Structure and Function in NSS transporters

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DESCRIPTION (provided by applicant): The neurotransmitter sodium symporter (NSS) family includes transporters responsible for the re-uptake of neurotransmitters GABA, glycine, norepinephrine, serotonin, and dopamine. They represent a key element in neurotransmission whereby the extracellular concentration of neurotransmitter is regulated at a low level, and transmitters are efficiently recycled. Also in this family are many transporters for amino acids not involved in neurotransmission, including many predicted prokaryotic proteins whose function has not yet been identified. A major limitation in understanding these proteins has been the difficulty in expressing and purifying significant amounts of protein for structural and functional studies. The recent demonstration that 1 of the prokaryotic NSS sequences encodes a functional tryptophan transporter (TnaT) has opened up new avenues for understanding the structure and function of all of these transporters. This proposal is designed to take advantage of these new approaches to understand TnaT and to relate these findings to mammalian neurotransmitter transporters. The proposed experiments include an examination of the bioenergetics of sodium-coupled tryptophan transport. These experiments will determine which ion gradients are coupled to tryptophan transport and will establish the stoichiometry of transport. The proposal also describes the use of genetic selection methods to estimate the physical proximity between different regions of the protein structure. Finally, there are experiments to evaluate the possibility that TnaT functions as a homo-oligomer and to gain structural information about the transporter using two-dimensional (2D) crystallography.