Deciphering the Molecular Mechanisms of Neurotransmitter Transport - Abstract A large and increasing number of people in the US are impacted by neurological disorders, such as major depression, anxiety, autism, Parkinson’s disease, and attention deficit disorder; moreover, the global burden of disease attributable to drug addiction continues to rise. Decades of research and drug development have shown that these diseases and disorders can be treated using drugs that modulate neurotransmitter availability. Many of these therapeutics block neurotransmitter reuptake or antagonize receptor signaling by acting on plasma membrane transporters and receptors. Unfortunately, many highly addictive substances such as amphetamines also bind to these proteins, but their mode of action is poorly understood and thus we are unable to combat their deadly effects. Synaptic vesicles (SVs) store neurotransmitter in presynaptic neurons, which are released into the synapse to propagate neuronal signaling. SV transport proteins are also required for neurotransmitter release as they load neurotransmitter into SVs, rendering them fundamental components in the regulation of neurotransmitter signaling. Despite their importance, we still know very little about the underlying molecular mechanisms of SV transporter function. Our work aims at providing a detailed and comprehensive understanding of the molecular mechanisms of SV transporter function. Unfortunately, nearly all SV transporters have been recalcitrant to biochemical and biophysical characterization and thus far, most have eluded high-resolution atomic investigations by any structural methodology. Vesicular monoamine transporter 2 (VMAT2) is the only transporter expressed in the central nervous system that loads biogenic amine containing neurotransmitters such as dopamine, serotonin, and norepinephrine into SVs. To tackle SV transport-associated diseases head-on, we will perform detailed structural and functional analysis on VMAT2, revealing its structure in several conformational states representing key intermediates in the transport cycle. The studies proposed here will illuminate the molecular mechanisms of VMAT2 in neurons and reveal the structural basis for how inhibitors and substrates bind. We will address these objectives by pursuing three specific aims: 1) Structural studies of VMAT2 in complex with the inhibitor tetrabenazine; 2) Determine the structural basis of reserpine inhibition; 3) Demonstrate how neurotransmitter and amphetamines are transported by VMAT2 by determining structures in various substrate-bound and apo conformational states. Our anticipated results promise to deepen our understanding of neurotransmitter binding and membrane transport mechanisms and provide necessary atomic information about this family of proteins, localization of the molecular determinants, and modes of drug interaction to guide the development of new inhibitors with fewer side effects and greater specificity.
