CHARACTERIZATION OF CA2+ CHANNEL CURRENTS IN CULTURED RAT CEREBELLAR GRANULE NEURONS

1. High-threshold voltage-gated calcium channel currents (I-Ba) were s tudied in cultured rat cerebellar granule neurones using the whole-cel l patch clamp technique with 10 mM Ba2+ as the charge carrier. The put ative P-type component of whole-cell current was characterized by util izing the toxin omega-agatoxin IVA (omega-Aga IVB) in combination with other blockers. 2. omega-Aga IVA (100 nM) inhibited the high voltage- activated (HVA) I-Ba by 40.9 +/- 3.4% (n = 27), and the dissociation c onstant K-d was 2.7 nM. Maximal inhibition occurred within a 2-3 min t ime course, and was irreversible. The isolated omega-aga IVA-sensitive current was non-inactivating. 3. omega-Aga IVA exhibited overlapping selectivity with both N- and L-channel blockers; omega-conotoxin GVIA (omega-CTX GVIA) (1 mu M) and the dihydropyridine (-)-202-709 (1 mu M) , respectively. Together these toxins reduced the o-Aga IVA-sensitive component to just 4.5 +/- 1.4% (n = 3). Thus only a small proportion o f the current can be unequivocally attributed to P-type current. Inhib ition of the HVA I,, by w-Aga IA also reduced the proportion of omega- Aga IVA-sensitive current to 28.0 +/- 3.2% (n = 3). 4. Application of omega-Aga IVA and a synthetic form of funnel-web toxin, N-(7-amino-4-a zaheptyl)-L-argininamide (sFTX-3.3; 10 mu M), produced an additive blo ck of the HVA I-Ba. Consequently these tyro toxins do not act on the s ame channel in cerebellar granule neurones. 5. omega-Aga IVA inhibitio n of low voltage-activated (LVB) I-Ba was studied in the ND7-23 neuron al cell line. omega-Aga IVA (100 nM) reduced the LVA current by 41.3 /- 3.2% (n = 17) in a fully reversible manner with no shift in the ste ady-state inactivation of the channel. 6. A component of current insen sitive to N-, L- and P-channel blockers remained unclassified in all o ur studies. This component, and also that remaining following block by omega-Aga IVA and omega-Aga IA, exhibited relatively rapid, although incomplete, inactivation compared to the other currents isolated in th is study. 7. In conclusion, omega-Aga IVA inhibits a component of curr ent in cultured cerebellar granule neurones which overlaps almost comp letely with that inhibited by L- and N-channel blockers. In addition, a large component of whole-cell current in these neurones still remain s unclassified.