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 Na
PaS) 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 Na
PaS establishes an important foundation for understanding function and disease mechanism of Na
and related voltage-gated calcium 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 Na
PaS 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 Na
PaS, 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 voltage-gated calcium (Casub.v) channels convert membrane electrical signals to intracellular Casup.2+-mediated events. Among the ten subtypes of Casub.v channel in mammals, Casub.v1.1 is ...specified for the excitationcontraction coupling of skeletal muscles. Here we present the cryo-electron microscopy structure of the rabbit Casub.v1.1 complex at a nominal resolution of 3.6. The inner gate of the ion-conducting 1-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 2-1-subunit, while the other side may attract cations through its negative surface potential. The intracellular III and IIIIV linker helices interact with the sub.1a-subunit and the carboxy-terminal domain of 1, respectively. Classification of the particles yielded two additional reconstructions that reveal pronounced displacement of sub.1a and adjacent elements in 1. The atomic model of the Casub.v1.1 complex establishes a foundation for mechanistic understanding of excitationcontraction coupling and provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Casub.v and Nasub.v channels.
Targeting sodium channelsVoltage-gated sodium (Nav) channels have been implicated in cardiac and neurological disorders. There are many subtypes of these channels, making it challenging to develop ...specific therapeutics. A core α subunit is sufficient for voltage sensing and ion conductance, but function is modulated by β subunits and by natural toxins that can either act as pore blockers or gating modifiers (see the Perspective by Chowdhury and Chanda). Shen et al. present the structures of Nav1.7 in complex with both β1 and β2 subunits and with animal toxins. Pan et al. present the structure of Nav1.2 bound to β2 and a toxic peptide, the µ-conotoxin KIIIA. The structure shows why KIIIA is specific for Nav1.2. These and other recently determined Nav structures provide a framework for targeted drug development.Science, this issue p. 1303, p. 1309; see also p. 1278The 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.
Structures of voltage-gated sodium channelsIn “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This ...difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery.Science, this issue p. eaau2486, p. eaau2596INTRODUCTIONThe nine subtypes of mammalian voltage-gated sodium (Nav) channels, Nav1.1 to Nav1.9, are responsible for the initiation and propagation of action potentials in specific excitable systems, among which Nav1.4 functions in skeletal muscle. Responding to membrane potential changes, Nav channels undergo sophisticated conformational shifts that lead to transitions between resting, activated, and inactivated states. Defects in Nav channels are associated with a variety of neurological, cardiovascular, muscular, and psychiatric disorders. In addition, Nav channels are targets for natural toxins and clinical therapeutics.Understanding the physiological and pathophysiological mechanisms of Nav channels requires knowing the structure of each conformational state. All eukaryotic Nav channels comprise a single polypeptide chain, the α subunit, that folds to four homologous repeats I to IV. Channel properties are modulated by one or two subtype-specific β subunits. Cryo–electron microscopy (cryo-EM) structures of two Nav channels, one from American cockroach and the other from electric eel, were resolved in two distinct conformations. However, the inability to record currents of either channel in heterologous systems prevented functional assignment of these structures. Structural elucidation of a functionally well-characterized Nav channel is required to establish a model for structure-function relationship studies.RATIONALEAfter extensive screening for expression systems, protein boundaries, chimeras, affinity tags, and combination with subtype-specific β subunits, we focused on human Nav1.4 in the presence of β1 subunit for cryo-EM analysis. The complex, which was transiently coexpressed in human embryonic kidney (HEK) 293F cells with BacMam viruses and purified through tandem affinity columns and size exclusion chromatography, was concentrated to ~0.5 mg/ml for cryo-EM sample preparation and data acquisition.RESULTSThe cryo-EM structure of human Nav1.4-β1 complex was determined to 3.2-Å resolution. The extracellular and transmembrane domains, including the complete pore domain, all four voltage-sensing domains (VSDs), and the β1 subunit, were clearly resolved, enabling accurate model building (see the figure).The well-resolved Asp/Glu/Lys/Ala (DEKA) residues, which are responsible for specific Na+ permeation through the selectivity filter, exhibit identical conformations to those seen in the other two Nav structures. A glyco-diosgenin (GDN) molecule, the primary detergent used for protein purification and cryo-EM sample preparation, penetrates the intracellular gate of the pore domain, holding it open to a diameter of ~5.6 Å. The central cavity of the pore domain is filled with lipid-like densities, which traverse the side wall fenestrations.Voltage sensing involves four to six Arg/Lys residues on helix S4 of the VSD. This helix moves “up” (away from the cytoplasm) in response to changes of the membrane potential, and this opens the channel finally. All four VSDs display up conformations. The movement of the gating charge residues is facilitated by coordination to acidic and polar residues on S1 to S3. The improved resolution allows detailed analysis of the coordination.The fast inactivation Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV inserts into a hydrophobic cavity enclosed by the S6 and S4-S5 segments in repeats III and IV. Analysis of reported functional residues and disease mutations corroborates our recently proposed allosteric blocking mechanism for fast inactivation.CONCLUSIONThe structure provides important insight into the molecular basis for Na+ permeation, electromechanical coupling, asynchronous activation, and fast inactivation of the four repeats. It opens a new chapter for studying the structure-function relationships of Nav channels, affords an accurate template to map mutations associated with diseases such as myotonia and periodic paralysis hyperkalemic, and illuminates a path toward precise understanding and intervention with specific Nav channelopathies.Voltage-gated sodium (Nav) 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 Nav channel has hampered mechanistic understanding. Here, we report the cryo–electron microscopy structure of the human Nav1.4-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 Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.
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 ryanodine receptors （RyRs） are intracellular calcium channels responsible for rapid release of Ca^2＋ 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 RyRI 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 A° resolution and an open state at 5.7 A°. 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.
Nav1.5, the primary voltage‐gated Na+ (Nav) channel in heart, is a major target for class I antiarrhythmic agents. Here we present the cryo‐EM structure of full‐length human Nav1.5 bound to ...quinidine, a class Ia antiarrhythmic drug, at 3.3 Å resolution. Quinidine is positioned right beneath the selectivity filter in the pore domain and coordinated by residues from repeats I, III, and IV. Pore blockade by quinidine is achieved through both direct obstruction of the ion permeation path and induced rotation of an invariant Tyr residue that tightens the intracellular gate. Structural comparison with a truncated rat Nav1.5 in the presence of flecainide, a class Ic agent, reveals distinct binding poses for the two antiarrhythmics within the pore domain. Our work reported here, along with previous studies, reveals the molecular basis for the mechanism of action of class I antiarrhythmic drugs.
The Cryo‐EM structure of human cardiac sodium channel Nav1.5 in complex with quinidine reveals the molecular basis for pore blockade of Nav1.5 by class Ic antiarrhythmic drugs. An advanced and comprehensive structural understanding of the mechanism of action of antiarrhythmic drugs will facilitate drug discovery targeting Nav channels.
Dysfunction of Na
v
1.5, the primary cardiac Na
v
channel, is associated with multiple arrhythmia syndromes, exemplified by type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). Establishment of ...the structure-function relationship and mechanistic understanding of the disease variants will facilitate the development of antiarrhythmic drugs. Here we report the cryo-EM structure of human Na
v
1.5-E1784K, the most common variant shared by LQT3 and BrS. Structural mapping of 91 LQT3-associated mutations reveal a hotspot that involves the fast inactivation segments. The high density of LQT3 mutation sites in this region can be reasonably interpreted by the “door wedge” model for fast inactivation, which was derived from our previous structural observations and is supported by a wealth of functional characterizations.
Na
v
1.5 is the primary voltage-gated Na
+
(Na
v
) channel in the heart. Mutations of Na
v
1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Na
v
1.5-E1784K variant at an overall resolution of 3.3 Å. The structure is nearly identical to that of the wild-type human Na
v
1.5 bound to quinidine. Structural mapping of 91- and 178-point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are clustered mainly to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our “door wedge” model for fast inactivation. The structural analysis presented here, with a focus on the impact of mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Na
v
1.5 channelopathies.
Voltage-gated sodium (Nav) channels govern membrane excitability by initiating and propagating action potentials. Consistent with their physiological significance, dysfunction, or mutations in these ...channels are associated with various channelopathies. Nav channels are thereby major targets for various clinical and investigational drugs. In addition, a large number of natural toxins, both small molecules and peptides, can bind to Nav channels and modulate their functions. Technological breakthrough in cryo-electron microscopy (cryo-EM) has enabled the determination of high-resolution structures of eukaryotic and eventually human Nav channels, alone or in complex with auxiliary subunits, toxins, and drugs. These studies have not only advanced our comprehension of channel architecture and working mechanisms but also afforded unprecedented clarity to the molecular basis for the binding and mechanism of action (MOA) of prototypical drugs and toxins. In this review, we will provide an overview of the recent advances in structural pharmacology of Nav channels, encompassing the structural map for ligand binding on Nav channels. These findings have established a vital groundwork for future drug development.
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