Intracellular calcium accumulation during depolarization in a molluscan neurone

1. The bursting pacemaker neurone R-15 of Aplysia was injected with the Ca2+ sensitive dye arsenzo III. Changes in absorbance were measured with a differential spectrophotometer to monitor changes in free intracellular Ca2+ during membrane depolarization under voltage clamp conditions. 2. Dye absorbance increased linearly for depolarizing pulse durations up to 100 msec and approximately linearly between 100 and 300 msec, but for longer durations the absorbance change decreased. 3. The absorbance change vs. voltage relation increased steeply between -20 and 0 mV (e-fold per 8.5 mV), peaked at +36 mV and declined non-linearly to an estimated null or suppression potential of about +139 mV. 4. TTX (5 x 10(-5 M) had no effect on the change in dye absorbance produced by brief or long duration stimuli whereas Ca2+ free ASW abolished all changes in dye absorbance. 5. The absorbance change saturated with increasing external Ca2+ concentrations. The relation between dye absorbance and external Ca2+ concentration was hyperbolic and for a small range of external Ca2+ concentration and membrane potentials could be fitted by a Michaelis--Menten expression where the dissociation constant and the maximum absorbance change are voltage dependent. 6. The absorbance change was reduced by external divalent ions which block the Ca2+ channel (e.g. Cd2+ and Ni2+). The suppression of dye absorbance was increased by membrane depolarization and suggests that there is a voltage dependent site within the Ca2+ channel which binds divalent ions. 7. The decline of the absorbance--voltage relation from its peak to the suppression potential showed a greater nonlinearity when longer duration voltage clamp pulses were used. The non-linearity can be explained if the accumulation of Ca2+ ions next to the inner surface of the membrane during depolarization reduces the driving force on Ca2+ ions and thus decreases Ca2+ ion influx. 8. The suppression potential estimated from the absorbance--voltage relation increased 29 mV per tenfold change in the external Ca2+ concentration and thus can be used to estimate the Ca2+ equilibrium potential. 9. The change in dye absorbance produced by brief depolarizing voltage clamp steps was inactivated at positive holding potentials (50% inactivation at about -14 mV). Our results suggest that the slow decrease in dye absorbance during prolonged depolarization is caused by inactivation of the Ca2+ channel.