INTRACELLULAR CALCIUM CLEARANCE IN PURKINJE-CELL SOMATA FROM RAT CEREBELLAR SLICES

1. The mechanisms governing; the return of intracellular calcium (Ca-i (2+)) to baseline levels following depolarization-evoked [Ca2+](i) ris es were investigated in Purkinje cell somata using tight-seal whole-ce ll recordings and fura-2 microfluorometry, for peak [Ca2+], ranging fr om 50 nM to 2 mu M. 2. Ca-i(2+) decay was well fitted by a double expo nential with time constants of 0.6 and 3 s. Both time constants were i ndependent of peak [Ca2+](i) but the contribution of the faster compon ent increased with [Ca2+](i). 3. Thapsigargin (10 mu M) and cyclopiazo nic acid (50 mu M) prolonged Ca-i(2+) decay indicating that sarco-endo plasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Ca-i( 2+) clearance. 4. A modest participation in clearance was found for th e plasma membrane Ca2+ (PMCA) pumps using 5,6-succinimidyl carboxyeosi n (40 mu M). 5. The Na+-Ca2+ exchanger also contributed to the clearan ce process, since replacement of extracellular Na+ by Li+ slowed C-i(2 +) decay. 6. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 mu M) a nd rotenone (10 mu M) increased [Ca2+], and elicited large inward curr ents at -60 mV. Both effects were also obtained with CCCP in the absen ce of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncoupl ers 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 Ca-i(2+) clearance. 7. The relative co ntribution of the clearance systems characterized in this study varied as a function of [Ca2+](i). At 0.5 mu M Ca-i(2+), SERCA pumps, PMCA p umps and the Na+-Ca2+ exchanger contribute equally to removal and acco unt for 78% of the process. Only 45% of the removal at 2 mu M Ca-i(2+) can be explained by these systems. In this high [Ca2+], range the maj or contribution is that of SERCA pumps (21%) and of the Na+-Ca2+ excha nger (18%), whereas the contribution of PMCA pumps is only 6%.