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.