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Selective and voltage dependent inhibition of N-type calcium channels by novel ω-Conotoxins CVIE and CVIF

G. Berecki,1,2 L. Motin,1,2 A. Haythornthwaite,2 N.L. Daly,3 P. Bansal,3 S. Vink,3 R. Drinkwater,4 R.J. Lewis,3,4 P.F. Alewood3 and D.J. Adams,1,2 1The Queensland Brain Institute, The University of Queensland, QLD 4072, Australia, 2The School of Biomedical Sciences, The University of Queensland, QLD 4072, Australia, 3The Institute for Molecular Biosciences, The University of Queensland, QLD 4072, Australia and 4Xenome Ltd, Indooroopilly, QLD 4069, Australia.

N-type Ca2+ channel selective ω-conotoxins have recently emerged as potential new drugs for the treatment of severe chronic pain. In this study, two new ω-conotoxins, CVIE and CVIF, were discovered and synthesized following a PCR screen of a Conus catus cDNA library. All animal experimentations were performed in accordance to guidelines of the University of Queensland Animal Ethics Committee. Both ω-conotoxins potently displaced 125I-GVIA binding to adult rat brain membrane. In Xenopus oocytes, CVIE and CVIF inhibited Ba2+ currents through recombinant N-type (α1B, α2δ1, and β3) channels with IC50 values of 2.6 ± 0.5 nM (n = 14) and 19.9 ± 3.2 nM (n = 16), respectively. Consistent with our previous study (Mould et al., 2004), both peptides exhibited increased affinity for N-type Ca2+ channels in the absence of auxiliary α2δ1 subunit. Neither CVIE nor CVIF had any effect on P/Q-, R- or L-type calcium channels. The voltage dependence and the time course of CVIE and CVIF block were analyzed and the current recovered completely from block at -125 mV but only partially at -80 mV, indicating that CVIE and CVIF have a higher affinity to Ca2+ channels in the inactivated state. The analogues [R10K]CVIE and [R10K]CVIF did not significantly alter the conserved ω-conopeptide backbone conformation, but increased the rates of onset and offset of ω-conotoxin action and improved recovery from block at -80 mV, compared to the native peptides. In DRG sensory neurons isolated from neonatal rats, CVIE and CVIF potently and selectively inhibited N-type Ca2+ channels and the reversibility of block was voltage dependent. The R10K substitution altered the kinetics of block and was associated with an increased rate and recovery from block at -80 mV, compared to the native peptide. In rat spinal cord slices, CVIE and CVIF inhibited reversibly excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. This result suggests the presence of an inactivation-resistant N-type Ca2+ channel population in the presynaptic nerve terminals. These N-type channel specific ω-conotoxins are potentially useful neurophysiological tools and inhibitors of nociceptive signaling with therapeutic implications.

Mould J, Yasuda T, Schroeder CI, Beedle AM, Doering CJ, Zamponi GW Adams DJ, Lewis RJ. (2004) Journal of Biological Chemistry, 279: 34705-14.