A conserved Na(+) binding site of the sodium-coupled neutral amino acid transporter 2 (SNAT2).
The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.
Zhang
Zhou
Z
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China.
Albers
Thomas
T
Fiumera
Heather L
HL
Gameiro
Armanda
A
Grewer
Christof
C
D000595:Amino Acid Sequence; D026924:Amino Acid Transport System A / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D019921:Biotinylation; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry
D026924:Amino Acid Transport System A; D002412:Cations; C498797:SLC38A2 protein, human; D012964:Sodium