Abstract
The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.
Highlights
A number of predatory species such as spiders, scorpions, and fish hunting mollusks have developed venoms to rapidly stun and kill their prey
Distinct Cav2.2 Channel Structural Determinants Underlie GVIA and MVIIA Block—We have recently shown that point mutations in a putative EF-hand region contained in the domain III S5–H5 region of the Cav2.2 ␣1 subunit regulate the blocking and unblocking rate constants for both -conotoxins MVIIA and GVIA [7]
Ellinor et al [6] reported that a set of five point mutations in the domain III S5–S6 region dramatically attenuated GVIA blocking of N-type calcium channels expressed in Xenopus oocytes
Summary
A number of predatory species such as spiders, scorpions, and fish hunting mollusks have developed venoms to rapidly stun and kill their prey Their venoms typically contain a mixture of potent peptide toxins that have evolved to bind to voltage- and ligand-gated ion channels [1]. Among calcium channel blocking peptides, -conotoxin GVIA (isolated from Conus geographus) and -conotoxin MVIIA (isolated from Conus magus) are perhaps the most widely known. Both of these toxins are thought to block the pore of N-type calcium channels from a variety of species. Our data show that manipulations of the domain III S5–S6 region in the rat Cav2.2 calcium channels differentially affect GVIA and MVIIA block.
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