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• W2016994955 abstract "Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. After cotransport of glutamic acid with Na+, the cycle is completed by countertransport of K+. We have identified an amino acid residue (glutamate 404) influencing ion coupling in a domain of the transporter implicated previously in kainate binding. Mutation of this residue to aspartate (E404D) prevents both forward and reverse transport induced by K+. Sodium-dependent transmitter exchange and a transporter-mediated chloride conductance are unaffected by the mutation, indicating that this residue selectively influences potassium flux coupling. The results support a kinetic model in which sodium and potassium are translocated in distinct steps and suggest that this highly conserved region of the transporter is intimately associated with the ion permeation pathway. Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. After cotransport of glutamic acid with Na+, the cycle is completed by countertransport of K+. We have identified an amino acid residue (glutamate 404) influencing ion coupling in a domain of the transporter implicated previously in kainate binding. Mutation of this residue to aspartate (E404D) prevents both forward and reverse transport induced by K+. Sodium-dependent transmitter exchange and a transporter-mediated chloride conductance are unaffected by the mutation, indicating that this residue selectively influences potassium flux coupling. The results support a kinetic model in which sodium and potassium are translocated in distinct steps and suggest that this highly conserved region of the transporter is intimately associated with the ion permeation pathway. The role of electrogenic (Na++ K+)-coupled glutamate transporters, located in the plasma membranes of nerve terminals and glial cells is to keep the extracellular concentration of the neurotransmitter below neurotoxic levels (1Kanner B.I. Schuldiner S. CRC Crit. Rev. Biochem. 1987; 22: 1-8Crossref PubMed Scopus (404) Google Scholar, 2Nicholls D.G. Attwell D. Trends Pharmacol. Sci. 1990; 11: 462-468Abstract Full Text PDF PubMed Scopus (974) Google Scholar). Furthermore, together with diffusion, they may help to terminate its action in synaptic transmission (3Mennerick S. Zorumski C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (291) Google Scholar, 4Tong G. Jahr C.E. Neuron. 1994; 13: 1195-1203Abstract Full Text PDF PubMed Scopus (306) Google Scholar). A glutamate transporter (GLT-1) has been purified to near homogeneity and reconstituted (5Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (186) Google Scholar, 6Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (373) Google Scholar). It represents around 0.6% of the protein of crude synaptosomal fractions and is one (7Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1140) Google Scholar) of four glutamate transporters expressed in brain cloned thus far (8Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1101) Google Scholar, 9Kanai Y. Hediger M.A. Nature. 1992; 360: 464-471Crossref PubMed Scopus (1200) Google Scholar, 10Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1018) Google Scholar, 11Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar). The stoichiometry of the electrogenic transport process (11Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar, 12Brew H. Attwell D. Nature. 1987; 327: 707-709Crossref PubMed Scopus (328) Google Scholar, 13Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (351) Google Scholar) is three sodium ions per glutamate (14Zerangue N. Kavanaugh M.P. Nature. 1996; 382: 634-637Crossref Scopus (711) Google Scholar), whereas potassium is transported in the opposite direction (11Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar). In addition, glutamate transport is accompanied by alkalinization of the external medium (15Bouvier M. Szatkowski M. Amato A. Attwell D. Nature. 1992; 360: 471-473Crossref PubMed Scopus (324) Google Scholar), and transport of glutamate can be driven by a pH gradient (16Nelson P.J. Dean G.E. Aronson P.S. Rudnick G. Biochemistry. 1983; 22: 5459-5463Crossref PubMed Scopus (50) Google Scholar). It is not clear whether a proton is cotransported with glutamate or an hydroxyl is antiported (17Billups B. Rossi D. Attwell D. J. Neurosci. 1996; 16: 6722-6731Crossref PubMed Google Scholar), but recent evidence favors cotransport with a proton (14Zerangue N. Kavanaugh M.P. Nature. 1996; 382: 634-637Crossref Scopus (711) Google Scholar). Mechanistic studies have shown that the transport process is ordered and that sodium and potassium are translocated in distinct steps. The sodium ions bind first, followed by glutamate. After their translocation and release on the inside, potassium binds on the inside and is translocated outwards to complete the translocation cycle (18Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (167) Google Scholar, 19Pines G. Kanner B.I. Biochemistry. 1990; 29: 11209-11214Crossref PubMed Scopus (91) Google Scholar). Because all substrates are charged molecules, conserved charged amino acids located in hydrophobic stretches of the transporter proteins are likely to be important for the binding and translocation of these substrates. Using site-directed mutagenesis, we have identified four amino acid residues (GLT-1 numbering) important for the transport process: histidine 326 (20Zhang Y. Pines G. Kanner B.I. J. Biol. Chem. 1994; 269: 19573-19577Abstract Full Text PDF PubMed Google Scholar) and the acidic amino acid residues aspartate 398, glutamate 404, and aspartate 470 (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In this report, we describe the in-depth characterization of a mutant in which glutamate 404 has been changed into aspartate. This mutant, E404D, appears to be able to catalyze the exchange of glutamate and aspartate but unable to carry out net flux of the acidic amino acids. We show that glutamate 404 is important for carrying out the potassium transporting limb of the cycle, suggesting that this residue may represent part of the potassium binding site. Capped mRNAs transcribed from the cDNAs encoding the rat brain glutamate transporter GLT-1 (7Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1140) Google Scholar) or its mutant E404D (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) were microinjected into state V-VI Xenopus oocytes (50 ng/oocyte), and membrane currents were recorded 3-6 days later (13Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (351) Google Scholar). Recording solution (Ringer) contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.4. In Na+ or Cl− substitution experiments, ions were replaced with equimolar choline or gluconate, respectively. Two microelectrode voltage-clamp recordings were performed at 22°C with a Geneclamp 500 interfaced to an IBM-compatible PC using a Digidata 1200 A/D control using the pCLAMP 6.0 program suite (Axon Instruments) and to a Macintosh using a MacLab A/D (ADInstruments). The currents were low pass-filtered between 10 Hz and 1 kHz and digitized between 20 Hz and 5 kHz. Microelectrodes were filled with 3 M KCl solution and had resistances of <1 megaohms. Offset voltages in Cl− substitution experiments were avoided by the use of a 3 M KCl-agar bridge from the recording chamber to a 3 M KCl reservoir containing an Ag/AgCl electrode. Current-voltage relations were determined either by measurement of steady-state currents in response to bath application of substrates or by off-line subtraction of control current records obtained during 200-ms voltage pulses to potentials between −100 and +40 mV from corresponding current records in the presence of substrate. Current measurements were made during superfusion of 100 μM [3H]D-aspartate (0.42 Ci/mmol; Amersham Corp.) onto oocytes voltage-clamped at various potentials for 100 s. Following washout of the bath (<20 s), oocytes were rapidly transferred into a scintillation tube, lysed, and measured for radioactivity. In control experiments, no significant efflux of radiolabel was detected during this time in oocytes injected with 100 pmol of [3H]D-aspartate (final concentration, 100 μM). Currents induced by 3H-labeled amino acids were recorded using Chart software (ADInstruments) and integrated off-line, followed by correlation of charge transfer with radiolabel flux in the same oocytes. HeLa cells were cultured in DMEM supplemented with 10% fetal calf serum (heat-inactivated), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. Infection with recombinant vaccinia/T7 virus vTF7-3 and subsequent transfection with plasmid DNA was done as described (22Keynan S. Suh Y.-J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar) using pT7-GLT-1 (shortened insert; 23Casado M. Bendahan A. Zafra F. Danbolt N.C. Aragon C. Gimenez C. Kanner B.I. J. Biol. Chem. 1993; 268: 27313-27317Abstract Full Text PDF PubMed Google Scholar), pT7-GLT-1 E404D (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), or vector alone (pBluescript SK−) at 2.8 μg/well (3-cm diameter). Cells from two wells were washed twice with 1 ml PBS and then taken up in 20 μl of PBS. The cell suspension was mixed with liposomes (4 μmol of asolectin and 0.7 μmol of brain phospholipids) and 0.9% cholate in a final volume of 220 μl of a solution of identical composition as the one used to equilibrate the spin columns, used for the subsequent reconstitution (24Radian R. Kanner B.I. J. Biol. Chem. 1985; 260: 11859-11865Abstract Full Text PDF PubMed Google Scholar). The columns were equilibrated with 5 mM Tris-SO4, pH 7.4, 1 mM MgSO4, 0.5 mM EDTA, 1% glycerol, and other ions and amino acids corresponding to the desired “in-medium” as detailed in the figure legends. In the case of exchange experiments, acidic amino acid, usually 10 mML-aspartate, was included in the equilibration buffer of the spin-column. The external L-aspartate was removed by passing these proteoliposomes over a spin column of identical composition but lacking the amino acid. The control liposomes (without entrapped L-aspartate) were also passed through a second spin column. Uptake of [3H]D-aspartate was measured as described (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) using 20 μl of proteoliposomes into 360 μl of 0.15 M NaCl and 1 μCi of [3H]D-aspartate (11.5 Ci/mmol) unless indicated otherwise in the figure legends. Uptake in intact cells was performed as described (22Keynan S. Suh Y.-J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar). When expressed in HeLa cells using a vaccinia/T7 recombinant virus, uptake of [3H]D-aspartate mediated by the rat brain glutamate transporter GLT-1 is similar to that in cells expressing a mutant GLT-1 transporter containing the conservative substitution E404→D (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Although similar levels of radiotracer uptake were also seen in voltage-clamped Xenopus oocytes injected with RNA transcribed from the corresponding wild-type and mutant cDNAs, membrane currents recorded simultaneously during radiolabel uptake were dramatically different in the two groups of cells (Fig. 1). While uptake of [3H]D-aspartate in the two groups was similar, the magnitude of currents associated with flux mediated by the mutant transporter was less than 5% of that seen in oocytes expressing the wild-type transporter (Fig. 1). These data show that the mutant E404D transporter mediates uptake of radiolabeled D-aspartate, but they suggest that this uptake is occurring in an essentially electroneutral fashion at −30 mV. Uptake of excitatory amino acids by all glutamate transporter clones thus far characterized results in activation of a current reflecting the sum of the inward current (resulting from cotransport of coupled ions such as sodium) together with a chloride current flowing through a thermodynamically uncoupled conductance pathway (10Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1018) Google Scholar, 25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar). The voltage dependence of the currents mediated by wild-type GLT-1 and the E404D mutant (GLT-1 E404D) has been examined by clamping oocytes, expressing the transporters, at potentials between +40 and −100 mV in the presence and absence of the transport substrate D-aspartate at 100 μM. In the wild-type GLT-1, inward currents are observed (Fig. 2A) that do not reverse (Fig. 2, A and C), similar to those observed in its human homolog (25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar). The behavior of E404D is quite different. In addition to the steady-state current, which is greatly diminished as compared with the wild-type, a relatively slow transient current is observed (Fig. 2B). Neither of these currents are observed in water-injected oocytes (13Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (351) Google Scholar). The very small steady-state current of E404D does, however, reverse at potentials more positive than −20 mV (Fig. 2, B and C). The aspartate-dependent transient currents appear to be capacitative because the charge movements, following hyperpolarizing or depolarizing pulses, are equal to those following the return to the original potential (Fig. 2B). Although we will speculate on the nature of these transient currents in the “Discussion,” it is important at this point to note that their magnitude indicates that the greatly reduced steady-state currents of E404D are not due to corresponding reduced expression levels of the mutant transporter. This is further supported by the fact that the much faster kainate-sensitive, sodium-dependent transients, which reflect sodium binding to the transporter or a subsequent conformational change (13Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (351) Google Scholar), are similar for wild-type and mutant transporters (data not shown). The acidic amino acid-dependent outward current observed in glutamate transporters expressed in oocytes is largely due to a sodium- and glutamate-dependent anion conductance not thermodynamically coupled to the transport cycle (25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar). The outward steady-state current in the E404D transporter (Fig. 2C) is largely carried by chloride ions moving into the oocyte because it is greatly attenuated when the external chloride concentration is reduced from 104 to 4.8 mM (Fig. 2D). Further support for this comes from the observations that much larger outward currents are observed when the external chloride is substituted by the highly permeant NO3− ion (Fig. 3).Fig. 3Anion selectivity of wild-type and E404D GLT-1 transporters. Voltage dependence of steady-state currents induced by application of 300 μMD-aspartate to representative oocytes expressing wild-type GLT-1 (A) or E404D (B) transporters with bath solution containing normal Ringer's solution (Cl−) and with Cl− substituted by I−, NO3−, or SCN−.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The anion conductance activated during uptake shows a selectivity sequence favoring chaotropic anions (25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 26Eliasof S. Jahr C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4153-4158Crossref PubMed Scopus (108) Google Scholar), and this selectivity sequence was unchanged in the E404D mutant (Fig. 3). Raising external potassium induces reverse transport (18Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (167) Google Scholar, 27Szatkowski M. Barbour B. Attwell D. Nature. 1990; 348: 443-446Crossref PubMed Scopus (645) Google Scholar). This phenomenon is observed in Xenopus oocytes, which have approximately 12 mM intracellular substrate concentrations (28Zerangue N. Kavanaugh M.P. J. Physiol. 1996; 493: 419-423Crossref PubMed Scopus (174) Google Scholar). The data presented suggest that the E404D transporter may be impaired in potassium binding. Evidence supporting this is presented in Fig. 4. Oocytes expressing the wild-type and E404D transporter have been clamped at 0 mV in Cl−-free Ringer's solution (substituted with nitrate). In the wild-type, superfusion with 100 μMD-aspartate induces the well characterized anion conductance of the transporter as measured by a large outward current, which is dependent on nitrate (Fig. 4A; this current is 20-fold lower in normal Ringer's solution; see also Fig. 2C). This anion conductance can also be induced by exposing the wild-type transporters to external potassium, which will cause the transporter to catalyze efflux of internal glutamate and/or aspartate (Fig. 4A). In the E404D transporters, this anion conductance can be activated by external aspartate but not by external potassium (Fig. 4B), indicating that potassium cannot interact with them. The data from the previous sections indicate that uptake of [3H]D-aspartate by the E404D transporters is due to its exchange with intracellular acidic amino acids but not to its net influx. To prove this, we have solubilized HeLa cells transiently expressing either wild-type or mutant transporters and reconstituted those transporters into liposomes. This system allows control of the composition of the external as well as the internal medium. Fig. 5A shows that potassium-loaded proteoliposomes inlaid with E404D transporters are virtually unable to accumulate [3H]D-aspartate. The accumulation in the wild-type is completely dependent on the presence of external sodium (data not shown). This accumulation is dependent on internal potassium and is not observed when it is replaced by Tris (Fig. 5B, closed triangles). If, however, 10 mML-aspartate is included in the internal medium together with Tris, significant accumulation of [3H]D-aspartate is observed with wild-type and E404D transporters (Fig. 5B) but not with liposomes inlaid with extracts of cells transfected with the vector alone (data not shown). Under these conditions, the accumulation is absolutely dependent on external sodium; similar results are also obtained when L-glutamate is present on the inside and also when sodium is used instead of Tris (data not shown). These experiments prove that E404D proteoliposomes catalyze exchange but cannot catalyze net influx, which is dependent on internal potassium. The ability of external potassium to interact with the mutant proteoliposomes has been investigated after their preloading with [3H]D-aspartate by exchange. After a 10-min accumulation of [3H]D-aspartate into the proteoliposomes by exchange, they have been diluted 10-fold into media containing sodium chloride and 100 μM of unlabeled L-glutamate or D-aspartate (Fig. 6). The two substrates induce a rapid efflux of the previously accumulated [3H]D-aspartate in both the wild-type (Fig. 6A) and the E404D transporter (Fig. 6B). Whereas dilution of the proteoliposomes inlaid with the wild-type transporter into sodium-containing media without anionic amino acids does not result in efflux, a rapid net efflux is obtained into a potassium-containing medium (Fig. 6A). However, no such net efflux is observed in the case of the mutant (Fig. 6B). Dilution into choline-containing media does not lead to efflux, neither in the wild-type nor in the mutant (data not shown).Fig. 6Efflux of [3H]D-aspartate from wild-type or E404D proteoliposomes it via exchange. For each time point 20 μl of proteoliposomes, containing 0.12 M NaPi and 10 mML-aspartate, were loaded with [3H]D-aspartate by dilution into 80 μl of 0.15 M NaCl and 1 μCi of label. After 10 min, the mixture was diluted into 1 ml of 0.15 M KCl (•) or 0.15 M NaCl containing the following additions: none (○); 100 μMD-aspartate (□); or 100 μML-glutamate (▵). At the times indicated on the abscissa, the reactions were terminated. A, wild type; B, E404D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further emphasize the selectivity of the defect of potassium-dependent net flux in the mutant, we have compared another important parameter, i.e. affinity for external sodium. Initial rates of sodium-dependent [3H]D-aspartate transport in HeLa cells expressing wild-type and mutant have a very similar dependence on the external sodium concentration (Fig. 7). Although in the experiment D-aspartate has been used at concentrations below its Km, similar results are obtained when saturating levels (200 μM) are used (data not shown). An identical sodium dependence of [3H]D-aspartate exchange has been observed in proteoliposomes inlaid with wild-type and E404D transporter (data not shown). Thus, E404D is selectively impaired in one or more of the interactions of potassium with the transporter. The transport cycle of glutamate, as it emerges from mechanistic studies (18Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (167) Google Scholar, 19Pines G. Kanner B.I. Biochemistry. 1990; 29: 11209-11214Crossref PubMed Scopus (91) Google Scholar), is illustrated in Fig. 8. On the outside, the three sodium ions have to bind to the transporter first (step 1). This is followed by the binding of the acidic amino acid (glutamate in this example; step 2), followed by translocation (step 3). Then the acidic amino acid debinds (step 4), followed by the three sodium ions (step 5). This leaves the binding sites facing inward. To start a new cycle, the transporter has to reorient these sites, and for this purpose, potassium is required. After it binds from the inside (step 6), it is translocated (step 7) and released from the outside (step 8). In the case of net efflux, this cycle is reversed, going clockwise, instead of counterclockwise. Exchange represents a partial reaction of this cycle (steps 1-4 or 5 and back), including the reversible translocation of the acidic amino acid. Net flux is dependent on cis-sodium is external sodium is required for influx and internal sodium for efflux. Also exchange requires sodium, but it does not matter on which of the two sides it is present. Although there are some differences in the relative permeabilities of glutamate and aspartate between E404D and wild-type (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the dominant feature is that E404D cannot carry out the whole cycle (Fig. 5A) but can exchange (Figs. 5B and 6). These data are in harmony with the electrophysiological data, which indicate that E404D is “locked” in the exchange mode (Fig. 1). Besides the ability to translocate sodium and the acidic amino acid, E404D exhibits an unimpaired substrate-dependent anion conductance (Figs. 2, 3, 4) and a similar affinity to external sodium as the wild-type, both in intact cells (Fig. 7) as well as in proteoliposomes (data not shown). Thus, we are not dealing here with some structural mutation altering the conformation of the transporter, but a specific step is affected. Two possibilities come to mind: 1) potassium binding and/or translocation is impaired; and 2) the affinity for sodium is increased such that the rate of unbinding is dramatically decreased (step 5 for influx, step 1 for efflux). This latter possibility can be excluded because potassium-dependent efflux in E404D liposomes is defective (Fig. 6), whereas the affinity for external sodium is the same as for the wild-type in this preparation. In addition, no marked differences in affinity for internal sodium between wild-type and mutant proteoliposomes are observed (data not shown). Furthermore, whereas in intact cells the affinity of the mutant transporter for external sodium is identical to that of the wild-type (Fig. 7), the mutant transporter has lost the ability to interact with external potassium (Fig. 4). Therefore, our results indicate that potassium binding and/or translocation is impaired in the E404D mutant from the inside as well as from the outside, and that perhaps glutamate 404 is one of the liganding groups for potassium. It is especially striking that the removal of a single methylene group is sufficient to cause this defect. In view of this extremely high specificity, it is perhaps not surprising that other potassium congeners, i.e. rubidium, ammonium, and cesium, which can replace potassium in the wild-type to varying extents, cannot enable net flux in the mutant transporter (data not shown). Voltage jumps in the presence and absence of external D-aspartate reveal that exchange mediated by the E404D mutant is accompanied by a marked transient current that decays with a time constant between 10 and 20 ms (Fig. 2). This transient current is significantly slower than the previously described sodium-dependent and kainate-sensitive transient current in EAAT2, the human homolog of GLT-1 (t = 3-4 msec; 13Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (351) Google Scholar). This latter current is thought to reflect sodium binding and/or state transitions involving charge movements subsequent to sodium binding. Such sodium-dependent conformational changes have recently been monitored experimentally in GLT-1 (29Grunewald M. Kanner B. J. Biol. Chem. 1995; 270: 17017-17024Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The slower transient currents associated with the exchange step may reflect reversible movement of sodium and glutamate across the electric field during this partial reaction cycle. The E404D mutant mediates a sodium- and aspartate-dependent anion conductance (Fig. 2-4). The selectivity of this conductance is unchanged from the wild-type transporter (SCN > NO3 > I > Cl; Fig. 3; see also 25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar and 26Eliasof S. Jahr C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4153-4158Crossref PubMed Scopus (108) Google Scholar). This result indicates that the anion-conducting state is likely to correspond to a sodium- and glutamate-occupied state (25Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (459) Google Scholar), rather than a state in the potassium-transporting limb of the transport cycle (Fig. 8). A similar conclusion has been reached with independent experiments (17Billups B. Rossi D. Attwell D. J. Neurosci. 1996; 16: 6722-6731Crossref PubMed Google Scholar). The current activated by aspartate in E404D reverses close to the reversal potential for Cl, consistent with electroneutral exchange with little or no net electrogenic transport. The residue E404D is conserved among all cloned mammalian glutamate transporters and is expected to fulfill a similar role in them. Mutation of the corresponding residue in EAAT3 (30Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar), the human homolog of EAAC1 (9Kanai Y. Hediger M.A. Nature. 1992; 360: 464-471Crossref PubMed Scopus (1200) Google Scholar), also abolished potassium-dependent efflux. 1M. P. Kavanaugh, unpublished observations. Interestingly, this residue is not conserved in the related neutral amino acid transporter ASCT-1 (31Arriza J.L. Kavanaugh M.P. Fairman W.A. Wu Y.-N. Murdoch G.H. North R.A. Amara S.G. J. Biol. Chem. 1993; 268: 15329-15332Abstract Full Text PDF PubMed Google Scholar, 32Shafqat S. Tamarappoo B.K. Kilberg M.S. Puranam R.S. McNamara J.O. Guadano-Ferraz A. Fremeau Jr., R.T. J. Biol. Chem. 1993; 268: 15351-15355Abstract Full Text PDF PubMed Google Scholar), and recent work shows that this transporter also mediates potassium-independent amino acid exchange (33Zerangue N. Kavanaugh M.P. J. Biol. Chem. 1996; 271: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Glu404 is located in the middle of a structural domain that influences binding of the glutamate analogue kainate (34Vandenberg R.J. Arriza J.L. Amara S.G. Kavanaugh M.P. J. Biol. Chem. 1995; 270: 17668-17671Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and is also near residue Asp398, which is required for transport (21Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Amino acid sequences in this region of the glutamate transporters are highly conserved and contain multiple short hydrophobic segments (7Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1140) Google Scholar, 8Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1101) Google Scholar, 9Kanai Y. Hediger M.A. Nature. 1992; 360: 464-471Crossref PubMed Scopus (1200) Google Scholar, 10Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1018) Google Scholar). Pore-forming domains in both voltage-gated and ligand-gated ion channels tend to be highly conserved in these proteins (35Jan L.Y. Jan Y.N. Cell. 1992; 69: 715-718Abstract Full Text PDF PubMed Scopus (190) Google Scholar) and often consist of relatively short loop structures (36MacKinnon R. Neuron. 1995; 14: 889-892Abstract Full Text PDF PubMed Scopus (222) Google Scholar). A similar role for the domain encompassing Glu404 in glutamate transporters is suggested both by its critical role in potassium permeation as well as its highly conserved structure." @default.
• W2016994955 created "2016-06-24" @default.
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• W2016994955 creator A5038910133 @default.
• W2016994955 creator A5060349010 @default.
• W2016994955 creator A5065754575 @default.
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• W2016994955 date "1997-01-01" @default.
• W2016994955 modified "2023-09-27" @default.
• W2016994955 title "Mutation of an Amino Acid Residue Influencing Potassium Coupling in the Glutamate Transporter GLT-1 Induces Obligate Exchange" @default.
• W2016994955 cites W1486976896 @default.
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