P-type Ca2+ channels are defined by their sensitivity to low nanomolar concentrations of ω-Agatoxin IVA, and by their insensitivity to dihydropyridines, such as the L-type Ca2+ channel agonist, Bay K 8644, and to the N-type Ca2+ channel antagonist, ω-conotoxin GVIA (Mintz et al. 1992a, b). However, we have recently reported (Tringham et al. 2001) that P-type channels are inhibited by the (S)-enantiomer of Bay K 8644 (IC50 = 22 nM) and by ω-conotoxin GVIA (IC50 = 2 µM). We have investigated if these discrepancies reflect the different ages of the animals or the different type of cell preparation used: mature Purkinje cells in cerebellar slices in our study and enzymatically dissociated immature Purkinje cells in previous studies.
To investigate developmental changes in P-type channels, we recorded currents (5 mM Ba2+, 22 °C) from somatic cell-attached patches of Purkinje cells, in parasagittal cerebellar vermis slices (250 µm) of mature (P40-50 days) and immature (P13-20 days) male Wistar rats (killed by cervical dislocation). To study the influence of enzymes, slices from immature rats were treated with protease XXIII (Sigma), according to a protocol mimicking that used to dissociate Purkinje cells (McDonough et al. 1996). Currents were evoked by depolarising ramps (0.53 mV ms-1), and were recorded with or without drug in the pipette. Values are means ± S.E.M., and were compared with Student’s unpaired t test (P < 0.05 taken as the significant level).
ω-Agatoxin (30 nM) blocked the Ca2+ channel currents by 92 ± 2 % (n = 24) and by 78 ± 10 % (n = 5) in mature and immature Purkinje cells, respectively, thus defining the majority of the currents as P-type currents. In contrast to the 82 ± 5 % inhibition (n = 11) produced by 300 nM (-)-(S)-Bay K 8644 in mature cells, there was a non-significant increase (P = 0.5) in current amplitude in immature cells (31 ± 17 %, n = 7). ω-Conotoxin GVIA (3 µM) inhibited the currents by 60 ± 9 % in mature cells (n = 30, P = 0.001) and by 80 ± 8 % in immature cells (n = 9, P = 0.02). Treatment of immature cerebellar slices with the protease reversed the inhibition by ω-conotoxin GVIA (19 ± 9 %, n = 6).
These results reveal developmental differences in the properties of native P-type Ca2+ channels expressed in cerebellar Purkinje cells. Furthermore, the pharmacology of the channels may be fundamentally altered by enzymatic treatment.
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