MOLECULAR BIOLOGY OF BRUSH BORDER TRANSPORT PROTEINS

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Active transport of organic solutes such as sugars, amino acids an+d small peptides across cell membranes of prokaryotes and eukaryotes proceeds via Na and H+-coupled transporters. In the past few years I have cloned, sequenced and characterized several-mammalian transporters. These include the intestinal and renal Na+-coupled glucose cotransporters SGLTI and SGLT2, the Na+ and K+-dependent neuronal and epithelial high affinity glutamate transporter EAAC1, the intestinal H+-coupled oligopeptide transporter PepT1 and its renal isoform hPepT2, and the renal facilitated urea transporter UT2. The present proposal focuses on the question of how uphill solute transport is linked to electrochemical ion gradients. I have chosen the Na+-coupled glucose transporters and the H+-coupled oligopeptide transporter as models to address this question. The most useful system to study these proteins has been expression in Xenopus oocytes followed by uptake studies and electrophysiological experiments. Previous studies of SGLT1 and preliminary experiments of PepT1 have provided interesting information on specific transport mechanisms displayed by these proteins which are distinct from those of EAAC1 and the GABA transporter GAT-1, yet have certain principles in common. It seems likely that, in combination with studies of transporters modified by genetic engineering, these results will yield an overall molecular view of how transporters link uphill solute transport to electrochemical ion gradients. Specifically, I propose to construct chimeras between SGLT1 and SGLT2 in order to assign functional properties to specific regions in SGLT1 and to use site-directed mutagenesis to pinpoint individual amino acid residues involved-in the Na+-coupling mechanism. To study the H+-coupling mechanism of PepT1 and hPepT2 I will first construct kinetic models based on electrophysiological studies and - isotopic flux measurements to understand how these proteins function. I will then compare these models with those of SGLT1, GAT-1 and EAAC1 and evaluate whether tire are common principles underlying these transport processes. Next, I will study the effect of individual amino acids of PepT1 which are conserved with two distantly related homologous transporters on transporter function. Finally, I will elucidate the role PepT1 plays in intestinal trans-epithelial transport of protein digestion end-products. The results from these studies should provide information of general importance to our understanding of how active solute transporters work, information which will be invaluable to the interpretation of tertiary structures when it becomes available.