Protease treatment of cerebellar purkinje cells renders omega-agatoxin IVA-sensitive Ca2+ channels insensitive to inhibition by omega-conotoxin GVIA.
The identification of currents carried by N- and P-type Ca(2+) channels in the nervous system relies on the use of omega-conotoxin (CTx) GVIA and omega-agatoxin (Aga) IVA. The peptide omega-Aga-IVA inhibits P-type currents at nanomolar concentrations and N-type currents at micromolar concentrations. omega-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 omega-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 Ca(2+) channel current (>/=85%). These showed that micromolar concentrations of omega-CTx-GVIA inhibited the current in P40-50 cells (66%, 3 microM; 78%, 10 microM) and in P13-20 Purkinje cells (86%, 3 muM; 89%, 10 microM). 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 omega-CTx-GVIA but not by omega-Aga-IVA (84%, 30 nM). Our finding that micromolar concentrations of omega-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 omega-CTx-GVIA but not by omega-Aga-IVA indicates a selective proteolytic action at site(s) on P-type channels with which omega-CTx-GVIA interacts. It also suggests that Ca(2+) channel pharmacology in neurons dissociated using protease may not predict that in neurons not exposed to the enzyme.
Tringham
Elizabeth W
EW
Department of Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Dupere
Jonathan R B
JR
Payne
C Elizabeth
CE
Usowicz
Maria M
MM
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020865:Calcium Channels, P-Type / Q000378:metabolism*; D008297:Male; D010447:Peptide Hydrolases / Q000494:pharmacology*; D011689:Purkinje Cells / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020867:omega-Agatoxin IVA / Q000494:pharmacology*; D020866:omega-Conotoxin GVIA / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D020865:Calcium Channels, P-Type; D020867:omega-Agatoxin IVA; D020866:omega-Conotoxin GVIA; D010447:Peptide Hydrolases