title: Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels.
abstract: OBJECTIVE
  ω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs).
  
  
  METHODS
  Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively.
  
  
  RESULTS
  R10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ω-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID.
  
  
  CONCLUSIONS
  R10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding.
authors.affiliation: Health Innovations Research Institute, RMIT University, Melbourne, Vic, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Berecki
authors.forename: N L
authors.initials: NL
authors.lastname: Daly
authors.forename: Y H
authors.initials: YH
authors.lastname: Huang
authors.forename: S
authors.initials: S
authors.lastname: Vink
authors.forename: D J
authors.initials: DJ
authors.lastname: Craik
authors.forename: P F
authors.initials: PF
authors.lastname: Alewood
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000700:Analgesics / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002121:Calcium Channel Blockers / Q000737:chemistry / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D014982:Xenopus laevis; D020960:omega-Conotoxins / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000700:Analgesics; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D020960:omega-Conotoxins
keywords: Cav2.2 channel; N-type VGCC; NMR; holding potential; serum stability; ω-conotoxin