The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

Abstract

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5‐trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA‐encoded phospholipase C, an eye‐specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein‐protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1‐S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.

Phototransduction and retinal degeneration in Drosophila

“…These findings demonstrate that in addition to serving as a Ca 2+ -permeable channel, TRP also plays an important role as a molecular anchor. This additional role may account for the observations that TRP is present in approximately stoichiometric proportions with INAD [196], which is a level tenfold higher than is necessary for a normal light-response [150]. The mutual requirement for TRP and INAD for localization reflects mutual roles in retention in the rhabdomeres, rather than for targeting [195,199].…”

“…The central protein in the signalplex is the scaffold protein, INAD, which is comprised of five PDZ protein interaction modules. The "core complex" [195] consists of INAD and three proteins that bind directly to INAD: TRP, INAC (PKC) and NORPA (PLC) [173,196,197]. Each of the three target proteins that are included in the core complex is expressed at similar levels [196] and may always be present in the complex as they depend on INAD for normal localization in the rhabdomeres and protein stability [173,198].…”

Calcium Transients in the Rhabdomeres of Dark- and Light-Adapted Fly Photoreceptor Cells

“…These large flies allow long-lasting and stable intracellular recordings of photoreceptor cells with intracellular electrodes that are blunt enough for iontophoretic injection of fluorescent C a 2ϩ indicators (see below), which might be difficult in Drosophila. Many genes of molecules involved in the phototransduction or in its regulation have been cloned in Calliphora and are found to be ϳ80% homologous to corresponding genes of Drosophila (Huber et al, 1996a(Huber et al, ,b, 1998. This indicates that the results obtained in Calliphora will be of direct relevance to the interpretation of data from Drosophila.…”

“…Because Ca 2ϩ influx in fly photoreceptor cells is a highly localized process, only occurring in the rhabdomere (Ranganathan et al, 1994;Huber et al, 1996a;Niemeyer et al, 1996), averaging the Ca 2ϩ -induced fluorescence across the entire cell might have obscured important kinetic details. We reasoned that it should be possible to record exclusively the fluorescence emanating from the rhabdomere by making use of the natural optics of the fly's eye, because the rhabdomere samples light only from a narrow angle of view, which in blowflies amounts to 1-2°(van Hateren, 1984).…”

“…In fly photoreceptors, these channels are highly permeable for Ca 2ϩ (Hardie and Minke, 1992;Reuss et al, 1997) and exclusively found in the rhabdomeres (Ranganathan et al, 1994;Huber et al, 1996a;Niemeyer et al, 1996). The rhabdomere consists of microvilli, tube-like protrusions of the plasma membrane (for review, see Hardie, 1985), which contain most known molecules of the transduction cascade as well as many molecules involved in the regulatory control of the cascade (for review, see Montell, 1998).…”