BUFFALO, N.Y. — When it comes to brain proteins, small changes can make a dramatic difference. Researchers studying NMDA (N-methyl-D-aspartate) receptors, which are essential for learning, memory and moment-by-moment consciousness, know that even slight changes in their activity level can mean the difference between normal function and serious neurological disorders.
Now, University at Buffalo researchers in a long-term collaboration with scientists at the Vollum Institute have captured for the first time and in exquisite detail pictures of receptors in a fully open conformation. The paper also describes for the first time the sequence of structural changes that transform receptors from being silent to being fully active.
Published today in Science Advances, the team's work expands the understanding of how NMDA receptors function and how their malfunction contributes to disease.
Gabriela Popescu, PhD, professor of biochemistry in the Jacobs School of Medicine and Biomedical Sciences at UB, is a corresponding author on the paper with Eric Gouaux, PhD, and Farzad Jalali-Yazdi, scientists at the Vollum Institute at Oregon Health & Science University. Lead authors are Jamie A. Abbott, PhD, research scientist in the Department of Biochemistry at UB, and Junhoe Kim, PhD, postdoctoral fellow at the Vollum Institute. Beiying Liu, PhD, is a co-author in the UB Department of Biochemistry.
Popescu has spent her career studying the biophysical properties of NMDA receptors, which generate electrical current in response to binding glutamate and help neurons communicate.
Why neurodegeneration happens
"NMDA receptors are key to ongoing brain activity," she says, "but when they are inappropriately active, they kill neurons, such as, for example, after a traumatic brain injury, after a stroke, or during progressive neurodegenerative diseases like Parkinson's disease and Alzheimer's disease."
For that reason, Popescu and her colleagues have been focused on trying to understand and track the sequence of events that transform resting, inactive receptors into active, electricity-generating receptors. A key part of assembling this sequence requires obtaining pictures of receptors in different functional states to identify which parts change position during activation, enabling the transition. While previous research has reported atomic-resolution structures for receptors in the resting state, Popescu says that structures for active, pore-open receptors have remained elusive.
Kinks in the helices
The researchers used cryogenic electron microscopy (cryo-EM), which involves flash-freezing samples of the receptors, then probing them with an electron beam to get a 3D view. The result is the first-ever, high-resolution image of a stable, open NMDA receptor. Unlike closed-pore receptors, in which the pore's four spiral structures, or helices, are straight, the researchers found that when the pore opens, the helices are bent or kinked.
It wasn't what they expected. "We thought that the channel would open simply by prying apart the four helical rods that prevent ion flow to widen the mouth of the pore," says Popescu. "Instead, we saw that all four rods are kinked outward and stabilized in this splayed conformation by new contacts with distant parts of the molecule."
Each kink, she explains, represents a region of interest in the receptor. "The identity of the amino acid at that position is essential as it must allow the kinking," she says. "Without the image, without knowing that the helix must bend, we would not understand why this particular residue is important."
The new observations will help researchers figure out the step-by-step process by which the receptor pore opens; they also provide insight into which other parts of the receptor may also play a role. There are many patients, Popescu notes, with single-point mutations in the DNA coding for this receptor. A point mutation is a change in a single amino acid in the receptor's structure. "The new information helps us better understand what these amino acid residues do, why they are important and what goes wrong when they are altered," she adds.
Structure-function findings
The researchers also reveal the first mechanism of NMDA receptor activation integrating functional evidence obtained in Popescu's lab from single-molecule electrophysiology recordings with structural evidence from single-particle cryo-EM obtained in the lab of co-senior author Eric Gouaux, PhD, senior scientist at the Vollum Institute.
"Before this study, we knew that the receptor must change its structure several times to transition from resting to primed to open," says Popescu. "Separately, structural studies produced pictures of receptors that looked a little different, but because we didn't know how the final open state looked, we couldn't arrange the pictures in the correct sequence."
Popescu is now able to connect the new images of the receptor with her previous findings on the rate with which receptors change conformation to complete the activation cycle. Knowing how the receptor looks at each point in the sequence helps isolate which regions play key roles in the transition and why and how drugs bind to these regions, as well as why mutations in these regions cause disease.
"The work that my lab has done over the past 20 years can tell us how drugs or mutations change the current that glutamate elicits when it binds to the NMDA receptor," she says. "Ultimately, the intensity, duration and frequency of this current — the tiny electrical signals that these receptors produce — determine how neurons communicate and whether our thoughts, feelings and emotions reflect reality in a way that helps us survive in a complex world. The new results show us why drugs and mutations change the current and can point us to ways to restore normal function."
The work was funded by the National Institutes of Health.
