2. Academic Validation
  3. Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels

Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels

  • Br J Pharmacol. 2014 Jul;171(13):3313-27. doi: 10.1111/bph.12686.
G Berecki 1,  N L Daly,  Y H Huang,  S Vink,  D J Craik,  P F Alewood,  D J Adams
Affiliations

Affiliation

  • 1 Health Innovations Research Institute, RMIT University, Melbourne, Vic, Australia.
PMID: 24628243 DOI: 10.1111/bph.12686
Abstract

Background and purpose: 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).

Experimental approach: 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.

Key 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 and implications: 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.

Keywords

Cav2.2 channel; N-type VGCC; NMR; holding potential; serum stability; ω-conotoxin.

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