Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. We have recently found that the TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Gαq, phospholipase Cβ and the TRP ion channel, or in proteins required for TRP function. Our data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, we found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca2+-influx in the translocation process. The critical role of Ca2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca2+.

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP 3 R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP 3 branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipidsignaling systems. To examine this hypothesis we applied the membrane-permeant InsP 3 R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP 3 R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP 3 -mediated Ca 2ϩ release and store-operated Ca 2ϩ entry in Xenopus oocytes at a stage operating after production of InsP 3 but before the opening of the surface membrane Cl Ϫ channels by Ca 2ϩ . We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP 3 branch of the inositol lipid-signaling pathway.

Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin–membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.