Properties of a High-threshold Voltage-activated Calcium Current in Rat Cerebellar Granule Cells

Postmitotic cerebellar granule cells, maintained for 5 - 6 days in Dulbecco's modified essential medium supplemented with 25 mM KCl, have been studied in whole-cell recording conditions to characterize calcium currents. With 10 mM Ba2+ as the divalent charge carrier, and using a pipette solution highly buffered for Ca2+ (30 mM EGTA, 100 mM HEPES - Tris, pH 7.2), only a high-threshold voltage-activated barium current was recorded from a holding potential of -90 mV. The addition of 1 mM ATP to the pipette medium allowed stable recording for an average duration of 10 min, compatible with pharmacological studies of the barium current. Ninety-six per cent of the current was half-inactivated at low negative holding potential (-76 mV). A total block of current was obtained with 1 microM Cd2+. Sixty-three per cent of the mean current was abolished by 3 microM omega-conotoxin (omega-CgTx; Ki=10 nM for a 15 min application), but individual cells showed either full sensitivity to this toxin or incomplete sensitivity. Seventy-eight per cent of the mean current was also abolished by 10 microM nicardipine but with a higher Ki of 0.5 microM. After exposure to omega-CgTx, BAY K 8644 had no effect on the remaining current, though it was suppressed by nicardipine. No sensitivity to diltiazem, desmethoxyverapamil or flunarizine could be detected. Our major conclusion is that at least half of the channels have a mixed pharmacology, showing sensitivity to both omega-CgTx and dihydropyridine antagonists.