Abstract
The identification of currents carried by N- and P-type Ca2+ channels in the nervous system relies on the use of ω-conotoxin (CTx) GVIA and ω-agatoxin (Aga) IVA. The peptide ω-Aga-IVA inhibits P-type currents at nanomolar concentrations and N-type currents at micromolar concentrations. ω-CTx-GVIA blocks N-type currents, but there have been no reports that it can also inhibit P-type currents. To assess the effects of ω-CTx-GVIA on P-type channels, we made patch-clamp recordings from the soma of Purkinje cells in cerebellar slices of mature [postnatal days (P) 40–50, P40–50] and immature (P13–20) rats, in which P-type channels carry most of the Ca2+ channel current (≥85%). These showed that micromolar concentrations of ω-CTx-GVIA inhibited the current in P40–50 cells (66%, 3 μM; 78%, 10 μM) and in P13–20 Purkinje cells (86%, 3 μM; 89%, 10 μM). The inhibition appeared to be reversible, in contrast to the known irreversible inhibition of N-type current. Exposure of slices from young animals to the enzyme commonly used to dissociate Purkinje cells, protease XXIII, abolished the inhibition by ω-CTx-GVIA but not by ω-Aga-IVA (84%, 30 nM). Our finding that micromolar concentrations of ω-CTx-GVIA inhibit P-type currents suggests that specific block of N-type current requires the use of submicromolar concentrations. The protease-induced removal of block by ω-CTx-GVIA but not by ω-Aga-IVA indicates a selective proteolytic action at site(s) on P-type channels with which ω-CTx-GVIA interacts. It also suggests that Ca2+ channel pharmacology in neurons dissociated using protease may not predict that in neurons not exposed to the enzyme.
Footnotes
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This study was supported by the Medical Research Council. E.W.T. received a University of Bristol Ph.D. scholarship. J.R.B.D. received a Wellcome Prize Ph.D. scholarship. C.E.P. received a Medical Research Council Ph.D. studentship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.130641.
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ABBREVIATIONS: CTx, conotoxin; Aga, agatoxin; P, postnatal day(s); TTX, tetrodotoxin; TEA, tetraethylammonium; CED, Cambridge Electronic Design; ANOVA, analysis of variance.
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↵1 Current affiliation: NeuroMed Pharmaceuticals Inc., Vancouver, British Columbia, Canada.
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↵2 Current affiliation: Pfizer Global Research and Development, Kent, United Kingdom.
- Received August 22, 2007.
- Accepted October 31, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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