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Fly photoreceptors are polarized cells, which have an extended interface between their cell body and the light signaling compartment, the rhabdomere. Upon intense illumination, rhabdomeric calcium concentration reaches millimolar levels that would be toxic if Ca2+ diffusion between the rhabdomere and cell body was not robustly attenuated. Yet, it is not clear how such effective attenuation is obtained. Here we show that Ca2+ homeostasis in the photoreceptor cell relies on the protein calphotin. This unique protein functions as an immobile Ca2+ buffer, which is localized along the base of the rhabdomere, separating the signaling compartment from the cell body. Generation and analyses of transgenic Drosophila strains, in which calphotin expression levels were reduced in a graded manner, showed that moderately reduced calphotin expression impaired Ca2+ homeostasis while calphotin elimination resulted in severe light dependent photoreceptor degeneration. Electron microscopy, electrophysiology and optical methods revealed that the degeneration was rescued by prevention of Ca2+ overload via overexpression of CalX, the Na+-Ca2+ exchanger. In addition, Ca2+ imaging experiments showed that reduced calphotin levels resulted in abnormally fast kinetics of Ca2+elevation in photoreceptor cells. Together, the data suggest that calphotin functions as a Ca2+ buffer; a possibility which we directly demonstrate by expressing calphotin in a heterologous expression system. We propose that calphotin-mediated compartmentalization and Ca2+ buffering constitute an effective strategy to protect cells from Ca2+ overload and light induced degeneration.

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The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was ϳ40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin.The intrinsically photosensitive retinal ganglion cells (ipRGC) 2 are a subclass of retinal ganglion cells expressing the visual pigment, melanopsin (OPN4), which calibrates by direct photic input the circadian pacemaker of the master circadian clock and supports some non-image forming light-dependent functions (reviewed in Ref. 1). There are difficulties in advancing understanding of ipRGC phototransduction. The main obstacle is the scarcity of ipRGC and the low expression levels of phototransduction proteins in these cells. This difficulty makes it nearly impossible to investigate phototransduction of the ipRGC by employing the same set of biochemical and electrophysiological approaches that proved successful in characterizing rhodopsin signaling processes in image-forming rod photoreceptor cells. Therefore, at present, the knowledge of phototransduction of ipRGC is still fragmented (1). A promising way to characterize the OPN4 photopigment arises from the apparent similarity between phototransduction of ipRGC and invertebrates. It has been well established that several features of ipRGC photosensitivity are also characteristic of invertebrate photoreceptor cells. (i)...

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