title: Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB.
abstract: OBJECTIVE
  Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na(v) 1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na(v) α-subtypes in neurons, Na(v) 1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na(v) 1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na(v) 1.8 and the TTX-S Na(v) 1.2 using a domain exchange strategy.
  
  
  METHODS
  Wild-type and chimeric Na(v) channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique.
  
  
  RESULTS
  MrVIB (1 µM) reduced Na(v) 1.2 current amplitude to 69 ± 12%, whereas Na(v) 1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na(v) 1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na(v) 1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na(v) 1.2.
  
  
  CONCLUSIONS
  Taken together, these results suggest that domain II of Na(v) 1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.
authors.affiliation: Health Innovations Research Institute, RMIT University, Melbourne, Vic, Australia.
authors.forename: O
authors.initials: O
authors.lastname: Knapp
authors.forename: S T
authors.initials: ST
authors.lastname: Nevin
authors.forename: T
authors.initials: T
authors.lastname: Yasuda
authors.forename: N
authors.initials: N
authors.lastname: Lawrence
authors.forename: R J
authors.initials: RJ
authors.lastname: Lewis
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020916:Conotoxins / Q000494:pharmacology*; D005260:Female; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000502:physiology*; D062559:NAV1.8 Voltage-Gated Sodium Channel / Q000502:physiology*; D009865:Oocytes; D021122:Protein Subunits; D013779:Tetrodotoxin / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D020916:Conotoxins; D062551:NAV1.2 Voltage-Gated Sodium Channel; D062559:NAV1.8 Voltage-Gated Sodium Channel; D021122:Protein Subunits; C568317:SCN10A protein, human; C094741:muO-conotoxin MrVIB; D013779:Tetrodotoxin