Sulfhydryl modification of cysteine mutants of a neuronal glutamate transporter reveals an inverse relationship between sodium dependent conformational changes and the glutamate-gated anion conductance.
In the central nervous system, glutamate transporters remove the neurotransmitter from the synaptic cleft. The electrogenic transport of glutamate is coupled to the electrochemical sodium, proton and potassium gradients. Moreover, these transporters mediate a sodium- and glutamate-dependent uncoupled chloride conductance. In contrast to the wild type, the uptake of radiolabeled substrate of the G283C mutant is inhibited by [2-(trimethylammonium)ethyl]methanethiosulfonate, a membrane impermeant sulfhydryl reagent. In the wild type and the unmodified mutant, substrate-induced currents are inwardly rectifying and reflect the sum of the coupled electrogenic flux and the anion conductance. However, the sulfhydryl-modified G283C mutant exhibits currents that are non-rectifying and reverse at the equilibrium potential for chloride. These properties are similar to those of the I421C mutant after sulfhydryl modification. Importantly, in contrast to I421C, the modification of G283C does not cause an increase of the magnitude of the anion conductance and a decrease of the apparent substrate affinity. Moreover, in the G283C/I421C double mutant the phenotype of I421C is dominant. Sulfhydryl modification of I421C, but not of G283C, abolishes the sodium dependent transient currents. The results indicate the existence of multiple transitions between the coupled transport cycle and anion conducting states.
Shachnai
Liat
L
Department of Biochemistry, Hebrew University Hadassah Medical School, P.O.B. 12272, Jerusalem 91120, Israel.
Shimamoto
Keiko
K
Kanner
Baruch I
BI
D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D005971:Glutamates / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008094:Lithium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008698:Mesylates / Q000494:pharmacology; D008845:Microinjections / Q000379:methods; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D012964:Sodium / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D014162:Transfection / Q000379:methods; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D005971:Glutamates; D008698:Mesylates; D013438:Sulfhydryl Compounds; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D001224:Aspartic Acid; D008094:Lithium; D012964:Sodium; D003545:Cysteine