Abstract
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 non-transported 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 alpha-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.
Highlights
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
At other positions in domain 10, cysteine substitutions were generally well tolerated: the Km values for the mutant transporters resemble those observed for the cysless parent (Km 48 Ϯ 2 M, n ϭ 6) and the wild type EAAT1 (Km 48 Ϯ 10 M) [24] with the exception of L448C (Km 219 Ϯ 49 M, n ϭ 5), which exhibited a reduced Vmax (17 Ϯ 1% of the cysless version) (Table I)
We focused on the I453C residue, because it has a fast rate of modification by MTS-ethyl sulfonate (MTSES) at 25 °C (378 Ϯ 9 MϪ1sϪ1, n ϭ 3) (Fig. 7A) and because it exhibits a significant reduction in the rate of MTS reagent modification in the presence of L-glutamate at 25 °C (74 Ϯ 10 MϪ1sϪ1, n ϭ 3) (Fig. 7A and Table II)
Summary
MTS reagent modification of these cysteine residues are consistent with an ␣-helical structure with one side being preferentially accessible to the extracellular aqueous environment. Substrates and inhibitors prevent the MTS reagent modification of domain 10 mutants and appear to do so at a step in the transport cycle that precedes major conformational changes required for substrate translocation. These data suggest that residues within domain 10 reside within a pore or binding pocket that becomes occluded when substrates and inhibitors bind to the transporter
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