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Original Article |
Correspondence to: Richard Payne, Department of Biology, University of Maryland, College Park, MD 20742. Fax:301-314-9358 E-mail:rp12{at}umail.umd.edu.
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 (Cai) after flashes delivering between 10 and 5 x 105 effective photons. Peak Cai increased with flash intensity to 138 ± 76 µM after flashes delivering ~104 effective photons, while the latent period of the elevation of Cai fell from ~140 to 21 ms. The onset of the light-induced elevation of Cai was always highly correlated with that of the receptor potential. The time for Cai to exceed 2 µM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 ± 6.4 ms). Cai 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 Cai 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 Cai and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Cai 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.
Key Words: cyclopiazonic acid, horseshoe crab, fluorescent indicator dye, receptor potential, phototransduction
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