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J. Biol. Chem., Vol. 281, Issue 15, 10263-10272, April 14, 2006
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From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136
Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na+ cotransport. To test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared with the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na+ in the glutamate-free transporter form.
Received for publication, October 2, 2005 , and in revised form, January 17, 2006.
* This work was supported by National Institutes of Health Grant R01-NS049335-02 and Deutsche Forschungsgemeinschaft Grants GR 1393/2-2,3 (to C. G.). 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.
The on-line version of this article (available at http://www.jbc.org) contains additional data and supplemental Fig. S1.
1 Supported by American Heart Association Fellowship 0525485B.
2 To whom correspondence should be addressed: 1600 NW 10th Ave., Miami, FL 33136. Tel.: 305-243-1021; Fax: 305-243-5931; E-mail: cgrewer{at}med.miami.edu.
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