Chilled brain molecules reveal epilepsy drug mechanism

Researchers at Johns Hopkins Medicine have discovered how perampanel, a widely-used epilepsy drug, works to reduce the excitability of brain cells and control seizures. This was achieved by supercooling a molecule on brain cells' surface to approximately minus 180 degrees Celsius, nearly twice as cold as Antarctica's coldest places. The research, published on June 4 in Nature Structural & Molecular Biology, elucidates how perampanel interacts with the AMPA receptor, a critical brain cell surface molecule, which could pave the way for developing new treatments for other neurological disorders like Alzheimer’s disease, schizophrenia, learning disabilities, glioblastoma, and chronic pain.

The AMPA receptor is essential for the neurotransmitter glutamate, which activates brain cells (neurons) by binding to specific proteins on the cell surface. This binding allows positively-charged ions to flood into the neuron, activating it. Edward Twomey, Ph.D., an assistant professor at Johns Hopkins University School of Medicine, notes that AMPA receptors and glutamate are crucial for learning, memory, and encoding experiences. Many neurological diseases are linked to issues with these receptors and glutamate.

Twomey collaborated with neuroscientist Richard Huganir, Ph.D., who has been studying AMPA receptors for four decades, to delve into the receptors' structure and their glutamate binding process. Overactivation of AMPA receptors is known to cause epilepsy. Perampanel is the only medication approved by the U.S. Food and Drug Administration (FDA) to target these receptors, although other pharmaceutical companies are developing similar compounds.

“This drug was initially discovered in the 1980s, and its precise mechanism has been a long-standing mystery,” Twomey explains. Huganir adds that earlier research showed where perampanel binds to AMPA receptors but not how it disrupts ion flow.

To investigate the mechanism, the team employed cryo-electron microscopy (cryoEM), a technique that has advanced over the past two decades, allowing the study of structures a million times smaller than a human hair. Johns Hopkins postdoctoral fellow W. Dylan Hale, Ph.D., conducted the majority of the experiments and analyses at the Beckman Center for CryoEM. They examined millions of images of AMPA receptors in mouse and rat brain cells interacting with the perampanel drug GYKI-52466, looking at binding with and without glutamate.

The researchers found that two of the four glutamate binding sites on the AMPA receptor are crucial in blocking ion flow with GYKI-52466. When glutamate binds to the receptor, it pulls a strand that opens the ion channel, similar to a pull chain releasing water from a showerhead. The drug prevents this action by decoupling the glutamate binding regions, putting the receptor into a desensitized state.

To complement the cryoEM images, they performed electrical recordings of ion flow and physiological studies in mice. They used artificial intelligence and machine learning to combine the cryoEM images into a 3D reconstruction of the receptor, providing a detailed understanding of how perampanel inhibits the ion channel.

Huganir and Twomey plan to use cryoEM to study mutated AMPA receptors, aiming to understand structural issues causing dysfunction. “In theory, we could develop drugs to make the receptor more active to treat conditions where its structure is altered,” Huganir says.

Contributors to this study included researchers from Johns Hopkins, the University of Texas Health Science Center at Houston, and funding from various institutions, including the Searle Scholars Program and the National Institutes of Health.