The α2δ Auxiliary Subunit Reduces Affinity of ω-Conotoxins for Recombinant N-type (Cav2.2) Calcium Channels

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

doi:10.1074/jbc.m310848200

Abstract

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(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 omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta 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 alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-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 omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
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
We show that the affinity of the ␻-conotoxins CVID and MVIIA to block N-type Ca2ϩ channel current is reduced in the presence of ␣2␦, and we identify position 10 in ␻-conotoxins as having an important influence on the extent of recovery from channel block

Summary

Introduction

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/). Multiple isoforms of the ␤ and ␣2␦ subunits exist that can interact with the ␣1 subunit to produce N-type Ca2ϩ channels with different gating properties, allowing the fine tuning of synaptic transmissions [11]. ␻-Conotoxins have been used as research tools to help define the distribution and physiological roles of specific N-type calcium channels (14 –17) and have potential therapeutic value as intrathecal treatments for pain. When expressed alone, ␣1 subunits produce functional channels with kinetic properties that differ substantially from those of the native channel [24, 25]. A negative regulatory effect of the overexpressed ␤3 subunit on N-type Ca2ϩ channel currents has been demonstrated in Xenopus oocytes [30]

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