The identification of currents carried by N-type and P-type Ca2+ channels in the nervous system relies on the use of ω-conotoxin GVIA (ω-CTx-GVIA) and ω-Agatoxin IVA (ω-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 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.
|Translated title of the contribution||Protease treatment of cerebellar Purkinje cells renders ω-Agatoxin IVA-sensitive Ca2+ channels insensitive to inhibition by ω-conotoxin GVIA|
|Pages (from-to)||806 - 814|
|Number of pages||9|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|Publication status||Published - Feb 2008|