title: Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.
abstract: Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.
authors.affiliation: Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem 91120, Israel.
authors.forename: Lihi
authors.initials: L
authors.lastname: Brocke
authors.forename: Annie
authors.initials: A
authors.lastname: Bendahan
authors.forename: Myriam
authors.initials: M
authors.lastname: Grunewald
authors.forename: Baruch I
authors.initials: BI
authors.lastname: Kanner
mesh_terms: D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004229:Dithiothreitol / Q000737:chemistry; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D011487:Protein Conformation
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D002104:Cadmium; D003545:Cysteine; D004229:Dithiothreitol