The molecular mechanisms of neutral amino acid transport by the alanine, serine, cysteine transporters

Abstract

ASCT1 and ASCT2 belong to the SLC1A family of transporters that also includes the human glutamate transporters. ASCTs utilise Na+ gradients to exchange small neutral amino acids across the membrane of various cell types. This thesis investigates the molecular determinants of neutral amino acid transport by ASCT1 and ASCT2. I have identified two residues that are essential in defining the neutral versus acidic amino acid binding sites in SLC1A transporters. Introducing an arginine residue of TM8 and a threonine residue in TM7 into ASCT1 (T459R and A382T) switches the substrate selectivity from neutral to acidic amino acids. Furthermore, I have identified two key differences within the substrate binding site of ASCT1 and ASCT2 that determine selectivity for glutamine and glutamine-based inhibitors. Using this information I have generated a prokaryotic model of ASCT-mediated neutral amino acid transport via a single point mutation in the prokaryotic homolog GltPh. Lastly, it was determined that ASCT1 has three Na+ binding sites, however only two of the three Na+ binding sites are required to be occupied for transporter function at any one time. This thesis has provided insights into the structural elements defining the molecular mechanisms of neutral amino acid transport by ASCTs.