Abstract
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.
Highlights
Transporters for small, neutral amino acids are essential for the shuttling of glutamine, the major nitrogen carrier in mammalian organisms, into and out of cells [1,2,3]
Asn82 is localized in the predicted transmembrane domain 1 (TMD1) (Fig. 1A), in which 87.5% of residues are conserved within the SLC38 transporters, suggesting that TMD1 may be very important for the function of SNAT2
Determination of the effect of Naϩ on transport currents showed that the affinities for Naϩ and, alanine were dramatically affected by the N82A mutation, whereas the more conservative mutation N82S resulted in affinities for both alanine and Naϩ that were in between SNAT2N82A and SNAT2WT
Summary
Molecular Biology and Transient Expression—The cDNA coding for rat SNAT2, which was kindly provided by H. For determining the voltage dependence of SNAT2 alanine transport, a combined voltage ramp/solution exchange protocol was used. In this protocol, the cell membrane was initially held at 0 mV before ramping the voltage to its final value (Ϫ150 to ϩ90 mV) within 2 s. Endogenous electrogenic alanine trans- tor 3 between different cells, depending on the expression levels port activity in HEK293T cells, as measured by current record- of each individual cell. Such changes in expression levels did not ing from nontransfected cells, is minimal (see “Results”). The Imax values were obtained by averaging the Imax values from these individual cells
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