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Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices

1 The mechanisms governing the return of intracellular calcium (Ca2+i) to baseline levels following depolarization‐evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight‐seal whole‐cell recordings and fura‐2 microfluorometry, for peak [Ca2+]i ranging from 50 nM to 2 μM. 2 Ca2+i decay was well fitted by a double exponential with time constants of 0.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i. 3 Thapsigargin (10 μM) and cyclopiazonic acid (50 μM) prolonged Ca2+i decay indicating that sarco‐endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Ca2+i clearance. 4 A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6‐succinimidyl carboxyeosin (40 μM). 5 The Na+‐Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Ca2+i decay. 6 Carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μM) and rotenone (10 μM) increased [Ca2+]i and elicited large inward currents at −60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Ca2+i clearance. 7 The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 μM Ca2+i, SERCA pumps, PMCA pumps and the Na+‐Ca2+ exchanger contribute equally to removal and account for 78 % of the process. Only 45 % of the removal at 2 μM Ca2+i can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21 %) and of the Na+‐Ca2+ exchanger (18 %), whereas the contribution of PMCA pumps is only 6 %.