title: Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.
abstract: 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 inXenopusoocytes 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kubota
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Durek
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.|Department of Chemistry, The University of Chicago, Chicago, IL 60637.|Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637.
authors.forename: Bobo
authors.initials: B
authors.lastname: Dang
authors.affiliation: Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1.|Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1.
authors.forename: Rocio K
authors.initials: RK
authors.lastname: Finol-Urdaneta
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.|Department of Chemistry, The University of Chicago, Chicago, IL 60637.|Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637.
authors.forename: Stephen B H
authors.initials: SB
authors.lastname: Kent
authors.affiliation: Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1; nanicorrea@uchicago.edu french@ucalgary.ca fbezanilla@uchicago.edu.|Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: French
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; nanicorrea@uchicago.edu french@ucalgary.ca fbezanilla@uchicago.edu.
authors.forename: Francisco
authors.initials: F
authors.lastname: Bezanilla
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; nanicorrea@uchicago.edu french@ucalgary.ca fbezanilla@uchicago.edu.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Correa
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001896:Boron Compounds / Q000737:chemistry; D004735:Energy Transfer / Q000187:drug effects*; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008564:Membrane Potentials / Q000187:drug effects*; D009124:Muscle Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000187:drug effects; D009865:Oocytes / Q000737:chemistry / Q000187:drug effects; D018408:Patch-Clamp Techniques; D051381:Rats; D012604:Scorpion Venoms / Q000737:chemistry / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry*; D061566:Voltage-Gated Sodium Channels / Q000187:drug effects* / Q000235:genetics; D014981:Xenopus / Q000235:genetics
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D028581:Lanthanoid Series Elements; D009124:Muscle Proteins; C466180:Scn4a protein, rat; D012604:Scorpion Venoms; D015222:Sodium Channels; D061566:Voltage-Gated Sodium Channels
keywords: relaxed state; slow inactivation; voltage gating; β-scorpion toxin; μ-conotoxin