Structure-function studies of human SLC36 amino acid transporters

The Solute Linked Carrier family 36 (SLC36) of integral membrane transport proteins has four members which are also known as the human Proton coupled Amino acid Transporters (hPATs).This is due to hPATs 1 and 2 having been functionally characterized as proton coupled transporters that bring about a 1:1 symport of H+ and an amino acid across the membrane (with optimum uptake at an extracellular pH of 5.5). Their main substrates were found to be the small neutral amino acids glycine, alanine and proline; however, in addition to these, PATs 1 and 2 were also observed to transport a range of other substrates including some pharmacologically active compounds. hPATs 3 and 4 were considered orphan transporters, with no information available except their mRNA localization in various tissues. Additionally there is no structural information available about the PAT family of transport proteins. The thesis has two broad aims. The first aim was to study the orphan PAT transporters and to investigate whether these proteins are also amino acids transporters and, if so, what their substrates are. The second aim was to use transmembrane domain software to predict the secondary structure of the PAT transporters using hPAT1 as the model protein. Epitope tags, luminometry and functional assays were used to test these predictions, and homology modelling then employed to gain a better understanding of the structure-function relationship of this transporter family. The results of both these areas of investigation are reported in this thesis. The orphan transporter hPAT4 was found to be a very high affinity electroneutral proline and tryptophan transporter when expressed in the Xenopus laevis oocyte system. Preliminary data for hPAT3 suggested uptake of proline at extracellular pH 7.4 in Xenopus laevis oocytes and in the mouse Sertoli cell line TM4. Membrane topology studies on hPAT1 provided experimental support for the prediction of 11 transmembrane domains for this transporter protein. Homology modelling indicates a region which might play a role in the substrate specificity of PAT proteins