Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons

We have investigated why fura-2 and Ca(2+)-sensitive microelectrodes report different values for the intracellular free calcium ion concentration ([Ca(2+)]i or its negative log, pCa(i)) of snail neurons voltage-clamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentrations similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca(2+)]i were used to calibrate both methods in vivo. In fura-2-loaded cells these pressure injections generated changes in [Ca(2+)]i that agreed well with those expected from the in vitro calibration. Thus, using fura-2 calibrated in vitro, the average resting [Ca(2+)]i was found to be 38 nM (pCa(i) 7.42 +/- 0.05). With Ca(2+)-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in the recorded microelectrode potential, as if the injection lowered [Ca2+]i. No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca(2+)-sensitive microelectrode gave an average resting [Ca2+]i of approximately 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when calibrated in vitro. We conclude that [Ca(2+)]i in snail neurons is approximately 40 nM and that Ca(2+)-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer.