Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1.
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.
Grunewald
Myriam
M
Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel.
Menaker
David
D
Kanner
Baruch I
BI
D019943:Amino Acid Substitution; D001710:Biotin / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000502:physiology*; D018698:Glutamic Acid / Q000031:analogs & derivatives; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship
D027342:Excitatory Amino Acid Transporter 2; D007202:Indicators and Reagents; D018698:Glutamic Acid; C020889:dihydrokainic acid; D001710:Biotin; D012964:Sodium; D003545:Cysteine; D007608:Kainic Acid