Every step we take begins with a burst of calcium inside our muscle cells, causing them to contract. Using cryo-electron microscopy (cryo-EM), researchers at the Max Delbrück Center have revealed how muscle cell calcium channels, known as type-1 ryanodine receptors (RyR1), open synchronously. The team, led by Vasilii Mikirtumov, PhD, a former doctoral student in the in situ structural biology lab of Misha Kudryashev, PhD, suggests that their findings may explain a mechanism behind serious muscle diseases and potentially point to therapeutic targets.

Kudryashev is senior and corresponding author of the scientists’ published paper in Nature Communications, titled “Ligand-induced activation of RyR1 in native membranes,” in which they conclude that their findings “… explain the mechanism of ligand-gated activation of RyR1 in native membranes and provide structural insights critical for understanding RyR1-associated skeletal muscle diseases and developing targeted therapeutic strategies.”

Skeletal muscle contraction relies on “… a precise interplay between electrical signals and Ca2+ release from internal stores,” the authors explained. Maintaining readiness for contraction, the cells keep calcium locked in this internal compartment, the sarcoplasmic reticulum (SR). Studding its membrane are thousands of RyR1 channels—the largest known ion channels—containing pores that release 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. “…  the physical mechanism orchestrating this cooperativity has remained unknown,” the authors noted in their paper.

Through their reported studies Mikirtumov, Kudryashev, and colleagues have captured the first high-resolution 3D images of RyR1 at six stages of opening inside the intact sarcoplasmic reticulum membrane. The structure of RyR1 has been described before, but 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-EM 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,” explained 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 team isolated the SR 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-EM 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,” explained 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.

The resulting images showed 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,” said Kudryashev. “Once one cog turns, it primes its neighbors to turn, too.”

In their paper the team wrote in summary, “The RyR1 structures in four distinct as well as two intermediate functional states induced by activating ligands showed that the activation of RyR1 includes a large rotation component in the membrane plane. The structures reveal direct, corner-to-corner physical contacts between receptors, providing a high-resolution view of the interface that mediates coupled gating.”

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.

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.

Mikirtumov added, “A lot of these mutations don’t seem to affect how a single channel opens, but rather how channels cooperate with their neighbors. 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. “We propose a mechanism for RyR1 activation, shifting the focus from an individual channel event to a cooperative process orchestrated by a mechanically coupled receptor lattice,” they stated.

The team is already testing the idea. “We need to prevent the channels from opening spontaneously,” said Kudryashev. “Now that we know how the inactive state is organized, we can design biologics or small molecules to stabilize this closed state.” The authors concluded, “These results emphasize the physiological importance of RyR1 arrays as integrated functional units and provide new perspectives on EC-coupling and the development of targeted therapeutics for RyR1-linked channelopathies.”