In a tour-de-force of cryo-electron microscopy (cryo-EM), UC San Francisco researchers have become to the first to solve the structure of a hard-working protein that helps reload neurons for repeated firing, enabling signals to be relayed along nerve pathways throughout the brain.
More importantly, the scientists were able to use the structure of the protein, a vesicular glutamate transporter (VGLUT2), to determine how it most likely works, potentially a first step in drug development.
The study, published online in Science on May 22, 2020, was led by first author Fei Li, PhD, a UCSF postdoctoral scholar, along with principal investigators Robert Edwards, MD, professor of neurology and physiology, and Robert Stroud, PhD, professor of biochemistry and biophysics.
Glutamate is an “excitatory” neurotransmitter that plays a role in essentially all functions of the brain. When it is released by vesicles within a firing neuron into the gap between neurons, called the synapse, the neurotransmitter makes it more likely that the downstream neuron also fires.
Whether you’re a jellyfish or a human, VGLUTs are so vital for recycling glutamate that even small mutations in their DNA are likely be incompatible with life. The transporters, by loading vesicles that continually fuse with the synaptic membrane to release the neurotransmitter – only to reform and begin the cycle over again – make it possible for the neuron to transmit electrical signals hundreds of times per second.
But too much firing of glutamate-driven nerve circuitry can result in epilepsy. Excessive release of glutamate during ischemic stroke can actually contribute to the death of neurons, a process called “excitotoxicity.” Excitotoxicity also is thought to play a role in chronic neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (ALS).
No specific role has been discovered yet for VGLUT proteins in these diseases, but the subtle mechanisms for regulating its activity identified by the researchers define the parameters of normal brain function, according to Edwards.
“We have identified mechanisms based on this structure which could regulate glutamate availability, and which upon further investigation might reveal a way to pharmaceutically manipulate the functioning of the transporter and vesicle to prevent glutamate from being released in the wrong place, in the wrong amount at the wrong time,” he said.
Li and her senior collaborators found that the complex mechanism involves two positively charged pieces of the VGLUT2 protein grasping onto negatively charged glutamate molecules. They also figured out how chloride moves through VGLUT2 in the opposite direction to power packaging glutamate into synaptic vesicles. In addition, they determined how all these actions depend on low vesicle pH, restricting VGLUT2 activity to the vesicle rather than the cell surface.
To resolve the protein’s structure in sufficient detail to make these discoveries, the researchers had to grapple with challenges posed by the protein’s small size, Stroud said. So did the fact that, like other proteins that transport chemicals across membranes, dynamic changes in the shape of VGLUT2 make it difficult to take a snapshot of a single state within the surrounding vesicle membrane. But advances in CryoEM technology developed by David Agard, PhD, and colleagues at UCSF ultimately proved up to the task.
“This is the first structural determination of a neurotransmitter transporter that loads synaptic vesicles and one of the very smallest structures ever determined by cryo-EM,” Stroud said. “By using detectors developed at UCSF that can directly detect single electrons, we can obtain pictures quickly and assemble them into movies. We use powerful algorithms to analyze these multiple images to correct for the very small motion caused by bombardment with electrons, yielding much greater resolution.”