Na?dependent neurotransmitter transporters or neurotransmitter sodium symporters (NSS) are integral membrane proteins that play a critical role in terminating synaptic transmission and, thus, in shaping the duration and magnitude of synaptic signaling. They work by coupling preexisting ion gradients to the thermodynamically unfavorable movement of small molecule neurotransmitters from the synapse to neuronal and glial cytoplasms. Pumps for the biogenic amines are of particular significance because their dysfunction has been implicated in a number of debilitating neuropsychiatric diseases and because they are the target of a broad array of psychoactive agents such as antidepressants and cocaine. Despite such clinical importance, atomic-level detail into the mechanism of substrate translocation and inhibition has remained elusive. Structure/function studies of LeuT, a prokaryotic NSS member, has revealed critical elements on both topics, but because the identity between this bacterial protein and the eukaryotic counterparts is so low, the comparisons will necessarily be limited. The goal of this application is to determine if the molecular models of transport and inhibition advanced for LeuT also apply to the eukaryotic NSS members. This will be accomplished by screening known biogenic amine transporters in heterologous expression systems for monodispersity and their propensity for crystallization. A representative member will then be crystallized, and its structure solved. Structure-based hypotheses of transport and inhibition will subsequently be tested with a combination of site-directed mutagenesis, cysteine crosslinking, steady-state kinetics, dissociation/association kinetics, and crystallography. While there is certainly much about LeuT that seems to be reflected in eukaryotic NSS counterparts, other areas fall short, so data from these experiments will fill a critical intellectual void. In addition, and perhaps more importantly, structural studies of a eukaryotic biogenic amine transporter will permit further investigation into specific antagonist binding sites and perhaps into the molecular basis for drug resistance, thereby opening the way to rational drug design efforts. When eventually coupled with in vivo work beyond the scope of this application, structure/function studies may also shed light on the molecular underpinnings of disease-causing polymorphisms. Achieving any one of these objectives would likely have significant impact in both the laboratory and in the clinic.
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