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Volume 16, Number 9, Issue of May 1, 1996 pp. 2924-2933
Copyright ©1996 Society for NeuroscienceINDO-1 Measurements of Absolute Resting and Light-Induced Ca2+ Concentration in Drosophila Photoreceptors
Received Sept. 27, 1995; revised Feb. 12, 1996; accepted Feb. 15, 1996.
Roger C. Hardie Cambridge University, Department of Anatomy, Cambridge CB2 3DY, United Kingdom
Absolute Ca2+ levels in dissociated Drosophila photoreceptors 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 µM Ca2+), 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 nM in normal Ringer's (1.5 mM Ca2+). 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 using ninaE mutants 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 <20 nM when extracellular Na+ was replaced with N-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.
Key words: calcium entry; inositol phosphates; phototransduction; calcium fluorimetry; INDO-1; Drosophila; photoreceptor; vision
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