کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5530479 | 1549311 | 2017 | 12 صفحه PDF | دانلود رایگان |
- 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 â¼1200 effectively absorbed photons and saturated (ÎF/F0 â¼Â 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (ÎF/F0 â¼4), long latency (â¼200 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 (ÎF/F0 â¼0.1), and unlikely to play any role in phototransduction.
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Journal: Cell Calcium - Volume 65, July 2017, Pages 40-51