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Structural basis for high-voltage activation and subtype-specific inhibition of human Na_v1.8.

ABSTRACT: The dorsal root ganglia-localized voltage-gated sodium (Na_v) channel Na_v1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Na_v1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Na_v1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSD_I) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSD_I and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6_IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6_I and Gly1406 on S6_III, allosterically modulate the channel's sensitivity to A-803467. Comparison of available structures of human Na_v channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

SUBMITTER: Huang X

PROVIDER: S-EPMC9335304 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8.

Huang Xiaoshuang X Jin Xueqin X Huang Gaoxingyu G Huang Jian J Wu Tong T Li Zhangqiang Z Chen Jiaofeng J Kong Fang F Pan Xiaojing X Yan Nieng N

Proceedings of the National Academy of Sciences of the United States of America 20220719 30

The dorsal root ganglia-localized voltage-gated sodium (Nav) channel Nav1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Nav1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Nav1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensin ...[more]

PMID: 35858452

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Project description:Pharmacologic approaches for the treatment of atrial arrhythmias are limited due to side effects and low efficacy. Thus, the identification of new antiarrhythmic targets is of clinical interest. Recent genome studies suggested an involvement of SCN10A sodium channels (NaV1.8) in atrial electrophysiology. This study investigated the role and involvement of NaV1.8 (SCN10A) in arrhythmia generation in the human atria and in mice lacking NaV1.8. NaV1.8 mRNA and protein were detected in human atrial myocardium at a significant higher level compared to ventricular myocardium. Expression of NaV1.8 and NaV1.5 did not differ between myocardium from patients with atrial fibrillation and sinus rhythm. To determine the electrophysiological role of NaV1.8, we investigated isolated human atrial cardiomyocytes from patients with sinus rhythm stimulated with isoproterenol. Inhibition of NaV1.8 by A-803467 or PF-01247324 showed no effects on the human atrial action potential. However, we found that NaV1.8 significantly contributes to late Na+ current and consequently to an increased proarrhythmogenic diastolic sarcoplasmic reticulum Ca2+ leak in human atrial cardiomyocytes. Selective pharmacological inhibition of NaV1.8 potently reduced late Na+ current, proarrhythmic diastolic Ca2+ release, delayed afterdepolarizations as well as spontaneous action potentials. These findings could be confirmed in murine atrial cardiomyocytes from wild-type mice and also compared to SCN10A-/- mice (genetic ablation of NaV1.8). Pharmacological NaV1.8 inhibition showed no effects in SCN10A-/- mice. Importantly, in vivo experiments in SCN10A-/- mice showed that genetic ablation of NaV1.8 protects against atrial fibrillation induction. This study demonstrates that NaV1.8 is expressed in the murine and human atria and contributes to late Na+ current generation and cellular arrhythmogenesis. Blocking NaV1.8 selectively counteracts this pathomechanism and protects against atrial arrhythmias. Thus, our translational study reveals a new selective therapeutic target for treating atrial arrhythmias.

Project description:Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

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Structural basis for high-voltage activation and subtype-specific inhibition of human Na_v1.8.

Publications

Structural basis for high-voltage activation and subtype-specific inhibition of human Na<sub>v</sub>1.8.

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