The ASCTs(alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and GltPh, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and GltPh. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly L-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of GltPh, with preference for L-aspartate over L-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH5.5; however, this was reversed by the additional mutation A382T.Wepropose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
CITATION STYLE
Scopelliti, A. J., Ryan, R. M., & Vandenberg, R. J. (2013). Molecular determinants for functional differences between alanine-serine-cysteine transporter 1 and other glutamate transporter family members. Journal of Biological Chemistry, 288(12), 8250–8257. https://doi.org/10.1074/jbc.M112.441022
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