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

Journal Article


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 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.

UOW Authors


  •   Kubota, Tomoya (external author)
  •   Durek, Thomas (external author)
  •   Dang, Bobo (external author)
  •   Finol Urdaneta, Rocio
  •   Craik, David J. (external author)
  •   Kent, Stephen B.H.. (external author)
  •   French, Robert J. (external author)
  •   Bezanilla, Francisco (external author)
  •   Correa, Ana M. (external author)

Publication Date


  • 2017

Citation


  • Kubota, T., Durek, T., Dang, B., Finol-Urdaneta, R. K., Craik, D. J., Kent, S. B.H., French, R. J., Bezanilla, F. & Correa, A. M. (2017). Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET. Proceedings Of The National Academy Of Sciences Of The United States Of America, 114 (10), E1857-E1865.

Start Page


  • E1857

End Page


  • E1865

Volume


  • 114

Issue


  • 10

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 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.

UOW Authors


  •   Kubota, Tomoya (external author)
  •   Durek, Thomas (external author)
  •   Dang, Bobo (external author)
  •   Finol Urdaneta, Rocio
  •   Craik, David J. (external author)
  •   Kent, Stephen B.H.. (external author)
  •   French, Robert J. (external author)
  •   Bezanilla, Francisco (external author)
  •   Correa, Ana M. (external author)

Publication Date


  • 2017

Citation


  • Kubota, T., Durek, T., Dang, B., Finol-Urdaneta, R. K., Craik, D. J., Kent, S. B.H., French, R. J., Bezanilla, F. & Correa, A. M. (2017). Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET. Proceedings Of The National Academy Of Sciences Of The United States Of America, 114 (10), E1857-E1865.

Start Page


  • E1857

End Page


  • E1865

Volume


  • 114

Issue


  • 10