Interactions of Key Charged Residues Contributing to Selective Block of Neuronal Sodium Channels by μ-Conotoxin KIIIA

McArthur, Jeffrey R.; Singh, Gulzar; McMaster, D.; Winkfein, Robert J.; Tieleman, D. Peter; French, Robert J.

doi:10.1124/mol.111.073460

Cited by 48 publications

(148 citation statements)

References 36 publications

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“…Thus, relative to the size of the functional Nav, their positions are defined well. Our experimental LRET data, from the use of multiple toxin-dye/LBT pairs, indicate locations of the bound toxins on Nav1.4 that are in good agreement with those locations in recently proposed toxin docking models (21,22). Positions of the bound Bodipy-labeled toxins are shown schematically in Fig.…”

Section: Discussionmentioning

confidence: 52%

“…For the smallest of these μ-conotoxins, KIIIA, blockade of single-channel conductance is less than complete, and simultaneous occupancy by the classic all-or-none Nav blocker, TTX, is possible. Homology models for Navs indicate that the larger, archetypal GIIIA functionally occludes the ion-conducting pore, whereas KIIIA's center of mass is slightly shifted toward the VSDs of DII and DIII (21,22). The positions of the Bodipy acceptor in our toxin-containing dyes are effectively constrained by site-specific conjugation to Ts1-W50X, to the N terminus of KIIIA-N3X, or to GIIIA-T5A-1X (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Conversely, KIIIA is a shorter peptide of only 16 aa that also blocks Na + ion conductance through Nav1.4. However, the blockage by KIIIA is incomplete, allowing guanidinium toxins like tetrodotoxin (TTX) access to a deeper pocket of site 1 of most Nav isoforms, as indicated by experiments where complete block is achieved when TTX and KIIIA are applied simultaneously (21,22,30,32).…”

Section: Resultsmentioning

confidence: 99%

“…Binding sites for Nav-specific toxins, as well as their binding characteristics, have been described functionally by electrophysiological assays, and theoretically by computational molecular modeling based on currently available crystal structures (21)(22)(23)(24)(25)(26). Current knowledge for the physical locations of the toxins, however, remains somewhat imprecise.…”

Section: Discussionmentioning

confidence: 99%

“…1B). The function and binding sites in mammalian Navs for these three toxins have been well characterized by electrophysiological experiments and computational modeling; therefore, they function as trustworthy references for dynamics of the donor movement (21)(22)(23)(24)(25)(26). We generated a total of 12 different Nav1.4 clones as LRET donors, which retained functional activity.…”

Section: Significancementioning

confidence: 99%

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Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Kubota

Durek

Dang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.V oltage-gated sodium channels (Navs) play an essential role in the generation and propagation of action potentials in excitable cells (1). Eukaryotic Navs are composed of a poreforming α subunit and auxiliary β subunits. The α subunit is a large single-polypeptide chain organized in four different domains (DI-DIV), each of which has a voltage-sensing domain (VSD; S1-S4 segments) and a pore-forming domain (S5-S6 segments). Each domain has a different amino acid composition, pointing to some level of functional specialization. Site-directed fluorimetry shows that the VSDs in DI, DII, and DIII of the rat skeletal muscle voltage-gated sodium channel (Nav1.4) are activated by depolarization faster than in DIV (2). From this observation, it has been hypothesized that DI-III VSDs control the pore opening of the mammalian Nav, whereas the DIV VSD governs its fast inactivation (2-5).Although mammalian Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. The crystal structures of several prokaryotic Navs have been solved recently; however, in contrast to the mammalian Navs, they are homotetrameric, and thus structurally more closely related to the organization of voltage-gated potassium channels (Kvs) (6-9). The structure of a human L-type voltage-gated calcium channel type 1.1 (Cav1.1), which is a large single polypeptide composed of four different domains similar to mammalian Navs, has been resolved using cryoelectron microscopy (10, 11). However, functional studies have shown that gating mechanisms of mammalian Cav channels are indeed different from gating mechanisms in mammalian Navs (12). Furthermore, such structural studies only provide static snapshots of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are nee...

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Section: Discussionmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Kubota

Durek

Dang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Re‐engineering the μ‐conotoxin SIIIA scaffold

2014

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Voltage-gated sodium (Nav) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Nav channel function and development of effective therapeutics. µ-Conotoxin SIIIA is a potent mammalian Nav 1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ-SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3-20) mutant with enhanced net positive charge showed a dramatic increase in its Nav 1.2 potency (IC50 of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Nav channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective NaV blockers.

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Selective Ligands and Drug Discovery Targeting the Voltage-Gated Sodium Channel Nav1.7

Payandeh¹,

Hackos²

2018

Handbook of Experimental Pharmacology

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The voltage-gated sodium (Nav) channel Nav1.7 has been the focus of intense investigation in recent years. Human genetics studies of individuals with gain-of-function and loss-of-function mutations in the Nav1.7 channel have implicated Nav1.7 as playing a critical role in pain. Therefore, selective inhibition of Nav1.7 represents a potentially new analgesic strategy that is expected to be devoid of the significant liabilities associated with available treatment options. Although the identification and development of selective Nav channel modulators have historically been challenging, a number of recent publications has demonstrated progression of increasingly subtype-selective small molecules and peptides toward potential use in preclinical or clinical studies. In this respect, we focus on three binding sites that appear to offer the highest potential for the discovery and optimization of Nav1.7-selective inhibitors: the extracellular vestibule of the pore, the extracellular loops of voltage-sensor domain II (VSD2), and the extracellular loops of voltage-sensor domain IV (VSD4). Notably, these three receptor sites on Nav1.7 can all be defined as extracellular druggable sites, suggesting that non-small molecule formats are potential therapeutic options. In this chapter, we will review specific considerations and challenges underlying the identification and optimization of selective, potential therapeutics targeting Nav1.7 for chronic pain indications.

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Interactions of Key Charged Residues Contributing to Selective Block of Neuronal Sodium Channels by μ-Conotoxin KIIIA

Abstract: Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias

Cited by 48 publications

References 36 publications

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Re‐engineering the μ‐conotoxin SIIIA scaffold

Selective Ligands and Drug Discovery Targeting the Voltage-Gated Sodium Channel Nav1.7

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