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NEUROPHARMACOLOGY

Protease Treatment of Cerebellar Purkinje Cells Renders -Agatoxin IVA-Sensitive Ca²⁺ Channels Insensitive to Inhibition by -Conotoxin GVIA

Department of Pharmacology, University of Bristol, Bristol, United Kingdom

The identification of currents carried by N- and P-type Ca²⁺ channels in the nervous system relies on the use of -conotoxin (CTx) GVIA and -agatoxin (Aga) IVA. The peptide -Aga-IVA inhibits P-type currents at nanomolar concentrations and N-type currents at micromolar concentrations. -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 -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 -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 -CTx-GVIA but not by -Aga-IVA (84%, 30 nM). Our finding that micromolar concentrations of -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 -CTx-GVIA but not by -Aga-IVA indicates a selective proteolytic action at site(s) on P-type channels with which -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.

Address correspondence to: Dr. Maria M. Usowicz, Department of Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, UK. E-mail: m.m.usowicz{at}bris.ac.uk