INTRACELLULAR CALCIUM AND ITS SODIUM-INDEPENDENT REGULATION IN VOLTAGE-CLAMPED SNAIL NEURONS

1. We have used both Ca2+-sensitive microelectrodes and, fura-2 to measure the intracellular free calcium ion concentration ([Ca2+](i) or its negative log, pCa(i)) of snail neurones voltage clamped to -50 or -60 mV. Using Ca2+-sensitive microelectrodes, [Ca2+](i) was found to be similar to 174 nar and pCa(i), 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+](i) was similar to 40 nM and pCa(i), 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+](i) which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+](i) was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+](i) in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+](i) could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a small increase in resting [Ca2+](i) and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+](i) or the rate of recovery of [Ca2+](i) following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+](i). Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+](i). 7. Intracellular orthovanadate caused an increase in resting [Ca2+](i) and prevented the full recovery of [Ca2+](i) following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+](i). We conclude that there is no Na+-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+](i) is maintained by an ATP-dependent Ca2+ pump.