THE RELATIONSHIP BETWEEN DEPLETION OF INTRACELLULAR CA2-CELLS( STORESAND ACTIVATION OF CA2+ CURRENT BY MUSCARINIC RECEPTORS IN NEUROBLASTOMA)

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca2+-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-1 15 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ s tores and the voltage-independent, receptor-regulated Ca2+ current rep resents a significant Ca2+ source for refilling. The time course of Ca 2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measur ed with nystatin patch voltage clamp. At the time of maximum current d ensity (0.18 +/- .03 pA/pF; n = 48), the Ca2+ content of the IFS-relea sable Ca2+ pool is reduced to 39 + 3 % (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15- 30 s. The activation of Ca2+ current is delayed by 10-15 s after the b eginning of Ca2+ release and continues to gradually increase for nearl y 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the pro duction and accumulation of a second messenger is the rate-limiting st ep in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ AT Pase. After 15 min in thapsigargin, IF3-releasable Ca2+ stores are dep leted by >90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 mu M; 30 mi n) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it a ctivates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current densi ty after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times gre ater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca2+-dependent inactivation.