Cysteine-scanning Mutagenesis Reveals a Conformationally Sensitive Reentrant Pore-Loop in the Glutamate Transporter GLT-1

Abstract

Removal of glutamate from the synaptic cleft by (Na ؉ ؉ K ؉ )-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30 -100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.Glutamate transporters in the brain ensure that the synaptic levels of the transmitter are kept below the neurotoxic levels (1-6). These transporters, located in the plasma membrane of nerve and glial cells, play an important role in limiting the duration of synaptic excitation (7-10). The uptake process is electrogenic (11-13), involving co-transport of three sodium ions, a proton, and a glutamate molecule and countertransport of a potassium ion (14 -16). In addition to the coupled flux, glutamate transporters mediate a thermodynamically uncoupled chloride flux activated by two of the molecules they transport, sodium and glutamate (17, 18).The five known eukaryotic glutamate transporters, GLT-1 (19), GLAST-1 (20), EAAC-1 (21), , and EAAT-5 (22), have an overall amino acid identity of ϳ50%. The homology is significantly higher in the carboxyl-terminal half of the transporters. Topology studies suggest that this region of the protein has an intriguing arrangement containing two oppositely oriented reentrant loops, the two transmembrane domains (TMs) 1 7 and 8, and an outward-facing hydrophobic region (Refs. 23-25, and see Fig. 1). Some of the features of the membrane topology remain under debate (26).Several amino acid residues critical for the function of glutamate transporters are located in the carboxyl-terminal half. Two adjacent amino acid residues of GLT-1, Tyr-403 and Glu-404, are located in TM7 (Fig. 1) and are conserved in all other glutamate transporters. Both were implicated in the binding of potassium ions (16,27) and appear to be close to one of the sodium binding sites (27). A conserved arginine residue, Arg...

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

“…10A) and 449 (Fig. 3B) and also at position 443 (36). These observations are in harmony with the TBOA-bound Glt Ph structure, which represents an "open" conformation where HP2 has moved toward (36), 448, and 449 and presumably also the intervening residues, although the latter could not be tested because the lack of activity of the cysteine mutants at positions 444 -447 (Fig.…”

“…3B, 5B, and 10 and Ref. 36), render the explanation of an overall a physical blockade of external accessibility of HP2 highly unlikely.…”

“…Previously, we have analyzed the effects of transporter ligands on the sensitivity of cysteine mutants introduced at positions 422-443 (36). This latter stretch encompasses around half of HP2, roughly corresponding to the part of HP2 referred to as HP2a in Glt Ph (11).…”

Mutation E252C Increases Drastically the K Value for Na+ and Causes an Alkaline Shift of the pH Dependence of NhaA Na+/H+ Antiporter of Escherichia coli

“…30). However, recently more and more examples (36,54) of residues in loops were found that, similar to E252C in NhaA, affect the kinetics and other parameters of the transporters. It is clear that atomic resolution of NhaA as of the other transporters is needed to get a glimpse as to how and, whether directly or indirectly, loops participate in the translocation mechanism.…”

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

“…Pore-loop structures are loop regions that fold back between the TMSs and are commonly observed in channel proteins where they function as selectivity filters (23,24). Well studied examples are the bacterial and neuronal glutamate transporters, GltT and GLT-1, in which pore-loops were shown to play an important role in the translocation event (17,18,25,26). Pore-loop structures are not observed in the LacY and GlpT structures, and it is unknown whether or not they represent a different translocation mechanism.…”