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 heart biology models and synthetic cell signaling circuits. 

“Biochemists have been studying ion channels for decades and debating about how they work for almost as long. We set out to build new versions to allow biologists to precisely control cellular signaling,” said David Baker, professor of biochemistry and director of the UW Medicine Institute for Protein Design, where the new calcium channels were developed.  

Naturally occurring calcium channels act as 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, heartbeat and neurotransmitter release. 

The messaging made possible by ion channels is one example of signal transduction, which enables 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 scholar at the UW Medicine Institute for Protein Design, led the effort to develop novel calcium channels.  

Natural calcium channels have been adapted as tools for research in the neurosciences, cardiology, toxicology and other fields. But these modified molecules are delicate and challenging to work with. Creating ion channels that are simpler and which achieve precise ion selectivity — showing preference in the chemical element it allows to pass through — has remained a major challenge. 

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

Unlike water-soluble proteins, which make up most known structures in protein databases, ion channels function within lipid membranes, the two-layer structure that surrounds a cell to separate its insides 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 engineering channels that can be precisely controlled, we hope to study, and eventually manipulate, cellular behaviors in entirely new ways,” said Liu. 

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

Cryoelectron microscopy, a method to make high-resolution 3D images of biological molecules, revealed that one functional channel assembled exactly as designed. The protein backbone matched the computational model with atomic precision.  

The team envisions applying their design approaches to explore the general physical principles underlying ion channels’ selectivity among ion channels. They are especially interested in creating synthetic channels for metal ions. These efforts could deepen understanding of how ions move through proteins embedded in membranes and how such mechanisms support complex processes like brain signaling and immune cell activation. 

UW Medicine neurobiologist and pharmacologist William A. Catterall, a leading figure internationally in the field of ion channel research, co-mentored his postdoc, Lui, and contributed to this study before his sudden passing. Over his more than 50-year career, Catterall made many fundamental discoveries about ion channels’ molecular structure and function, their role in conditions like epilepsy, autism and heart arrhythmias, and how they operate under treatments, such as anesthesia and poison antidotes. 

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

The research was funded by The Audacious Project, Howard Hughes Medical Institute, Gates Foundation (INV-043758), Wu Tsai Protein Innovation Fund, Open Philanthropy, Alexandria Venture Investments Translational Investigator Fund, Nan Fung Life Sciences Translational Investigator Fund, BASF Corporation, and United States Air Force Office of Scientific Research (FA9550-22-1-0506), Department of Energy (DE-SC0018940), and National Institutes of Health (R01HL112808, R01AG063845). 

This news release was adapted from a UW Medicine Institute for Protein Design blog written by Ian C. Haydon.