The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

The voltage-gated calcium (Ca) channels convert membrane electrical signals to intracellular Ca-mediated events. Among the ten subtypes of Ca channel in mammals, Ca1.1 is specified for the excitation-contraction coupling of skeletal muscles. Here we present the cryo-electron microscopy structure of the rabbit Ca1.1 complex at a nominal resolution of 3.6 Å. The inner gate of the ion-conducting α1-subunit is closed and all four voltage-sensing domains adopt an 'up' conformation, suggesting a potentially inactivated state. The extended extracellular loops of the pore domain, which are stabilized by multiple disulfide bonds, form a windowed dome above the selectivity filter. One side of the dome provides the docking site for the α2δ-1-subunit, while the other side may attract cations through its negative surface potential. The intracellular I-II and III-IV linker helices interact with the β-subunit and the carboxy-terminal domain of α1, respectively. Classification of the particles yielded two additional reconstructions that reveal pronounced displacement of β and adjacent elements in α1. The atomic model of the Ca1.1 complex establishes a foundation for mechanistic understanding of excitation-contraction coupling and provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Ca and Na channels.

Voltage-gated sodium (Na) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Na channel has hampered mechanistic understanding. Here, we report the cryo-electron microscopy structure of the human Na1.4-β1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the β1 subunit, providing insight into the molecular basis for Na permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Na channels. The structure provides a path toward mechanistic investigation of Na channels and drug discovery for Na channelopathies.

Voltage-gated sodium (Na) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Na channel from American cockroach (designated NaPaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSD, and a carboxy-terminal domain binds to the III-IV linker. The structure of NaPaS establishes an important foundation for understanding function and disease mechanism of Na and related voltage-gated calcium channels.

The voltage-gated calcium channel Ca(v)1.1 is engaged in the excitation-contraction coupling of skeletal muscles. The Ca(v)1.1 complex consists of the pore-forming subunit α1 and auxiliary subunits α2δ, β, and γ. We report the structure of the rabbit Ca(v)1.1 complex determined by single-particle cryo-electron microscopy. The four homologous repeats of the α1 subunit are arranged clockwise in the extracellular view. The γ subunit, whose structure resembles claudins, interacts with the voltage-sensing domain of repeat IV (VSD(IV)), whereas the cytosolic β subunit is located adjacent to VSD(II) of α1. The α2 subunit interacts with the extracellular loops of repeats I to III through its VWA and Cache1 domains. The structure reveals the architecture of a prototypical eukaryotic Ca(v) channel and provides a framework for understanding the function and disease mechanisms of Ca(v) and Na(v) channels.

The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo–electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the μ-conotoxin KIIIA, in the presence of an auxiliary subunit, β2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of β2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.

Animal toxins that modulate the activity of voltage-gated sodium (Na) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Na channel NaPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSD and the pore of NaPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Na channel drugs.

The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca2+ from the sarcoplasmic/endoplasmic reticulum (SR/ER) to the cytoplasm, which is essential for the excitation-contraction (E-C) coupling of cardiac and skeletal muscles. The near-atomic resolution structure of closed RyR1 revealed the molecular details of this colossal channel, while the long-range allosteric gating mechanism awaits elucidation. Here, we report the cryo-EM structures of rabbit RyR1 in three closed conformations at about 4 Å resolution and an open state at 5.7 Å. Comparison of the closed RyR1 structures shows a breathing motion of the cytoplasmic platform, while the channel domain and its contiguous Central domain remain nearly unchanged. Comparison of the open and closed structures shows a dilation of the S6 tetrahelical bundle at the cytoplasmic gate that leads to channel opening. During the pore opening, the cytoplasmic “O-ring” motif of the channel domain and the U-motif of the Central domain exhibit coupled motion, while the Central domain undergoes domain-wise displacement. These structural analyses provide important insight into the E-C coupling in skeletal muscles and identify the Central domain as the transducer that couples the conformational changes of the cytoplasmic platform to the gating of the central pore.