INDO-1 Measurements of Absolute Resting and Light-Induced Ca2+Concentration inDrosophilaPhotoreceptors

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Abstract
Absolute Ca2+levels in dissociatedDrosophilaphotoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca2+influx that saturated the dye (>10 μmCa2+), but lagged the electrical response by 2.8 msec. Resting Ca2+levels in the dark, measured during the latent period before the response, averaged 160 nmin normal Ringer’s (1.5 mmCa2+). Ca2+increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca2+during the latent period; and (2) by usingninaEmutants with greatly reduced rhodopsin levels. Ca2+rose linearly as a function of the time integral of the light-sensitive current with a slope of 2.7 nm/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nm/pC, indicating a 2.5-fold reduction in the fractional Ca2+current. From these data, it can also be estimated that >99% of the Ca2+influx is effectively buffered by the cell. In Ca2+-free Ringer’s, resting cytosolic Ca2+was reduced (to 30–70 nm), but contrary to previous reports, significant light-induced increases (∼250 nm) could be elicited. This rise was reduced to +was replaced withN-methyl-d-glucamine, suggesting that it could be attributed to Na+influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nm.

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