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J. Biol. Chem., Vol. 279, Issue 33, 34705-34714, August 13, 2004

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The ₂ Auxiliary Subunit Reduces Affinity of -Conotoxins for Recombinant N-type (Ca_v2.2) Calcium Channels^*

Jorgen Mould¶, Takahiro Yasuda¶, Christina I. Schroeder, Aaron M. Beedle||**, Clinton J. Doering||, Gerald W. Zamponi||, David J. Adams, and Richard J. Lewis¶¶

From the Institute for Molecular Bioscience and the School of Biomedical Sciences, The University of Queensland, Queensland 4072, Australia and the ^||Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada

The -conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The -conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The and ₂-1 auxiliary subunits influence the expression and characteristics of the _1B subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the -conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the 3 subunit had little influence on the on- and off-rates of -conotoxins, coexpression of ₂ with _1B significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The ₂ also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of ₂ was not affected by oocyte deglycosylation. The extent of recovery from the -conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA had greater recovery from the block, whereas [K10R]CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of -conotoxin reversibility. Recovery from CVID block was reduced in the presence of ₂ in human embryonic kidney cells and in oocytes expressing _1B-b. These results may have implications for the antinociceptive properties of -conotoxins, given that the ₂ subunit is up-regulated in certain pain states.

Received for publication, October 2, 2003 , and in revised form, May 26, 2004.

The atomic coordinates and structure factors (codes 1TTK, 1TT3, 1TTL, and 1TR6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the National Health and Medical Research Council of Australia, an Australian Research Council Special Research Centre for Functional and Applied Genomics Discovery Grant, and operating grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, the Northwest Territories, and Nunavut (to G. W. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

^** Holder of studentship support from the Alberta Heritage Foundation for Medical Research and the Natural Science and Engineering Research Council of Canada.

Supported by an AHFMR studentship.

Canadian Institutes of Health Research Investigator and a Senior Scholar of the Alberta Heritage Foundation for Medical Research.

^¶¶ To whom correspondence should be addressed. Tel.: 61-7-33462984; Fax: 61-7-33462101; E-mail: r.lewis{at}imb.uq.edu.au.

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