An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates
2020; Elsevier BV; Volume: 295; Issue: 44 Linguagem: Inglês
10.1074/jbc.ra120.013704
ISSN1083-351X
AutoresBettina Kolen, Daniel Kortzak, Arne Franzen, Christoph Fahlke,
Tópico(s)Photoreceptor and optogenetics research
ResumoExcitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle. Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle. After release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by a family of glutamate transporters, the excitatory amino acid transporters (EAATs) (1Danbolt N.C. Glutamate uptake.Prog. 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For EAAT/GltPh, it ensures strict stoichiometric coupling of glutamate to Na+, K+, and H+ transport by permitting translocation only for certain ligation states of the transporter (21Kanner B.I. Sharon I. Active transport of l-glutamate by membrane vesicles isolated from rat brain.Biochemistry. 1978; 17 (708689): 3949-395310.1021/bi00612a011Crossref PubMed Scopus (261) Google Scholar, 22Kanner B.I. Bendahan A. Binding order of substrates to the sodium and potassium ion coupled l-glutamic acid transporter from rat brain.Biochemistry. 1982; 21 (6129891): 6327-633010.1021/bi00267a044Crossref PubMed Scopus (166) Google Scholar, 23Zerangue N. Kavanaugh M.P. Flux coupling in a neuronal glutamate transporter.Nature. 1996; 383 (8857541): 634-63710.1038/383634a0Crossref PubMed Scopus (706) Google Scholar, 24Kortzak D. Alleva C. Weyand I. Ewers D. Zimmermann M.I. Franzen A. Machtens J.P. Fahlke C. Allosteric gate modulation confers K+ coupling in glutamate transporters.EMBO J. 2019; 38 (31506973): e10146810.15252/embj.2019101468Crossref PubMed Scopus (19) Google Scholar). Elevator-like transport is also the basis of the anion channel function of this class of glutamate transporters. In atomistic molecular dynamics simulations, the lateral movement of the transport domain and subsequent water entry in the cleft between transport and trimerization domain generates a selective anion conduction pathway (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). 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It thus accounts for the existence of specialized glutamate transporters and low-capacity transporters with predominant anion channel function (4Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. An excitatory amino-acid transporter with properties of a ligand-gated chloride channel.Nature. 1995; 375 (7791878): 599-60310.1038/375599a0Crossref PubMed Scopus (1013) Google Scholar, 5Wadiche J.I. Amara S.G. Kavanaugh M.P. Ion fluxes associated with excitatory amino acid transport.Neuron. 1995; 15 (7546750): 721-72810.1016/0896-6273(95)90159-0Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 30Mim C. Balani P. Rauen T. Grewer C. The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.J. Gen. Physiol. 2005; 126 (16316976): 571-58910.1085/jgp.200509365Crossref PubMed Scopus (72) Google Scholar). To further test the predictions of this model, we searched for point mutations that modify anion channel open probabilities without altering glutamate transport rate. In a series of experiments in which EAAT residues that are homologous to anion pore-forming residues in GltPh (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) were substituted by proline (because of its specific conformational rigidity), we found that L46P substantially increases macroscopic anion current amplitudes of EAAT2. We did not observe changes in the voltage and substrate dependence of mutant anion currents, suggesting that the glutamate transport cycle remained unaffected by this mutation. We here performed a detailed investigation of the consequences of this mutation on glutamate transport and anion conduction of EAAT2. Fig. 1A depicts an alignment of amino-terminal sequences of mammalian SLC1 transporters and prokaryotic homologues. Bacterial transporters usually exhibit shorter amino-terminal domains, and leucine 46 is not conserved among the family. At present, no three-dimensional structure exists for EAAT2, and we therefore mapped the homologous residue, leucine 54, to outward- (31Yu X. Plotnikova O. Bonin P.D. Subashi T.A. McLellan T.J. Dumlao D. Che Y. Dong Y.Y. Carpenter E.P. West G.M. Qiu X. Culp J.S. Han S. Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation.eLife. 2019; 8 (31580259): e4812010.7554/eLife.48120Crossref PubMed Scopus (33) Google Scholar) and inward-facing conformation (32Garaeva A.A. Oostergetel G.T. Gati C. Guskov A. Paulino C. Slotboom D.J. Cryo-EM structure of the human neutral amino acid transporter ASCT2.Nat. Struct. Mol. Biol. 2018; 25 (29872227): 515-52110.1038/s41594-018-0076-yCrossref PubMed Scopus (84) Google Scholar, 33Garaeva A.A. Guskov A. Slotboom D.J. Paulino C. A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2.Nat. Commun. 2019; 10 (31366933): 342710.1038/s41467-019-11363-xCrossref PubMed Scopus (51) Google Scholar) of ASCT2, the only mammalian SLC1 transporter with known structures in both conformations (Fig. 1, B and C). Leucine 54 is close to the amino-terminal end of the trimerization domain and does not undergo movements during the isomerization from outward- to inward-facing conformations. The open channel conformation has so far not been described for ASCT2, and we therefore mapped the homologous valine 12 to GltPh in its open anion channel conformation (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), where it projects its side chain into the water-filled anion permeation pathway as pore-lining residue (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) (Fig. 1D). Fig. 2, A and B depict representative recordings from cells transiently transfected with WT (Fig. 2A) or L46P EAAT2 (Fig. 2B). In these experiments, cells were dialyzed with KNO3-based internal solution and then perfused either with NaNO3-based external solutions without or with 1.0 mm l-glutamate, or with an external solution in which NaNO3 was completely substituted with KNO3. EAAT2 transports glutamate in the presence of external Na+ and l-glutamate (21Kanner B.I. Sharon I. Active transport of l-glutamate by membrane vesicles isolated from rat brain.Biochemistry. 1978; 17 (708689): 3949-395310.1021/bi00612a011Crossref PubMed Scopus (261) Google Scholar, 22Kanner B.I. Bendahan A. Binding order of substrates to the sodium and potassium ion coupled l-glutamic acid transporter from rat brain.Biochemistry. 1982; 21 (6129891): 6327-633010.1021/bi00267a044Crossref PubMed Scopus (166) Google Scholar, 23Zerangue N. Kavanaugh M.P. Flux coupling in a neuronal glutamate transporter.Nature. 1996; 383 (8857541): 634-63710.1038/383634a0Crossref PubMed Scopus (706) Google Scholar). The use of NO3− that is more permeant than Cl− (34Wadiche J.I. Kavanaugh M.P. Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel.J. Neurosci. 1998; 18 (9742136): 7650-766110.1523/JNEUROSCI.18-19-07650.1998Crossref PubMed Google Scholar) increases EAAT2 anion currents to levels greatly exceeding uptake currents (5Wadiche J.I. Amara S.G. Kavanaugh M.P. Ion fluxes associated with excitatory amino acid transport.Neuron. 1995; 15 (7546750): 721-72810.1016/0896-6273(95)90159-0Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 35Melzer N. Biela A. Fahlke C. Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels.J. Biol. Chem. 2003; 278 (14506254): 50112-5011910.1074/jbc.M307990200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) (Fig. 2, C and D). Almost complete block upon application of TBOA, which prevents opening of EAAT anion channels by blocking inward movement of the transport domain (36Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.Nature. 2007; 445 (17230192): 387-39310.1038/nature05455Crossref PubMed Scopus (394) Google Scholar), demonstrates that WT and mutant EAAT2 anion currents are significantly larger than background currents (Fig. 2, C and D) and can thus be directly measured without subtraction procedures for many experimental conditions (35Melzer N. Biela A. Fahlke C. Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels.J. Biol. Chem. 2003; 278 (14506254): 50112-5011910.1074/jbc.M307990200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Torres-Salazar D. Fahlke C. Neuronal glutamate transporters vary in substrate transport rate but not in unitary anion channel conductance.J. Biol. Chem. 2007; 282 (17908688): 34719-3472610.1074/jbc.M704118200Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). WT as well as L46P EAAT2 anion currents are small and without appreciable time and voltage dependence in NaNO3-based external solutions lacking glutamate (Fig. 2). Application of l-glutamate increases anion currents for WT and mutant transporters (Fig. 2, C and D). EAAT2 also mediates large anion current amplitudes under conditions, in which extracellular Na+ is completely substituted by K+ (38Leinenweber A. Machtens J.P. Begemann B. Fahlke C. Regulation of glial glutamate transporters by C-terminal domains.J. Biol. Chem. 2011; 286 (21097502): 1927-193710.1074/jbc.M110.153486Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 39Divito C.B. Borowski J.E. Glasgow N.G. Gonzalez-Suarez A.D. Torres-Salazar D. Johnson J.W. Amara S.G. Glial and neuronal glutamate transporters differ in the Na+ requirements for activation of the substrate-independent anion conductance.Front. Molec. Neurosci. 2017; 10: 15010.3389/fnmol.2017.00150Crossref PubMed Scopus (8) Google Scholar). WT and mutant currents displayed similar time, voltage, and substrate dependences. However, L46P EAAT2 anion currents are much larger than WT currents. Moreover, whereas WT EAAT2 anion current amplitudes were highest in external Na+ and l-glutamate, we observed maximum mutant current under KNO3-based external solutions (Fig. 2D). To test whether increased macroscopic L46P EAAT2 currents arise from changes in subcellular localization or in expression levels, we employed confocal imaging and protein biochemistry (Fig. S1). WT and L46P EAAT2 display predominant surface membrane insertion in confocal images from HEK293T cells (Fig. S1, A and B). Fig. S1C depicts fluorescent scans of SDS-PAGE from membrane preparation of transfected HEK293T cells, indicating core and complex glycosylated WT and L46P EAAT2 (40Gendreau S. Voswinkel S. Torres-Salazar D. Lang N. Heidtmann H. Detro-Dassen S. Schmalzing G. Hidalgo P. Fahlke C. A trimeric quaternary structure is conserved in bacterial and human glutamate transporters.J. Biol. Chem. 2004; 279 (15265858): 39505-3951210.1074/jbc.M408038200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Individual bands were quantified with ImageJ software and revealed similar expression levels (Fig. S1C). To exclude the possibility that changes in the number of transfected cells compensate for altered expression levels, we also determined transfection rates by manually counting transfected and untransfected cells. We did not observe differences between WT and mutant transporters (Fig. S1D) and conclude that L46P leaves expression levels and subcellular distributions of EAAT2 unaffected. Because expression levels of WT and mutant transporters are similar, the observed differences in L46P EAAT2 macroscopic current amplitude might be because of increased mutant anion channel conductances or open probabilities. We employed nonstationary noise analysis to determine unitary current amplitudes of WT and L46P EAAT2 anion channels (41Schneider N. Cordeiro S. Machtens J.P. Braams S. Rauen T. Fahlke C. Functional properties of the retinal glutamate transporters GLT-1c and EAAT5.J. Biol. Chem. 2014; 289 (24307171): 1815-182410.1074/jbc.M113.517177Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To perform such experiments with comparable WT and mutant current amplitudes, we generated inducible stable Flp-In T-REx 293 cell lines for WT as well as for L46P EAAT2 (40Gendreau S. Voswinkel S. Torres-Salazar D. Lang N. Heidtmann H. Detro-Dassen S. Schmalzing G. Hidalgo P. Fahlke C. A trimeric quaternary structure is conserved in bacterial and human glutamate transporters.J. Biol. Chem. 2004; 279 (15265858): 39505-3951210.1074/jbc.M408038200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In WT cells, anion currents with amplitudes sufficient for noise analysis were only rarely observed, even under maximum tetracycline concentration and induction periods. Flp-In T-Rex 293 cells differ from HEK293T in stable expression of the lacZ-Zeocin fusion gene and the Tet repressor, modifications that are not expected to modify the function of EAAT2, making the comparison of noise analysis results from both types of cells possible. We tested potential effects of the expression system by comparing noise analysis results on WT EAAT2 anion currents in HEK293T and Flp-In T-REx 293 cells (Fig. S2). Both systems provided the same results, and we pooled results from both expression systems for the analysis of WT EAAT2. For L46P EAAT2, noise analysis was exclusively performed in inducible Flp-In T-REx 293 cells. Fig. 3, A and B show representative time courses of mean macroscopic currents and corresponding current variances for WT (A) or L46P (B) EAAT2 elicited by repetitive voltage steps to −140 mV in symmetrical NaNO3 and 0.5 mm external l-glutamate. Fig. 3C provides pooled noise analysis data from 20 cells expressing WT (red circles) and 17 cells expressing L46P EAAT2 (blue circles). We plotted ratios of current variances by mean current amplitudes against normalized mean current amplitudes at various time points. A fit of the resulting relationship with a linear function (Equation 3) provides the single-channel current amplitudes as y axis intercept (42Alekov A. Fahlke C. Channel-like slippage modes in the human anion/proton exchanger ClC-4.J. Gen. Physiol. 2009; 133 (19364886): 485-49610.1085/jgp.200810155Crossref PubMed Scopus (47) Google Scholar). Bootstrap analysis was used to simulate the error-generating process of data sampling from a larger population and to get an approximation of the true error of fitting parameters. 50,000 bootstrap samples (each containing 1000 WT or 850 mutant EAAT2 data points with replacements) were randomly generated from the original dataset (41Schneider N. Cordeiro S. Machtens J.P. Braams S. Rauen T. Fahlke C. Functional properties of the retinal glutamate transporters GLT-1c and EAAT5.J. Biol. Chem. 2014; 289 (24307171): 1815-182410.1074/jbc.M113.517177Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 43Efron B. Tibshirani R. An Introduction to the Bootstrap. Chapman & Hall/CRC, Boca Raton, FL1993Crossref Google Scholar) and individually fitted with linear functions (Fig. 3C). Unitary current amplitudes were determined for each bootstrap sample from linear fits, providing single channel amplitudes of 25 ± 2 femtoampere (fA) (mean ± 95% CI; n = 20) for WT and 29 ± 2 fA (mean ± 95% CI; n = 17) for mutant EAAT2 (p < 0.05) (Fig. 3D). We conclude that L46P slightly increases the unitary current amplitude of EAAT2 anion channels. EAAT anion channels conduct a variety of anions; they select, however, against large anions by size (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Wadiche J.I. Kavanaugh M.P. Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel.J. Neurosci. 1998; 18 (9742136): 7650-766110.1523/JNEUROSCI.18-19-07650.1998Crossref PubMed Google Scholar, 35Melzer N. Biela A. Fahlke C. Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels.J. Biol. Chem. 2003; 278 (14506254): 50112-5011910.1074/jbc.M307990200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The minimum pore size of the GltPh anion channel is around 5–6 Å (25Machtens J.P. Kortzak D. Lansche C. Leinenweber A. Kilian P. Begemann B. Zachariae U. Ewers D. de Groot B.L. Briones R. Fahlke C. Mechanisms of anion conduction by coupled glutamate transporters.Cell. 2015; 160 (25635461): 542-55310.1016/j.cell.2014.12.035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), only slightly smaller than the minimum size required for gluconate permeation (6.9 Å) (44Linsdell P. Hanrahan J.W. Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel.J. Gen. Physiol. 1998; 111 (9524141): 601-61410.1085/jgp.111.4.601Crossref PubMed Scopus (124) Google Scholar). To test for potential changes in selectivity of L46P EAAT2 anion channels, we recorded currents in cells dialyzed with NaNO3 and perfused with solutions containing mixtures of NaNO3 and Na-gluconate or of NaNO3 and choline-NO3 (Fig. 4). In these experiments, accurate separation of EAAT2-mediated currents from endogenous current amplitudes is especially important. We therefore measured background currents by adding TBOA at the end of each experiment and corrected WT and mutant EAAT2 anion currents for such current components. Outward currents were virtually nonexistent in cells expressing WT transporters perfused with NO3−-free external solutions (Fig. 4, A and C), in full agreement with a negligible gluconate− permeability. In contrast, we observed significant outward currents for L46P EAAT2 (Fig. 4, B and D). Gluconate− permeability of L46P EAAT2 suggests that the mutations widen the selectivity filter and permit passage of anions that are usually too large to permeate. Plotting current reversal potentials versus external
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