Highly conserved asparagine 82 controls the interaction of Na+ with the sodium-coupled neutral amino acid transporter SNAT2

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, Asn(82) in the predicted transmembrane domain 1 (TMD1), Tyr(337) in TMD7, and Arg(374) 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 Asn(82) 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 Asn(82) in Na+ coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.