Proteins critical to cell electrical signaling built from scratch

The design of new calcium channels, built bottom-up from scratch, was reported last week in Nature.

Ion channels generate electrical impulses produced by living cells. Synthetic ion channels could serve as tools for biomedical research, from neuroscience experiments to cardiac biology models and synthetic cell signaling circuits.

“Biochemists have studied ion channels for decades and debated how they work for almost as long. We decided to create new versions to allow biologists to precisely control cell signaling,” said David Baker, professor of biochemistry and director of the UW Medicine Institute for Protein Design, where the new calcium channels were developed.

Natural calcium channels act like pores on the membranes of excitable cells, such as those found in nerves and muscles. The channels drive electrical activity by controlling the influx of charged particles called calcium ions. The channels regulate signals that direct muscle contraction, heart rate, and neurotransmitter release.

Messaging enabled by ion channels is an example of signal transduction, which allows cells to sense and respond to their environment. Signal transduction is one of the fundamental processes that makes a living thing alive.

The calcium channel design project demonstrated that even complex biochemical functions that remain only partially understood can now be built from first principles using artificial intelligence. Yulai Liu, a postdoctoral researcher at the UW Medicine Institute for Protein Design, led the effort to develop new calcium channels.

Natural calcium channels have been adapted as research tools in neuroscience, cardiology, toxicology and other fields. But these modified molecules are delicate and difficult to use. Creating simpler ion channels that achieve precise ion selectivity, showing a preference for the chemical element they let through, remains a major challenge.

The team used RFdiffusion, an artificial intelligence program, to construct calcium channels starting with the selective filter. This structure allows calcium ions to flow while blocking other ions like sodium. The researchers then generated supporting protein structures from this feature.

Unlike water-soluble proteins, which make up most of the structures known in protein databases, ion channels function within lipid membranes, the two-layer structure that surrounds a cell to separate its interior from its environment. The team had to adapt their protein design tools to generate appropriate amino acid sequences to form a chain that would then reliably fold into transmembrane ion channel proteins.

“By designing channels that can be precisely controlled, we hope to study and eventually manipulate cellular behaviors in entirely new ways,” Liu said.

The designed calcium channels were produced in insect cells and studied using patch-clamp electrophysiology, an established laboratory method for assessing ion channel function. Several of the designs conducted calcium as expected and achieved calcium selectivity: they were able to transmit approximately five times more current for calcium ions than for sodium ions.

Cryo-electron microscopy, a method for making high-resolution 3D images of biological molecules, revealed that a functional channel assembled exactly as expected. The protein backbone matched the computer model with atomic precision.

The team plans to apply its design approaches to explore the general physical principles underlying ion channel selectivity among ion channels. They are particularly interested in creating synthetic channels for metal ions. These efforts could deepen the understanding of how ions move across proteins embedded in membranes and how these mechanisms support complex processes such as brain signaling and immune cell activation.

UW Medicine neurobiologist and pharmacologist William A. Catterall, an international leader in the field of ion channel research, co-supervised his postdoctoral fellow, Lui, and contributed to this study before his sudden passing. During his career spanning more than 50 years, Catterall has made many fundamental discoveries about the molecular structure and function of ion channels, their role in conditions such as epilepsy, autism and cardiac arrhythmias, and how they function under treatments such as anesthesia and poison antidotes.

The Catterall laboratory in the Department of Pharmacology at the University of Washington School of Medicine provided electrophysiology expertise that guided the characterization of the designed proteins.