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 pH 5.5; however, 
this was reversed by the additional mutation A382T. We propose that these 
residues differences in TM7 and TM8 combine to determine differences in 
substrate selectivity between members of the SLC1 family.