A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

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CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na ؉ ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na ؉ . Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MT-SET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na ؉ at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membranecytoplasm interface.The secondary citrate transporter CitS of Klebsiella pneumoniae is a member of the bacterial 2-hydroxycarboxylate transporter (2HCT) 1 family. Members of the 2HCT family transport substrates that contain the 2-hydroxycarboxylate motif, as in citrate, malate, lactate, etc. (1). The family contains H ϩ and Na ϩ symporters that couple the translocation of the substrate to the co-ion as well as precursor/product exchangers that couple the uptake of the substrate to the excretion of a metabolic end product (2, 3). CitS is a Na ϩ symporter. It obligatorily couples the translocation of the divalent citrate anion (Hcit 2Ϫ ) to the translocation of two sodium ions and one proton (4, 5). The CitS protein is an integral membrane protein containing 446 amino acid residues (47.5 kDa). The protein is believed to consist of a bundle of 11 hydrophobic transmembrane segments (TMSs) with the N and C terminus located in the cytoplasm and periplasm, respectively ( Fig. 1) (6 -9). An additional hydrophobic segment, predicted to be transmembrane as well, was shown to reside in the periplasm between TMSs V and VI. The segment, termed Vb, and a stretch ...

mentioning

confidence: 99%

mentioning

confidence: 99%

Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Sobczak

Lolkema

2004

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“…The five known eukaryotic glutamate transporters, glutamate transporter-1 (2), glutamate/aspartate transporter-1 (3), excitatory amino acid carrier-1 (EAAC-1) 1 (4), and excitatory amino acid transporters 4 and 5 (5,6), have an overall identity of ϳ50%. A detailed experimental determination of the topological organization of the carboxyl terminal half revealed two oppositely oriented reentrant loops and an extracellular-facing hydrophobic linker region interspersed between two transmembrane domains (7)(8)(9), although some disagreement remains on this issue (10,11). The first insights into how the transporter folds upon itself are provided by a recent study showing that the two reentrant loops are in close proximity (12).…”

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confidence: 99%

Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1

Borre¹,

Kanner

2004

The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium-and potassiumcoupled glutamate transporter EAAC-1, the substratebinding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436 -37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of L-[ 3 H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for L-glutamate at ؊60 mV is ϳ30-fold higher than that of the wild type. Also, with D-aspartate, R445S exhibits an ϳ15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.Glutamate is the major excitatory neurotransmitter in the central nervous system and is removed from the synapse and its surroundings by glutamate transporters. Indeed, investigation of glutamate transporter knockout mice indicates that they play a central role in preventing both hyperexcitability and excitotoxicity (1). The five known eukaryotic glutamate transporters, glutamate transporter-1 (2), glutamate/aspartate transporter-1 (3), excitatory amino acid carrier-1 (EAAC-1) 1 (4), and excitatory amino acid transporters 4 and 5 (5, 6), have an overall identity of ϳ50%. A detailed experimental determination of the topological organization of the carboxyl terminal half revealed two oppositely oriented reentrant loops and an extracellular-facing hydrophobic linker region interspersed between two transmembrane domains (7-9), although some disagreement remains on this issue (10, 11). The first insights into how the transporter folds upon itself are provided by a recent study showing that the two reentrant loops are in close proximity (12).Glutamate uptake is an electrogenic process (13,14) in which the transmitter is cotransported with three sodium ions and one proton (15, 16), followed by the countertransport of one potassium ion (17)(18)(19). Glutamate transporters mediate a stoichiometric (coupled) current and a non-stoichiometric (uncoupled) current (20). The uncoupled current, activated in the presence of Na ϩ and Glu Ϫ , is carrie...

“…The homology is significantly higher in the carboxyl-terminal half which has a non-conventional topology containing two reentrant loops, two transmembrane domains, 7 and 8, as well as an outward facing hydrophobic region (22)(23)(24). Some of the features of the topology remain under debate as a different study assigns transmembrane domain 7 as a reentrant loop (25).…”

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confidence: 99%

Coupled, but Not Uncoupled, Fluxes in a Neuronal Glutamate Transporter Can Be Activated by Lithium Ions

Borre

Kanner

2001

In the central nervous system a family of related (Na ؉ -K ؉ )-coupled glutamate transporters remove the transmitter from the cleft and prevent its neurotoxic actions. In addition to this coupled uptake, these transporters also mediate a sodium-and glutamate-dependent uncoupled anion conductance. Most models assume that the initial steps for both processes are the same, leading to the anticipation that both may exhibit a similar requirement for cations. In this study we have tested this idea in the neuronal glutamate transporter EAAC-1. We report that in this transporter lithium can replace sodium in the coupled uptake. Strikingly, the glutamatedependent gating of the uncoupled conductance mediated by EAAC-1 has a strict requirement for sodium; lithium cannot substitute for it. Moreover, we describe two mutants, T370S and G410S, in which the cation selectivity of the two processes is affected differently. In both mutants sodium, but not lithium, can support coupled transport. On the other hand, the sodium selectivity of the gated anion conductance in oocytes expressing the mutant transporters is not affected. Our observations indicate that although both the coupled and the uncoupled fluxes are sodium-dependent, the conformation gating the anion conductance is different from that during substrate translocation.