Relationship between intracellular calcium and its muffling measured by calcium iontophoresis in snail neurones.

1. We have measured intracellular free calcium ion concentration ([Ca2+]i) with fura-2, and intracellular chloride with chloride-sensitive microelectrodes, in voltage-clamped snail neurones. By making iontophoretic injections of CaCl2 we have investigated calcium muffling, the sum of the processes which minimize the calcium transient, at different values of [Ca2+]i. 2. By injection of calcium into cell-sized droplets of buffer we measured the calcium transport index. It was stable over the range pCa 6-7.4 (0.48 +/- 0.06 measured at pCa 6.70 +/- 0.12, n = 5). 3. Measurement of intracellular chloride activity during a series of fura-2-KCl pressure injections revealed a nearly linear relationship between fura-2 Ca(2+)-insensitive fluorescence and the sum of the increments in intracellular chloride. This allowed us to calculate the intracellular fura-2 concentration ([fura-2]i). 4. The rate of recovery of [Ca2+]i following a depolarization-induced load was increased by low [fura-2]i (10-20 microM) but decreased by higher [fura-2]i (40-80 microM). These effects are consistent with the addition of a mobile buffer to the cytoplasm. 5. Iontophoresis of Ca2+ at various membrane potentials allowed us to calculate the intracellular calcium muffling power (the amount of calcium required to cause a transient tenfold increase in [Ca2+]i per unit volume) and calcium muffling ratio (number of Ca2+ ions injected divided by the maximum increase in [Ca2+]i per unit volume) at different values of [Ca2+]i. 6. Calcium muffling power at resting [Ca2+]i was approximately 40 microM Ca2+ (pCa unit)-1, (about 250 times less than for hydrogen ions). It increased linearly about fivefold with [Ca2+]i over the range 20-120 nM (10 cells, 153 measurements) and therefore exponentially with decreasing pCa. 7. The calcium muffling ratio appeared to be constant (361 +/- 14, n = 10 cells, 130 measurements) over the range 20-120 nM Ca2+. 8. In three experiments we modelled the additional calcium buffering power produced by multiple pressure injections of fura-2 into voltage-clamped snail neurones. Back-extrapolation of the increases in calcium buffering power allowed us to calculate the calcium muffling power of the neurones. 9. Small increases in [fura-2]i (approximately 10 microM) significantly increased intracellular calcium muffling power in individual experiments. However, the variability among neurones in intracellular calcium muffling power was large enough to obscure the additional buffering produced by fura-2 in pooled experiments.