HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 39, 26391-26400, September 26, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/39/26391    most recent M802401200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Qu, S. Articles by Kanner, B. I. Search for Related Content PubMed PubMed Citation Articles by Qu, S. Articles by Kanner, B. I. Social Bookmarking                      What's this?

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2^*

From the Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel

To explore rearrangements of the reentrant loop HP2 relative to transmembrane domains (TMs) 7 and 8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. The pairs were introduced around 10–15Å "above" the residues, which make contact with substrate in the related archaeal homologue Glt_Ph. Transport by the double mutants M449C/L466C (HP2/TM 8), L453C/I463C (HP2/TM 8), and I411C/I463C (TM 7/TM 8) was inhibited by copper(II)(1,10-phenanthroline)₃ (CuPh) and by Cd²⁺. Inhibition was only observed when the two cysteines were present in the same construct, but not with the respective single cysteine mutants or when only one cysteine was paired with a mutation to another residue. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of M449C/L466C by CuPh. The non-transportable analogues kainate and D, L-threo-β-benzyloxyaspartate are expected to stabilize an outward-facing conformation, but only the latter potentiated the effect of CuPh on M449C/L466C. However, both analogues increased the aqueous accessibility of the cysteines introduced at positions 449 and 466 to a membrane-impermeant sulfhydryl reagent. Inhibition of L453C/I463C by CuPh was protected not only by glutamate but also by the two analogues. In contrast, these ligands had very little effect on the inhibition of I411C/I463C by CuPh. Our results are consistent with the proposal that HP2 serves as the extracellular gate of the transporter and indicate that glutamate and the two analogues induce distinct conformations of HP2.

Received for publication, March 27, 2008 , and in revised form, July 25, 2008.

^* This work was supported, in whole or in part, by NINDS, National Institutes of Health Grant NS16708. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 972-2-6758506; Fax: 972-2-6757379; E-mail: kannerb{at}cc.huji.ac.il.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?