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

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Abstract
The metallochromic indicator dye, arsenazo III, was injected into somata of molluscan neurons from Archidoris montereyensis. Membrane current and dye absorbance change were simultaneously monitored under voltage clamp. Absorbance measured at 660 nm increased during positive-going voltage steps large enough to activate membrane conductances. In situ difference spectra were qualitatively similar to dye, dye-Ca difference spectra recorded in vitro. The absorbance change was abolished by either a thorough removal of external Ca or internal chelation of Ca by EGTA [ethyleneglycol-bis(.beta.-aminoethylether)N,N,N''N''-tetraacetic acid]. The absorbance increase primarily reflected changing internal Ca concentration and the Ca entered from the outside. Dye absorbance increased in a nearly linear fashion during voltage clamp pulses of 100-300 ms duration. This is in qualitative agreement with electrical studies which demonstrated only fractional inactivation of Ca conductance during such periods. Plots of absorbance change vs. Vm [maximum velocity] 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 around +110 mV. Evidence is presented that Ca influx was in some cases sufficient to cause sizeable changes in its equilibrium potential. During multisecond voltage clamps the slope of the absorbance change showed a large decline. Where Ba substituted for Ca 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 Ba influx. Part of the slope decline might result from processes related to Ca uptake and not to membrane conductance decrease. Dye absorbance change during normal and TEA [tetraethylammonium] action potentials were measured. Comparison of these changes with voltage clamp records indicated that Ca influx during a spike was capable of raising concentration by roughly 2 .times. 10-7 M if the cell were considered to be a uniform sphere with no buffering capacity. Ca 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 K+ conductance systems. There was no evidence found for facilitation of the Ca conductance. Following a moderate influx of Ca it required 20-60 s, depending on the neuron, for the dye absorbance to return to base line (at 9.degree. 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. At 610 nm the rapid phase was more prominent than at 660 nm and there was a relatively greater excursion below the baseline absorbance. For .lambda. > 660 nm the fast phase and the baseline undershoot were negligible. This wavelength dependence can be explained on a qualitative basis by a pH decrease subsequent to Ca entry.