How calcium channels in muscle cells open together

Every step we take begins with a burst of calcium inside our muscle cells, causing them to contract. To prepare for action, the cells keep calcium locked in an internal compartment, the sarcoplasmic reticulum. Studding its membrane are thousands of RyR1 channels—the largest known ion channels—that contain pores that release the calcium. For a muscle to contract properly, RyR1 channels must open synchronously via a mechanism known as “coupled gating.” How they accomplish this has been unclear since it was described almost 30 years ago.

A team led by Dr. Vasilii Mikirtumov, a former doctoral student in the In Situ Structural Biology Lab of Dr. Misha Kudryashev at the Max Delbrück Center, has now captured the first high-resolution 3D images of RyR1 at six stages of opening inside the intact sarcoplasmic reticulum membrane. The work is published in Nature Communications.

Although the structure of RyR1 has been described before, previous studies used channels that had been removed from the membrane. Instead, the Kudryashev lab studied the structure of RyR1 in its natural environment using the advanced imaging techniques of cryo-electron microscopy and tomography.

“Because RyR1 is a membrane protein, you have to pull it out with detergents to purify it. But that environment can be disruptive to such a sensitive protein,” says Mikirtumov, who is now a postdoctoral researcher in the lab of Christian Spahn at Charité – Universitätsmedizin Berlin. “We wanted to capture the structure of the channel in its native membrane and find out whether its opening mechanism looks different there.”

The researchers found that it does. The images show that neighboring channels remain in contact with each other when transitioning from the closed to open states. This contact, or interface, mediates coupled gating: As one channel rotates open, it strains the interface with its neighbor, making it easier for that channel to rotate and open too. “It’s like the cogs in a clock,” says Kudryashev, senior author of the paper. “Once one cog turns, it primes its neighbors to turn, too.”

Six snapshots of an opening channel

The team isolated the sarcoplasmic reticulum from rabbit muscle and imaged it at the Core Facility for Cryo-Electron Microscopy, which is run jointly by Charité – Universitätsmedizin Berlin, the Max Delbrück Center and the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP). The Kudryashev lab has specialized expertise in cryo-electron microscopy and tomography and the computational tools necessary to analyze the resulting data.

“We shoot electrons through the sample and take thousands of pictures, each with many copies of the same protein,” explains Mikirtumov. “Then we average them all together, and that gives us a high-resolution 3D reconstruction.”

By adding small molecules to initiate channel opening, the researchers caught RyR1 at six stages between fully closed and fully open. Comparing the structures revealed the full opening motion: The bulky outer part rotates within the plane of the membrane, like turning the ring of a camera lens, while the pore in the channel widens to roughly twice its original width.

Using cryo-electron tomography, the team also imaged pairs of neighboring channels at five stages of opening. They found neighboring channels were more likely to be synchronized and that two interacting closed channels were more stable than two closed channels in isolation. These findings support coupled gating and suggest that channels hold each other shut.

A target for treating muscle disease

Mutations in the RYR1 gene cause malignant hyperthermia, a life-threatening reaction to some anesthetics, and congenital myopathies that weaken muscles. Many of these mutations alter the channel exactly where it touches its neighbor.

“A lot of these mutations don’t seem to affect how a single channel opens, but rather how channels cooperate with their neighbors,” says Mikirtumov. “We mapped several of them onto the interface, and we think that in these cases, it’s the cooperation between channels that breaks down.”

The researchers propose that disruption at the interface between channels makes them leaky, releasing calcium when they should retain it. This makes the interface itself a target for therapies.

The team is already testing the idea. “We need to prevent the channels from opening spontaneously,” says Kudryashev. “Now that we know how the inactive state is organized, we can design biologics or small molecules to stabilize this closed state.”

Who’s behind this story?

Sadie Harley

BSc Life Sciences & Ecology. Microbiology lab background with pharmaceutical news experience in oil, gas, and renewable industries.

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Robert Egan

Bachelor’s in mathematical biology, Master’s in creative writing. Well-traveled with unique perspectives on science and language.

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