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 wi thin 4 mu m of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentr ation (Ca-i) after flashes delivering between 10 and 5 x 10(5) effective ph otons. Peak Ca-i increased with flash intensity to 138 +/- 76 mu M after fl ashes delivering similar to 10(4) effective photons, while the latent perio d of the elevation of Ca-i fell from similar to 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 mu M was approxima tely equal to that for the receptor potential to exceed 8 mV (mean differen ce; 2.2 +/- 6.4 ms). Ca-i was also measured during steps of light deliverin g similar to 10(5) effective photons/s to photoreceptors that had been blea ched with hydroxylamine so as to reduce their quantum efficiency. Elevation s 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 devel op. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca-i and the receptor potential still ro se at approximately the same time, indicating a shared process generating t he latent period. Light-induced elevations of Ca-i resulted from Ca2+ relea se from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca2+ pumps, but not by removal of ext racellular Ca2+ ions. CPA also greatly diminished and slowed the receptor p otential elicited by dim flashes. The results demonstrate a rapid release o f Ca2+ ions that appears necessary for a highly amplified electrical respon se to dim flashes.