title: Calcium signalling in Drosophila photoreceptors measured with GCaMP6f.
abstract: 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 10-25ms, reached 50% Fmaxwith ∼1200 effectively absorbed photons and saturated (ΔF/F0∼10-20) with 10000-30000 photons. In Ca2+free bath, smaller (ΔF/F0∼4), long latency (∼200ms) light-induced Ca2+rises were still detectable. These were unaffected in InsP3receptor 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 <10nM (ΔF/F0∼0.1), and unlikely to play any role in phototransduction.
authors.affiliation: Cambridge University, Department of Physiology Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Asteriti
authors.affiliation: Cambridge University, Department of Physiology Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK.
authors.forename: Che-Hsiung
authors.initials: CH
authors.lastname: Liu
authors.affiliation: Cambridge University, Department of Physiology Development and Neuroscience, Downing Street, Cambridge, CB2 3EG, UK. Electronic address: rch14@cam.ac.uk.
authors.forename: Roger C
authors.initials: RC
authors.lastname: Hardie
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D008856:Microscopy, Fluorescence; D017956:Photoreceptor Cells, Invertebrate / Q000166:cytology / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D010738:Type C Phospholipases
keywords: Calcium imaging; Genetically encoded calcium indicators; InsP(3); Na/Ca exchanger; Phospholipase C; Phototransduction; TRP channels