Timing of Ca²⁺ Release from Intracellular Stores and the Electrical Response of Limulus Ventral Photoreceptors to Dim Flashes

Light-induced release of Ca²⁺ from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca²⁺ indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 μm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca_i) after flashes delivering between 10 and 5 × 10⁵ effective photons. Peak Ca_i increased with flash intensity to 138 ± 76 μM after flashes delivering ∼10⁴ effective photons, while the latent period of the elevation of Ca_i fell from ∼140 to 21 ms. The onset of the light-induced elevation of Ca_i was always highly correlated with that of the receptor potential. The time for Ca_i to exceed 2 μM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 ± 6.4 ms). Ca_i was also measured during steps of light delivering ∼10⁵ effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca_i were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca_i and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca_i resulted from Ca²⁺ release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca²⁺ pumps, but not by removal of extracellular Ca²⁺ ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca²⁺ ions that appears necessary for a highly amplified electrical response to dim flashes.