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J. Biol. Chem., Vol. 283, Issue 18, 12284-12292, May 2, 2008

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Highly Conserved Asparagine 82 Controls the Interaction of Na⁺ with the Sodium-coupled Neutral Amino Acid Transporter SNAT2^*

Zhou Zhang, Armanda Gameiro^¶, and Christof Grewer^¶1

From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136, the ^¶Department of Chemistry, Binghamton University, Binghamton, New York 13902, and the College of Life and Environment Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China

The neutral amino acid transporter 2 (SNAT2), which belongs to the SLC38 family of solute transporters, couples the transport of amino acid to the cotransport of one Na⁺ ion into the cell. Several polar amino acids are highly conserved within the SLC38 family. Here, we mutated three of these conserved amino acids, Asn⁸² in the predicted transmembrane domain 1 (TMD1), Tyr³³⁷ in TMD7, and Arg³⁷⁴ in TMD8; and we studied the functional consequences of these modifications. The mutation of N82A virtually eliminated the alanine-induced transport current, as well as amino acid uptake by SNAT2. In contrast, the mutations Y337A and R374Q did not abolish amino acid transport. The K_m of SNAT2 for its interaction with Na⁺, K_Na⁺, was dramatically reduced by the N82A mutation, whereas the more conservative mutation N82S resulted in a K_Na⁺ that was in between SNAT2_N82A and SNAT2_WT. These results were interpreted as a reduction of Na⁺ affinity caused by the Asn⁸² mutations, suggesting that these mutations interfere with the interaction of SNAT2 with the sodium ion. As a consequence of this dramatic reduction in Na⁺ affinity, the apparent K_m of SNAT2_N82A for alanine was increased 27-fold compared with that of SNAT2_WT. Our results demonstrate a direct or indirect involvement of Asn⁸² in Na⁺ coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.

Received for publication, August 14, 2007 , and in revised form, March 3, 2008.

^* This work was supported by Florida Department of Health Grant 04NIR-07 and National Institutes of Health Grant R01-NS049335-02 (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 supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed: Dept. of Chemistry, Binghamton University, 4400 Vestal Pkwy. E., Binghamton, NY 13902. Tel.: 607-777-3250; Fax: 607-777-4478; E-mail: cgrewer{at}binghamton.edu.

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