Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

•Fruitflies generated expressing GCaMP6f in photoreceptors using the opsin promoter.•Quantitative in vivo Ca2+ imaging in completely intact flies as well as dissociated cells.•Ca2+ rise in Ca2+ free bath abolished in Na+/Ca2+ exchanger mutants.•Ca2+ free rise due to re-equilibration of Na+/Ca2+ exchange following Na+ influx.•No significant light-induced release of Ca2+ from internal stores.

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25 ms, reached 50% Fmax with ∼1200 effectively absorbed photons and saturated (ΔF/F0 ∼ 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (ΔF/F0 ∼4), long latency (∼200 ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F0 ∼0.1), and unlikely to play any role in phototransduction.

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