Neurotransmitter Transporter’s Structures Suggest New Drug Strategies

Scientists at St. Jude Children’s Research Hospital have drawn on structural biology expertise to determine structures of vesicular monoamine transporter 2 (VMAT2), a key component of neuronal communication, which acts to package neurotransmitters into vesicles prior to their transfer to the synapse. The ability to visualize VMAT2 in different states will help scientists better understand how it functions and how the different shapes the protein takes influence drug binding. This could be critical information for the development of drugs to treat hyperkinetic (excess movement) disorders such as Tourette syndrome.

Chia-Hsueh Lee, PhD, at St. Jude department of structural biology, and colleagues reported on their findings in Nature, in a paper titled “Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.”

Neurons talk to each other using chemical signals called neurotransmitters. Monoamines, which include dopamine, serotonin and adrenaline, are neurotransmitters that play a central role in neuronal communication, the authors explained. These molecules affect how the brain works, controlling our emotions, sleep, movement, breathing, circulation and many other functions. “Dysfunction in these circuits can lead to a range of psychiatric or neurodegenerative disorders, including depression, Parkinson’s disease and chorea.”

Monoamines synthesized in presynaptic neurons are packaged into synaptic vesicles by VMATs. These vesicles are cellular compartments that store the neurotransmitters before they are released at the synapses, which are the junctions across which chemical signals pass from one neuron to another. The vesicles can be thought of as the cargo ships of the neuronal cell.

VMATs are proteins on the membrane of these vesicles that move the monoamines into the space within. Once packaged with their neurotransmitter cargo the vesicles are then transferred to where they need to go. “VMATs are transporters that are required for packing these monoamine neurotransmitters into synaptic vesicles,” explained co-corresponding author Lee. Once the VMAT has packed the vesicle with monoamines, the vesicle can then move toward the synaptic gap, into where it releases the chemical compounds.

VMATs belong to the SLC18 family of transporters. There are two known types of VMAT, designated VMAT1 and VMAT2. VMAT1 is more specialized, and found only in neuroendocrine cells, whereas VMAT2 is found throughout the neuronal system and has significant clinical relevance. It acts as “… the main transporter responsible for monoamine packaging in neurons,” the investigators noted. “We knew that VMAT2 is physiologically very important,” Lee said. “This transporter is a target for pharmacologically relevant drugs used in the treatment of hyperkinetic disorders such as chorea and Tourette syndrome.”

Despite the importance of VMAT2, its structure—which would allow researchers to investigate more fully how it works—has remained elusive. “Despite the biological and clinical relevance of these transporters, the molecular basis of substrate transport and drug inhibition of VMATs remains unclear,” the team pointed out. “… detailed structural information about these processes is still lacking, which is key to understanding the mechanism of monoamine neurotransmitter packaging and to developing new therapeutic agents.”

For their reported study, Lee and his team used cryo-electron microscopy (cryo-EM) to obtain structures of VMAT2 bound to the monoamine serotonin and to the drugs tetrabenazine and reserpine, which are used to treat chorea and hypertension, respectively.

This was a significant achievement. “VMAT2 is a small membrane protein,” explained co-first author Yaxin Dai, PhD, at St. Jude department of structural biology. “This makes it a very challenging target for cryo-EM structure determination.”

Despite the difficulty, the team’s specialized cryo-EM approach, coupled with functional studies, allowed the researchers to capture multiple structures of VMAT2,  tease out how the protein functions, and investigate exactly two drugs targeting VMAT2 work. “VMAT transporters adopt multiple conformations [shapes] while transporting their substrate,” explained co-first author Shabareesh Pidathala, PhD, at St. Jude department of structural biology. “This is called alternating access transport, where the protein is either “outward” or “inward” facing … To completely gain mechanistic understanding at an atomic level, we needed to capture multiple conformations of this transporter.”

Through their studies the investigators confirmed that reserpine and tetrabenazine bind two different conformations of VMAT2. The discovery of this dynamic mechanism could open up multiple opportunities for drugs to bind. Previous studies had shown that the two drugs inhibit VMAT2 differently, the authors noted in their paper. “TBZ acts as a non-competitive inhibitor, whereas reserpine is a competitive inhibitor. Moreover, whereas TBZ is specific to VMAT2, reserpine can inhibit both isoforms.”

Pidathala added, “30 or 40 years of pharmacological research had suggested that these two drugs bind to the transporter in different ways, but nobody knew the atomic details of how this works. Our structures nicely demonstrate that these two drugs stabilize two different conformations of the transporter to block its activity.”

More specifically, the authors explained, “Our structural and functional analyses reveal how TBZ and reserpine selectively target different conformations of VMAT2, inhibiting its function by interfering with distinct steps of the transport cycle. TBZ traps VMAT2 in a lumen-facing occluded conformation, probably preventing its progression to the lumen-facing … On the other hand, reserpine binds VMAT2 only when the transporter vestibule faces the cytosol …”

The structure of VMAT2 with serotonin bound also allowed the researchers to pinpoint specific amino acids that interact with the neurotransmitter and drive transport. “We believe this is a common mechanism that this transporter uses to engage all the monoamines,” said Lee. The team added, “Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.”

While the reported results offers a huge leap forward in understanding monoamine transport, Lee and his team are delving deeper into its mechanism. For example, the intake of monoamines into vesicles is fueled by protons moving in the other direction. “We identified amino acids that are important for this proton-dependent process,” Lee said, “but we still don’t know how exactly protons drive this transport. Determining this mechanism is our future direction, which will help us to fully appreciate how this transporter works.” Nevertheless, the authors concluded, “These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.”