INACTIVATION OF CALCIUM CURRENTS IN GRANULE CELLS CULTURED FROM MOUSE CEREBELLUM

1. Cells dissociated from mouse cerebellum were grown in vitro Ca2+ channel currents were recorded from granule cells with the patch-clamp technique under conditions which suppressed currents through Na+ and K+ channels and minimized run-down of current through Ca2+ channels. 2. A strong depolarizing voltage step from a hyperpolarized holding potential produced inward Ca2+ channel current that decayed exponentially to a non-zero level. Inward current decayed to approximately 40% of its peak amplitude (range 20-90%). 3. The inward current increased in amplitude when Ca2+ was replaced with Ba2+ or after raising the concentration of extracellular Ba2+, but the rate of decay of current was unaffected. 4. The current-voltage (I-V) relation showed that peak or sustained current increased with voltage pulses more positive than approximately -30 mV, reached a maximum amplitude near +20 mV and became progressively smaller with larger depolarizations. 5. The tail currents produced after rapidly repolarizing the membrane potential to -70 mV from a positive test pulse decayed along a single exponential time course with a time constant of approximately 0.5 ms. The amplitude of tail current measured at a fixed repolarization potential increased as the pre-pulse was made more positive and reached a maximum with pre-pulse more positive than +40 mV. A plot of normalized amplitude of the tail current as a function of the pre-pulse potential was fitted with a Boltzmann relation with V1/2 = approximately +8 mV and steepness k = 14 mV. 6. Shifting the holding potential to more positive potentials reduced the amplitude of the Ca2+ channel current elicited by the fixed voltage step and abolished the decay of the inward current. The peak current was normalized to the maximum peak current elicited from a very negative holding potential and plotted as a function of holding potential. The points were fitted with a Boltzmann relation for inactivation with V1/2 = approximately -57 mV and steepness k = 14 mV. 7. The onset of inactivation was studied in two-pulse experiments in which the duration of conditioning pre-pulse was varied. Increasing the duration of a pre-pulse to a fixed potential reduced the peak inward current evoked by the second test pulse. Plotting normalized current as a function of pre-pulse duration showed that inactivation developed along a double exponential time course. Both fast and slow time constants decreased as the pre-pulse potential was made more positive. 8. Two-pulse experiments in which the duration of the interpulse interval was varied following a pre-pulse that elicited maximal current showed that the amplitude of Ca2+ channel current elicited by the second test pulse recovered along a double exponential time course. 9. The dihydropyridine agonist + (S)-202-791 increased the amplitude of the current in response to a positive test pulse, shifted the activation curve to more negative potentials, and slowed deactivation. 10. The experimental results show that cerebellar granule cells possess a dihydropyridine-sensitive Ca2+ current which has fast and slow components to the time course of inactivation. The mechanism of inactivation and the role of Ca2+ channels in the function of granule cells are discussed.