Measurement of calcium influx under voltage clamp in molluscan neurones using the metallochromic dye arsenazo III.

1. The metallochromic indicator dye, arsenazo III, was injected into somata of molluscan neurones from Archidoris monteryensis. Membrane current and dye absorbance change were simultaneously monitored under voltage clamp. 2. Absorbance measured at 660 nm increased during positive-going voltage steps large enough to activate membrane conductances. In situ idifference spectra were qualitatively similar to dye, dye-calcium difference spectra recorded in vitro. The absorbance change was abolished by either a thorough removal of external calcium or internal chelation of calcium by EGTA. It was concluded that the absorbance increase primarily reflected changing internal calcium concentration and that the calcium entered from the outside. 3. Dye absorbance increased in a nearly linear fashion during voltage clamp pulses of 100--300 msec duration. This is in qualitative agreement with electrical stuidies which demonstrated only fractional inactivation of calcium conductance during such periods. Plots of absorbance change vs. Vm peaked at +30 to +40 mV and fell off sharply until approximately +70 mV where the slope became less steep. A null or reversal of the absorbance change was generally observed aroung +110 mV. Evidence is presented that calcium influx was in some cases sufficient to cause sizeable changes in its equilibrium potential. 4. During multisecond voltage clamps the slope of the absorbance change showed a large decline. Where barium substituted for calcium as the influx species in identical clamps, the absorbance at 660 nm also increased but in a much more linear fashion. Except for a slight effect on the initial few pulses, the absorbance signal did not recover after a period of barium influx. These results suggest that part of the slope decline might result from processes related to calcium uptake and not to membrane conductance decrease. 5. Dye absorbance changes during normal and TEA action potentials were measured. Comparison of these changes with voltage clamp records indicated that calcium influx during a spike was capable of raising concentration by roughly 2 X 10(-7) M if the cell were considered to be a uniform sphere with no buffering capacity. Calcium influx during action potentials was increased dramatically by TEA, primarily as a result of a prolonged plateau phase. The existence and duration of the plateau was controlled mainly by potassium conductance systems, however. There was no evidence found for facilitation of the calcium conductance. 6. Following a moderate influx of calcium it required 20--60 sec, depending on the neurone, for the dye absorbance to return to base line (at 9 degrees C). The recovery time course showed a marked difference when examined at different wavelengths. For lambda = 660 nm there was an initial period in which the absorbance decreased rapidly, followed by a slower phase which generally carried the absorbance below the initial (prepulse) value...