Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

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 Ca(2+) influx in response to light, but whether Ca(2+) is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca(2+) 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% F(max) with ∼1200 effectively absorbed photons and saturated (ΔF/F(0) ∼ 10–20) with 10000–30000 photons. In Ca(2+) free bath, smaller (ΔF/F(0) ∼4), long latency (∼200 ms) light-induced Ca(2+) rises were still detectable. These were unaffected in InsP(3) receptor mutants, but virtually eliminated when Na(+) was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca(2+) free rises were also eliminated in Na(+)/Ca(2+) exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca(2+) free rises are strictly dependent on Na(+) influx and activity of the exchanger, suggesting they reflect re-equilibration of Na(+)/Ca(2+) exchange across plasma or intracellular membranes following massive Na(+) influx. Any tiny Ca(2+) free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F(0) ∼0.1), and unlikely to play any role in phototransduction.