Lipids modulate open probability of RyR1 under cryo-EM conditions

Abstract Ryanodine receptors (RyRs) are intracellular tetrameric ion channels responsible for Ca 2+ release from the sarcoplasmic and endoplasmic reticulum. Among the three known mammalian RyR isoforms, RyR1 is critical for muscle contraction and has been studied most extensively. The cytoplasm-exposed multidomain fragment of RyRs integrates multiple cellular signals that regulate channel gating and small deviations from the physiological open probability of RyRs leads to life-threatening diseases. While cryo-EM has been instrumental in revealing near-atomic details of RyR gating mechanisms, the open probability of RyR1 under cryo-EM conditions is notably lower than that observed in electrophysiological studies, complicating structural investigations of RyR1 gating modulation. Here, we present a cryo-EM study examining the open probability of RyR1 solubilized in CHAPS with varying lipid concentrations. We found that increasing lipid concentration from 0.001% to 0.05% raised the RyR1 open probability from 16 to 84%; however, RyR1 reconstituted into lipid nanodiscs remained closed. We modelled 72 lipid molecules in the map reconstructed at the highest lipid concentration. These findings demonstrate critical role of lipids in modulating RyR1 gating under cryo-EM conditions and suggest optimal lipid-mimetics for structural studies of RyR1 gating modulation.