Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

Abstract

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryo-EM of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly-knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for seamless transition from inactivated to closed conformations.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

“…S9 C ). Although these densities were not visible beyond 4σ and hence are most likely to be water molecules, the periodicity of their appearance is striking and corresponds to densities that we observed in other RyR1 WT reconstructions ( 26 ) as well as in other ion channels ( 27 ).…”

“…The Ca 2+ binding site of RyR1 lies at the interface of CD and CTD with negatively charged and polar residues from both domains (i.e., Glu3893, Glu3967 from CD and Thr5001, His5002 from CTD). Coordination of Ca 2+ closes the cavity around the cation and triggers opening ( 21 , 26 ). Upon Ca 2+ binding, the carbonyls of E3967 and Thr5001 approach from 10.1 Å to 8.1 Å.…”

“…The ATP binding site is located in a nook created by the U-motif, CTD, and the S6 helix ( 21 , 26 ). The ATP binding site in the RyR1 Y523S quasi-open map (obtained in the absence of ATP) was empty, whereas the RyR1 Y523S open + map clearly displays the density corresponding to the added ATP ( Fig.…”

“…Although the densities were not strong enough to be modeled, they appear to correspond to long alkyl chains of a lipid molecule, which was likely 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) that was used in the reconstitution in nanodiscs. We previously modeled phosphatidyl choline in the same crevice for RyR1 WT under inactivated conditions ( 26 ). Of note, this density is not visible in the RyR1 Y523S open + structure with the crevice appearing empty, which is also in agreement with our previous data indicating absence of lipids in open RyR1 ( 26 ).…”

“…We previously modeled phosphatidyl choline in the same crevice for RyR1 WT under inactivated conditions ( 26 ). Of note, this density is not visible in the RyR1 Y523S open + structure with the crevice appearing empty, which is also in agreement with our previous data indicating absence of lipids in open RyR1 ( 26 ).…”