Ca2+ feedback control of TRP/TRPL channels in Drosophila photoreceptors

Projektinformationen

FLYghtCaRe

ID Finanzhilfevereinbarung: 658818

DOI

10.3030/658818

Projekt abgeschlossen

EK-Unterschriftsdatum 24 Juni 2015

Startdatum 1 November 2015

Enddatum 31 Oktober 2017

Finanziert unter

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Gesamtkosten

€ 183 454,80

EU-Beitrag

€ 183 454,80

Koordiniert durch

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - FLYghtCaRe (Ca2+ feedback control of TRP/TRPL channels in Drosophila photoreceptors)

Berichtszeitraum: 2015-11-01 bis 2017-10-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Vision begins in specialised cells (photoreceptors) where incident photons of light are converted into an electrical response (light-induced current) by a process known as phototransduction. Photoreceptors in both vertebrates and invertebrates must continuously adjust their sensitivity to cope with the huge changes in illumination over day and night. Invertebrate photoreceptors possess the most efficient light adapting mechanisms known. For example, photoreceptors in the fruitfly Drosophila can detect single photons, generating discrete amplified responses called ‘quantum bumps’, yet still respond reliably in full daylight. In contrast, quantum bumps in vertebrate photoreceptors (rods) are smaller and slower than in flies, whilst rods saturate already at medium light intensities. Calcium ions (Ca2+) are crucial for the fly’s impressive performance, leading to: (i) quantum bump amplification, (ii) fast responses, and (iii) gain control in light adaptation. Much of this is achieved by sequential positive and negative feedback of the light-induced currents mediated by Ca2+ entering the cell via ion channels known as TRP and TRPL. Both channels are negatively modulated by Ca2+, but positive feedback is specific to TRP.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

To unravel the molecular basis for this we generated flies with chimeric channels in which different regions of the TRP and TRPL channels were swapped, and then measured their responses to see which parts of the channels were required for positive and negative feedback. Our results demonstrate that the TRP channel itself is a key target in the positive feedback and identify specific regions of the channel that are required for this.

We also measured the dynamic changes in Ca2+ underlying these processes using genetically encoded fluorescent Ca2+ indicators (GECIs) expressed in the photoreceptors. Fluorescent signals were measured both from dissociated cells and also in vivo in intact flies by imaging the rhabdomeres through the fly’s own optics. We thus provided the first quantitative measurements of Ca2+ in Drosophila photoreceptors in both wild-type flies and in a range of mutants in which Ca2+ homeostasis is altered.

Most of the Ca2+ signal derives from influx through the TRP and TRPL ion channels; however, in Ca2+ free bath, smaller and slower light-induced Ca2+ rises are still detectable in dissociated photoreceptors. The source of this has been debated for years and was recently proposed to be due to light-induced release from internal Ca2+ stores. By expressing our GECIs in different mutants we showed conclusively that this was not the case. Instead, we found it was due to a membrane transporter (“sodium/calcium exchanger”) which normally protects the cells from Ca2+ overload by extruding Ca2+ in exchange for sodium, (usually highest outside the cell). However, in Ca2+ free bath the lack of negative feedback results in a massive influx of sodium ions, which drives the transporter in reverse and paradoxically increases Ca2+.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Overall, these results bring a new level to our understanding of the mechanisms underpinning one of the most interesting and important aspects of insect visual performance: the Ca2+ dependent modulation of the light response. Moreover, they provide unique quantitative data on the dynamics of Ca2+ signalling in Drosophila photoreceptors, and resolve a longstanding debate about the putative role of internal Ca2+ stores in phototransduction.

Genetically encoded Ca2+ indicator imaged in the deep pseudopupil of the living fly