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

Abstract

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 × 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.

Chemical calcium indicators

“…It has been used to measure Ca 2+ in photoreceptors of invertebrates (99), gastric myocytes (80), cardiac myocytes (100), skeletal muscle fibers (101). …”

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+release and the opening of cGMP-gated channels

“…Excitation by light or InsP 3 is blocked by the InsP 3 receptor antagonist heparin [18,29]. Direct measurements show that Ca 2+ release is sufficiently fast to activate the light-dependent conductance [14,45]. The InsP 3 receptor is localized in the endoplasmic reticulum adjacent to the base of the rhodopsin-containing microvilli at the site of Ca 2+ release [46].…”

“…In the photoreceptors of Limulus ventral eye (for review see [13]), the cascade involves PLC, InsP 3 , Ca 2+ and cGMP. Light produces an InsP 3 -induced Ca 2+ elevation that precedes the onset of the receptor potential [14]. Furthermore, intracellular injection of Ca 2+ mimics the light response [15-17] and buffering intracellular Ca 2+ inhibits it [16,18].…”

Polycarboxylate Fluorescent Indicators as Ion Concentration Probes in Biological Systems

“…The indicators respond to Ca 2+ binding by an increase in fluorescence, however they do not exhibit a shift in either excitation or emission wavelength. Replacement of the two chlorine substituents in Fluo-3 with fluorine atoms yielded Fluo-4 [39][40][41], with similar spectral profile, whereas introduction of a nitro group at the 5'-position of Fluo-4 produced Fluo-5N, and fluorine substitution at the 5'-and 5', 6'-positions of Fluo-4 yielded Fluo-5F and Fluo-4FF, respectively, with similar spectral profiles to the parent molecule. Introduction of the Fluo-4 fluorophore to an APTRA molecule yielded Mag-Fluo-4.…”