Neurons message each other with the help of excitatory or inhibitory neurotransmitters. But to actually deliver those messages, the neurotransmitters must dock with protein receptors embedded in the cell membranes of adjacent neurons. Of particular interest in the human brain is the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), which binds to several different receptors, including GABAA, producing sedative effects. These receptors turn out to be hugely important: malformed GABAA receptors can cause neurological disorders such as epilepsy, insomnia, anxiety, depression, and schizophrenia.
Not knowing the exact structure and mechanism of GABAA receptor activation has made it difficult to improve upon or develop new pharmacological treatments. But a pair of papers published January in Nature build on recent advances in atomic-level imaging techniques to start to solve the mystery of how neurotransmitters and receptors normally bind and how therapeutic drugs might mitigate dysfunctional binding.
“The detailed structural insights reported in the two papers … will probably provide a solid framework for structure-guided drug design and open up new avenues for drug-discovery research,” writes Michaela Jansen, a biophysicist at Texas Tech University Health Sciences Center in a Nature News and Views article on the two papers.
In the first study, researchers used cryo-electron microscopy (cryo-EM) to produce high-resolution 3D images of one of the most common types of GABAA receptor found in the human brain, a type called α1β3γ2 based on the composition of its protein subunits. Within the last few years, the development of a direct electron detector and the software algorithms used to convert images into 3D structures vaulted the resolution of cryo-EM technology into the atomic scale. X-ray crystallography had been the go-to method for determining the structure of biomolecules, but heteromeric proteins with asymmetry, like GABAA, don’t crystallize well, if at all.
Cryo-EM doesn’t require crystallization, so it allowed the researchers to examine fully intact GABAA receptors in a physiologically relevant state by embedding them in surrogate lipid bilayers made from a whole brain lipid extract. “If I had to list one reason why I find this study so impactful, that would the lipid context they are in,” says Jansen.
To aid 3D imaging and reconstruction, the researchers also used a brand new approach in which they attached an enlarged antibody molecule, called a megabody, to the GABAA receptor. The weighty megabody throws the GABAA molecules off balance when they are deposited onto an electron microscope sampling grid so that they can be imaged from a variety of angles. “This approach can be applied to any protein,” says study author Radu Aricescu, a structural biologist at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, and the University of Oxford.
The resulting structure revealed previously unattainable details about GABAA conformation in a lipid bilayer, providing clues about where GABA and other ligands that affect neurotransmission—including general anesthetics and barbiturates—may bind and how the binding sites may interact.
In the second study, members of the research team used the same methods to investigate a variety of real-world examples of ligands binding to GABAA, including the neurotransmitter GABA, the GABAA channel blocker picrotoxin, the compound bicuculline (which competes with GABA for the same binding site but has an opposite effect), and the benzodiazepines alprazolam (Xanax) and diazepam (Valium). Cryo-EM revealed the precise binding sites of the different ligands as well as conformational changes that this binding induced at other sites, thus altering the potential for neurotransmission.
“This study provides high confidence snapshots of how some of the most popular and historically famous prescription drugs bind to their target in the brain,” says Ryan Hibbs, a biophysicist at the University of Texas Southwestern Medical Center who was not involved in the study.
In one case, the team unexpectedly identified two binding sites for diazepam—one of which coincides with the drug’s dominant anti-anxiety effect, shown in previous research, and another associated with undesirable anesthetic side-effects, including fatigue, dizziness, and nausea. Aricescu believes that this demonstrates the method’s potential usefulness for pharmacological discovery. “Just by simply looking at this structure, we know how to modify the diazepam molecule so that it won’t be able to bind anymore to the anesthetic site,” he says.
But before researchers can go after the full array of viable drug targets, Aricescu says they need to first understand the entire signaling cycle of the GABAA molecule they started with, including the mechanisms that cause the receptor’s ion channel to open, and perhaps one or two other GABA receptor subtypes. This will provide “a reference framework for future mechanistic investigations of GABAergic signaling and pharmacology,” he says.
“These structures provide a launching point for studies on neurotransmitter receptor protein dynamics, which will further flesh out the mechanisms by which the GABAA channel opens, and by which benzodiazepines and other positive modulators potentiate that process,” Hibbs says.
Jansen adds that she would like to see better resolution of GABAA’s intracellular domain. “The intracellular domain is by far the most diverse domain of these channels,” she notes. “So, if one wanted to make very selective drugs, that would be the domain to aim for.”