ω‐Conotoxin CVIB differentially inhibits native and recombinant N‐ and P/Q‐type calcium channels

Abstract

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.

Analgesic ω-Conotoxins CVIE and CVIF Selectively and Voltage-Dependently Block Recombinant and Native N-Type Calcium Channels

“…5B), which is similar to that reported previously for N-type-selective -conotoxins CVID, MVIIA, or GVIA (Motin et al, 2007). The residual I Ba in the presence of these -conotoxins (1 M) represents non-N-type current through other (mostly L-, P/Q-, and R-type) VGCCs, which can be selectively inhibited (Motin et al, 2007). Bath application of CVIB (500 nM) and nifedipine (10 M) to inhibit P/Q-and L-type VGCC currents, respectively, demonstrated that neither CVIE nor CVIF affected these current components (Fig.…”

“…The maximum inhibition of inward Ba 2ϩ current produced by 100 nM CVIE or CVIF in DRG neurons was ϳ50% (Fig. 5B), which is similar to that reported previously for N-type-selective -conotoxins CVID, MVIIA, or GVIA (Motin et al, 2007). The residual I Ba in the presence of these -conotoxins (1 M) represents non-N-type current through other (mostly L-, P/Q-, and R-type) VGCCs, which can be selectively inhibited (Motin et al, 2007).…”

“…A detailed analysis of the effect of MVIIA on the N-type channel confirmed that block was neither voltage-nor frequency-dependent, but recovery from block strongly depended on the HP (Feng et al, 2003). In contrast, our previous studies of -conotoxin CVID demonstrated that hyperpolarization does not significantly enhance the extent of recovery (Mould et al, 2004); CVIB block and recovery also seemed to be largely voltage-independent (Motin et al, 2007). Although the degree of N-type channel inactivation seems to be a key factor modulating the -conotoxin ion channel binding site interaction, several unknown mechanisms also contribute in determining the relative proportion of reversibly and irreversibly blocked channels (Feng et al, 2003).…”

“…Primary Culture of DRG Neurons and Patch-Clamp Recording. DRG neurons were enzymatically dissociated from the ganglia of 7-to 14-day-old Wistar rats, as described previously (Motin et al, 2007), and used for experiments within 24 to 48 h. Cells were transferred into a small-volume (ϳ200 l) recording chamber constantly perfused with a solution containing 150 mM tetraethylammonium chloride, 2 mM BaCl 2 , 10 mM D-glucose, and 10 mM HEPES, pH 7.4 (with NaOH). Borosilicate glass electrodes were filled with an internal solution containing 140 mM CsCl, 1 mM MgCl 2 , 5 mM MgATP, 0.1 mM sodium GTP, 5 mM BAPTA-Cs 4 , and 10 mM HEPES, pH 7.3 (with CsOH) and had resistances of 1.5 to 2.5 M⍀.…”

Conus Venom Peptide Pharmacology

“…CONOPEPTIDE PHARMACOLOGY ␣2␦ subunits can significantly alter their channel affinity (Lewis et al, 2000;Mould et al, 2004;Motin et al, 2007 …”