Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

Zhang, Joel Z.; Yarov‐Yarovoy, Vladimir; Scheuer, Todd; Karbat, Izhar; Cohen, Lior; Gordon, Dalia; Gurevitz, Michael; Catterall, William A.

doi:10.1074/jbc.m112.370742

Cited by 69 publications

(65 citation statements)

References 31 publications

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“…Ts1 is a β-scorpion toxin isolated from the Brazilian scorpion, Tityus serrulatus (28). Such scorpion toxins bind to Navs through the S1-S2 and the S3-S4 linkers of the VSD in DII and the S5-S6 pore linker in DIII (25,26). Their mechanism of action has been described as a "voltage sensor trapping effect," wherein the β-scorpion toxin traps the DII VSD in an activated state, facilitating Nav opening (23,24).…”

Section: Resultsmentioning

confidence: 99%

“…Our results provide details about the locations of toxin-binding sites. For instance, mutations in the voltage sensor of DII, which alter the function of toxin Ts1, imply that this region may be part of the toxin-binding site (25,26). However, some of the effects of the mutations may be due to long-range allosteric modulations of a distant toxinbinding site.…”

Section: Discussionmentioning

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%

“…From mutagenesis-based structure/function studies, the Navbinding site for the Ts1 toxin has been proposed to be formed mainly by the DII VSD as mentioned above, with a minor contribution from the DIII pore linker (25,26). The μ-conotoxins are thought to bind to the outer vestibule of the channel and to occlude the Na + ion-conducting pathway physically (30).…”

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: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

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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Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

et al. 2014

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Ts1 toxin is a protein found in the venom of the Brazilian scorpion Tityus serrulatus. Ts1 binds to the domain II voltage sensor in the voltage-gated sodium channel Nav and modifies its voltage dependence. In the work reported here, we established an efficient total chemical synthesis of the Ts1 protein using modern chemical ligation methods and demonstrated that it was fully active in modifying the voltage dependence of the rat skeletal muscle voltage-gated sodium channel rNav1.4 expressed in oocytes. Total synthesis combined with click chemistry was used to label the Ts1 protein molecule with the fluorescent dyes Alexa-Fluor 488 and Bodipy. Dye-labeled Ts1 proteins retained their optical properties and bound to and modified the voltage dependence of the sodium channel Nav. Because of the highly specific binding of Ts1 toxin to Nav, successful chemical synthesis and labeling of Ts1 toxin provides an important tool for biophysical studies, histochemical studies, and opto-pharmacological studies of the Nav protein.

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Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

et al. 2014

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Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

Cited by 69 publications

References 31 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

Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

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