Summary The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.

Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+.

Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents were oxidized, cysteine-nitrosylated, and depleted of the channel stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability leading to intracellular calcium leak in skeletal muscle. Similarly, six-month old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month old wild type mice. Treating aged mice with S107, stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle specific force and exercise capacity. Taken together these data indicate that leaky RyR1 contribute to age-related loss of muscle function.

Ryanodine receptors (RyRs)/Ca2+ release channels, on the endoplasmic and sarcoplasmic reticulum of most cell types, are required for intracellular Ca2+ release involved in diverse cellular functions, including muscle contraction and neurotransmitter release. The large cytoplasmic domain of the RyR serves as a scaffold for proteins that bind to and modulate the channel's function and that comprise a macromolecular signaling complex. These proteins include calstabins [FK506-binding proteins (FKBPs)], calmodulin (CaM), phosphodiesterase, kinases, phosphatases, and their cognate targeting proteins. This review focuses on recent progress in the understanding of RyR regulation and disease mechanisms that are associated with channel dysfunction.

The VDAC (voltage-dependent anion channel) plays a central role in apoptosis, participating in the release of apoptogenic factors including cytochrome c. The mechanisms by which VDAC forms a protein-conducting channel for the passage of cytochrome c are not clear. The present study approaches this problem by addressing the oligomeric status of VDAC and its role in the induction of the permeability transition pore and cytochrome c release. Chemical cross-linking of isolated mitochondria or purified VDAC with five different reagents proved that VDAC exists as dimers, trimers or tetramers. Fluorescence resonance energy transfer between fluorescently labelled VDACs supports the concept of dynamic VDAC oligomerization. Mitochondrial cross-linking prevented both permeability transition pore opening and release of cytochrome c, yet had no effect on electron transport or Ca 2+ uptake. Bilayer-reconstituted purified cross-linked VDAC showed decreased conductance and voltage-independent channel activity. In the dithiobis(succinimidyl propionate)-cross-linked VDAC, these channel properties could be reverted to those of the native VDAC by cleavage of the cross-linking. Cross-linking of VDAC reconstituted into liposomes inhibited the release of the proteoliposome-encapsulated cytochrome c. Moreover, encapsulated, but not soluble cytochrome c induced oligomerization of liposome-reconstituted VDAC. Thus the results indicate that VDAC exists in a dynamic equilibrium between dimers and tetramers and suggest that oligomeric VDAC may be involved in mitochondriamediated apoptosis.

SummaryThe type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca 2+ ) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca 2+ , ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca 2+ alone induce conformational changes in the cytoplasmic assembly ('priming'), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP /PS1). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca) leak. RyR-mediated ER Ca leak activates Ca-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.