A hydrophobic domain in glutamate transporters forms an extracellular helix associated with the permeation pathway for substrates
A hydrophobic domain in glutamate transporters forms an extracellular helix associated with the permeation pathway for substrates
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DOI：
10.1074/jbc.m202508200
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发表时间：
2002-08-16
影响因子：
4.8
通讯作者：
Amara, SG
中科院分区：
文献类型：
--
作者：
Leighton, BH;Seal, RP;Amara, SG
Recent work has shown that cysteine residues introduced into domain 10, a highly hydrophobic segment in the excitatory amino acid transporter 1, react readily when hydrophilic sulfhydryl-modifying reagents are applied extracellularly. To investigate the functional contributions of this region, we mutated each residue in domain 10 (Ala(446)-Gly(459)) to cysteine and assessed the transport kinetics and inhibitor sensitivities of the mutant carriers. Modification of the introduced sulfhydryl group with membrane-impermeant methanethiosulfonate derivatives inhibited substrate transport by all but one functional cysteine mutant. Substrates and/or nontransported inhibitors block thiol modification of most mutants within this region, implying that access to the domain becomes restricted as a consequence of the binding of substrates and substrate analogs. An examination of the temperature dependence of substrate protection for one mutant (I453C) indicates that substrates prevent modification at a step prior to the large conformational changes associated with translocation. When superimposed on a helical model, mutants with similar attributes are positioned in close proximity. Our data are consistent with a model in which domain 10 exists as an a-helix at an aqueous interface of the translocation pathway, which can be directly occluded by substrates and inhibitors at an early step in the transport cycle.
