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J. Biol. Chem., Vol. 279, Issue 4, 2513-2519, January 23, 2004

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Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1^*

Lars Borre and Baruch I. Kanner

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

The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium- and potassium-coupled glutamate transporter EAAC-1, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436–37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of L-[³H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for L-glutamate at –60 mV is 30-fold higher than that of the wild type. Also, with D-aspartate, R445S exhibits an 15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.

Received for publication, October 19, 2003 , and in revised form, October 30, 2003.

^* This work was supported by U.S.-Israel Binational Science Foundation Grant 9900123, NINDS/National Institutes of Health Grant NS16708, and the Bernard Katz Minerva Center for Cellular Biophysics. 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.

Recipient of a Danish Research Agency fellowship.

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

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