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J. Biol. Chem., Vol. 277, Issue 33, 29847-29855, August 16, 2002

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A Hydrophobic Domain in Glutamate Transporters Forms an Extracellular Helix Associated with the Permeation Pathway for Substrates^*

Barbara H. Leighton^§, Rebecca P. Seal^¶, Keiko Shimamoto, and Susan G. Amara^§**

From the  Vollum Institute, ^§ Howard Hughes Medical Institute, Oregon Health & Sciences University, Portland, Oregon 97201, the ^¶ Departments of Physiology and Neurology, Medical Center, University of California, San Francisco, California 97143, and the  Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan

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 (Ala⁴⁴⁶-Gly⁴⁵⁹) 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 -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.

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