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

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.