected the three MTSET-sensitive transporters GlyT1b, GlyT2a-A223C, and GlyT2a-EL1. Na+ or Cl− alone (choline or gluconate substitution) did not protect, suggesting a strong allosteric interaction between the Na+- and Cl−-binding sites. The addition of glycine to Na+ and Cl− further protected both GlyT1b and GlyT2a-EL1. Altogether, these results indicate that the conformational changes occurring in the transport cycle modify the EL1 cysteine accessibility. In addition, the conformation of this loop appears to differ between GlyT1b and GlyT2a, maybe as a consequence of the structural constraints imposed by the extra Na+-binding site of GlyT2a. We thank Philippe Ascher and Boris Barbour for critical comments and suggestions on the manuscript." @default.
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• W2079382471 title "The Glial and the Neuronal Glycine Transporters Differ in Their Reactivity to Sulfhydryl Reagents" @default.
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