Calcium signalling in $\textit{Drosophila}$ photoreceptors measured with GCaMP6f

Abstract

$\textit{Drosophila}$ 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 $\textit{in vivo}$ by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% $\textit{F}$max with $\sim$ 1200 effectively absorbed photons and saturated ($\textit{ΔF/F}$${0}$ $\sim$ 10–20) with 10000–30000 photons. In Ca$^{2+}$ free bath, smaller ($\textit{ΔF/F}$${0}$ $\sim$ 4), long latency ($\sim$ 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 ($\textit{ΔF/F}$${0}$ $\sim$ 0.1), and unlikely to play any role in phototransduction.