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.
Zhang
Zhou
Z
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136, USA.
Gameiro
Armanda
A
Grewer
Christof
C
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D004553:Electric Conductivity; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D051381:Rats; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship
D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D050505:Mutant Proteins; C412568:Slc38a2 protein, rat; D001216:Asparagine; D012964:Sodium; D000409:Alanine