Sudha Chakrapani and her team of researchers understand that exploring the smallest molecular structures requires advanced instrumentation, big data and state-of-the-art shared resources. Recently appointed Director of the Cleveland Center for Membrane and Structural Biology (CCMSB), Chakrapani’s team is discovering breakthroughs in the new Cryo-EM Core facility, home to the first cryo-electron microscope in the Greater Cleveland area.
Cryo-electron microscopy has only recently become available for small protein targets and was the focus of the 2017 Nobel prize in chemistry. Chakrapani spearheaded CWRU’s initiatives to establish a high-resolution Cryo-EM Core facility that became operational in the summer of 2019.
The team’s research program focuses on understanding the molecular mechanisms that underlie synaptic transmission and neuronal excitability. The ion channels involved in these processes are important for physiological functions such as gastrointestinal motility and secretion, motor coordination, and pain transmission.
Over the last several months, her team resolved the first structures of full-length serotonin receptors and glycine receptors functioning naturally and with drugs regulating their function using Cryo-EM. These findings, which were recently published in the scientific journals Nature and Nature Communications, are paving the way for developing better therapies to treat certain neurological conditions. By combining powerful modalities and methods, researchers can zoom-in on fine details of various biological macromolecular drug targets and spur advances in structure-based drug design.
“Think of the brain as a conductor and the organ systems as the musicians in the orchestra,” said Chakrapani. “During the course of daily activities and sleep, the electrical activity in the brain needs to be orchestrated so that every organ in the body functions properly. For this coordination to happen effectively, the brain’s electrical activity must first be converted to chemical signals with the help of chemical messengers called neurotransmitters. Each neurotransmitter works through a unique receiver protein called a receptor. The receptor represents the communication portal and the neurotransmitter regulates opening and closing of this portal.”
One such portal is the glycine receptor, and glycinergic synapses play a central role in motor control and pain processing in the central nervous system. The glycine receptor has two gates: 1) the activation gate, and 2) the desensitization gate. When there is no message to be relayed, both the gates remain closed and the receptor is said to be in a resting state. The arrival and binding of the neurotransmitter glycine open both gates and the electrical message is allowed to pass through. This represents the open state of the receptor. This portal has checkpoints called selectivity filters which allow certain ions to pass and blocks other ions. All this happens in a millisecond, less than the blink of an eye.
The improper functioning of the glycine receptor is associated with neurological disorders such as hyperekplexia and epilepsy and can cause chronic pain. Mutations in glycine receptors lead to stiff person syndrome. In addition, during strychnine poisoning, the glycine receptor portal is irreversibly blocked and can result in death due to asphyxiation caused by paralysis of the neural pathways that control breathing.
In their recent work, the Cryo-EM Core team has isolated glycine receptors in their entirety and then assembled the purified receptor in membrane mimetics called nanodiscs. Nanodiscs are circular discs composed of lipids and proteins and allow studying of glycine receptors in native environments. The samples containing assembled nanodiscs were imaged in the presence and absence of neurotransmitter glycine under high-end electron microscopes. In these multi-million-dollar microscopes, the samples are imaged under extremely low (cryogenic) temperatures using very high-powered electron beams. Several thousands of images were collected and computationally aligned to generate three-dimensional models of the full-length receptor in distinct states.
These findings represent an important step towards a better understanding of maladies at the molecular level and will assist in discovering precision drug development to mitigate pain and suffering.
This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research, and the Stanford-SLAC Cryo-Electron Microscopy Facility. This work was supported by the National Institutes of Health grants R01GM131216, R35GM134896 to Sudha Chakrapani and the AHA postdoctoral Fellowship to Arvind Kumar. (20POST35210394) and Sandip Basak (17POST33671152).
Collaborators Shanlin Rao and Prof. Mark Sansom are at the University of Oxford.