Cryo-EM structures of apo and antagonist-bound human Cav3.1

Zhao, Yanyu; Huang, Guangli; Wu, Qiurong; Wu, Kun; Li, Ruiqi; Lei, Jianlin; Pan, Xiaojing; Yan, Nieng

doi:10.1038/s41586-019-1801-3

Cited by 151 publications

(192 citation statements)

References 74 publications

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“…Similar to the 3D EM reconstruction of human Cav3.1 (22), the intracellular segments, including CaM, are largely invisible, despite addition of CaM. Instead, the map of Nav1.5 contains an extra appendage contiguous with VSDI, reminiscent of the amino-terminal domain (NTD) in NavPaS (15,20) ( Figure S2C).…”

Section: Cryo-em Analysis Of Human Na V 15mentioning

confidence: 77%

“…The two structures will hereafter be referred to as Nav1.5T and Nav1.5Q for complexes with TTX and quinidine, respectively. Unlike Cav1.1 and Cav3.1, which undergo main chain shift of the pore-forming S6 segments when binding to pore blockers such as diltiazem, verapamil, and Z944 (22,23), the two structures of Nav1.5 remain nearly identical the root-mean-square deviation (RMSD) of 0.16 Å over 771 Cα atoms ( Figure 2A).…”

Section: Pore Blockade By Quinidinementioning

confidence: 99%

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Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Jin

Huang

et al. 2019

Preprint

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Among the nine subtypes of human voltage-gated Na + (Na v ) channels, tetrodotoxin (TTX)-resistant Na v 1.5 is the primary one in heart. More than 400 point mutations have been identified in Na v 1.5 that are associated with cardiac disorders exemplified by type 3 long QT syndrome and Brugada syndrome, making Na v 1.5 a common target for class I antiarrythmic drugs. Here we present the cryo-EM structures of Na v 1.5 bound to quinidine and TTX with resolutions at 3.2 Å and 3.3 Å, respectively, for the pore domain. The structures allow mapping of 278 disease-related mutations in the resolved region, establishing the framework for mechanistic investigation of these pathogenic mutations. Quinidine is positioned right below the selectivity filter and coordinated by residues from repeats I, III, and IV. Pore blockade is achieved through both direct obstruction of the ion permeation path and induced rotation of a Tyr residue that leads to intracellular gate closure. Structural comparison of Na v 1.5 with our previously reported Na v 1.2/1.4/1.7 reveals the molecular determinant for the different susceptibility to TTX by TTX-resistant and -sensitive Na v subtypes, and highlights the functional significance of the underexplored and least conserved extracellular loops.

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Section: Cryo-em Analysis Of Human Na V 15mentioning

confidence: 77%

Section: Pore Blockade By Quinidinementioning

confidence: 99%

Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Jin

Huang

et al. 2019

Preprint

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“…Earlier studies defined critical residues of the high-affinity metal binding site of Cav3.2, including an Asp189-Gly190-His191 motif in IS3-S4 and an additional Asp residue in IS2 (23) with His191 being a key residue absent in other Cav3 subunits (and any other VGCC -subunits). Highresolution cryo-EM structure of human Cav3.1 has been solved recently (35). Since Cav3.1 is not highly sensitive to zinc or redox-mediated modulation due to the absence of the critical histidine residue in its IS3-S4 loop, we obtained Cav3.2 structure using homology modeling and analyzed putative metal binding site ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These cysteines may be involved in disulfide bonds or oxidised to cysteine sulfinic (Cys-SO2H) or sulfonic (Cys-SO3H) acids (44). Cryo-EM structure of Cav3.1 revealed a disulphide bond between the C104 in IS1-IS2 and C889 on IIS5-IIS6 pore loop, which is unique to Cav3 channels (35). This bond was hypothesised to be important for the unique redox sensitivity of T-type Ca 2+ channels (35).…”

Section: Discussionmentioning

confidence: 99%

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

Huang

Shi

Liang

et al. 2020

Journal of Biological Chemistry

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T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

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“…Owing to the technological advancement of electron cryomicroscopy (cryo-EM), we were able to resolve the atomic structures of Cav and Nav channels from different species and in complex with distinct auxiliary subunits, animal toxins, and FDA-approved drugs (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Among these, human Nav1.7 is of particular interest to the pharmaceutical industry for the development of novel pain killers; mutations in Nav1.7 are associated with various pain disorders, such as indifference to pain or extreme pain syndrome (21)(22)(23)(24)(25)(26)(27)(28).…”

Section: Introductionmentioning

confidence: 99%

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na⁺channels in nanodisc

Gao

Valinsky

et al. 2020

Preprint

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NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the cryo-EM analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Navs, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaces the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 Å and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ~ 6 Å to ~ 4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.

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Cryo-EM structures of apo and antagonist-bound human Cav3.1

Cited by 151 publications

References 74 publications

Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na⁺channels in nanodisc

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Cryo-EM structures of apo and antagonist-bound human Cav3.1

Cited by 151 publications

References 74 publications

Structural basis for pore blockade of the human cardiac sodium channel Nav1.5 by tetrodotoxin and quinidine

Structural basis for pore blockade of the human cardiac sodium channel Nav1.5 by tetrodotoxin and quinidine

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+channels in nanodisc

Contact Info

Product

Resources

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Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na⁺channels in nanodisc