INDO-1 measurements of absolute resting and light-induced Ca2+ concentration in Drosophila photoreceptors.
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 microM 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 (approximately 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.
Hardie
R C
RC
Cambridge University, Department of Anatomy, United Kingdom.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003624:Darkness; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D007211:Indoles; D008027:Light; D009154:Mutation; D009994:Osmolar Concentration; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism* / Q000528:radiation effects*; D011930:Reaction Time
D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium