Aequorin response facilitation and intracellular calcium accumulation in molluscan neurones

Abstract

When molluscan neural somata are filled with the Ca-indicating photoprotein aequorin and subjected to a 1 Hz train of depolarizing pulses (0.3 s duration to + 15 mV) under voltage clamp, the successive photo-emissions due to Ca influx facilitate. The origin of this phenomenon was investigated in identified neurons from the abdominal ganglion of Aplysia californica. Since outward currents inactivate cumulatively in successive pulses, the effective depolarization increases due to a series resistance error. Elimination of this error by electronic compensation or pharmacological block of outward current reduced aequorin response facilitation by only .apprx. 30%, on the average. When voltage-dependent Na and K currents are blocked in tetraethylammonium (TEA)-substituted zero-Na sea water, the remaining inward Ca currents display no facilitation. A slow decline during a pulse and a slight progressive depression in successive pulses are observed. Ba-substitution for Ca in the same medium eliminates a small residual K current insensitive to external TEA. The remaining inward Ba currents also display depression instead of facilitation. A non-pharmacological separation of Ca current was accomplished by measuring tail currents at the K equilibrium potential following depolarizing pulses. Increments of optical absorbance in neurons filled with the Ca-sensitive dye arsenazo III show a depression rather than facilitation to successive depolarizations in a train. Calibration of arsenazo III response amplitude indicates that the dye reports only about 1% of the Ca concentration increment expected from knowledge of cell vol and the charge carried by Ca current during a depolarizing pulse. This suggests that cytoplasmic buffering of free Ca must occur rapidly, on a time scale comparable to the response time of arsenazo III (about 1 ms) or more rapidly. The slow K tail current following a depolarizing pulse is Ca-dependent and probably provides an approximate index of the internal submembrane Ca concentration. Since neither Ca currents nor the concentration transients show facilitation, aequorin response facilitation is due to the non-linear dependence of aequorin photo-emissions on Ca concentration. The different kinetics of arsenazo III responses and aequorin responses can be reconciled by a simple model representing Ca accumulation and known response properties of the 2 indicator substances. In a train of impulses evoked by injecting depolarizing current into a neuron, the successive action potentials grow in duration. A nearly constant Ca influx signalled by arsenazo III accompanies broadening action potentials.