Introduction Transmembrane signalling cascades initiated by G-protein- coupled receptors are a widely used mechanism for signalling the detection of many sensory modalities. These cascades end with the activation of plasma-membrane ion channels whose activity alters membrane potential and initiates synaptic transmission of a signal to the central nervous system. Several different families of ion channels have been implicated in this process. Historically, the oldest and best characterized are cyclic-nucleotide-gated channels, whose role in vertebrate visual and olfactory transduction is well established (Matulef and Zagotta, 2003). More recently, members of the TRP family of ion channels have been implicated in the transduction of several sensory modalities in both vertebrate and invertebrate systems. These include light (Drosophila TRPC), pheromones (rodent TRPC2), taste (rodent TRPM), physical stimuli and temperature (Drosophila and mammal TRPV, TRPA and TRPN) (Montell et al., 2002). Currently, a crucial factor limiting our understanding of how TRP channels encode sensory modalities is the lack of information about how these channels are activated. In several cases, only a few transduction components have been identified and the inability to perform in vivo analysis of channel activation has been a major obstacle in revealing how TRP channels are activated. The Drosophila phototransduction cascade is historically the oldest and to date the best understood model for the analysis of a TRP channel involved in sensory transduction (Hardie and Raghu, 2001). In the fly eye, rhodopsin, a seven- transmembrane-span G-protein-coupled receptor, activates phospholipase Cβ (PLCβ) (Bloomquist et al., 1988) via Gq (Scott et al., 1995). This initiates a biochemical cascade that ends with the opening of two classes of calcium- and cation- selective TRPC channels, TRP and TRPL (Niemeyer et al., 1996). Several key elements of the transduction cascade have been identified including Gq, PLCβ and protein-kinase C. Several of these components, along with the TRP channel, are clustered into a macromolecular signalling complex by the multivalent PDZ-domain protein INAD (Tsunoda et al., 1998). The INAD complex is thought to increase the speed and specificity of the light response (Montell, 1998; Tsunoda et al., 1998). However, despite this wealth of detail about the components of the transduction cascade, the mechanism of activation of TRP and TRPL remains poorly understood, and is one of the outstanding problems in both sensory neurobiology and intracellular calcium signalling. Although the essential role of PLCβ in the activation of TRP and TRPL is well established (Bloomquist et al., 1988), the biochemical events initiated by this enzyme that lead to channel activation remain unclear. Inositol-1,4,5-trisphosphate (IP 3 ), the best-understood second messenger generated from phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] hydrolysis by PLCβ (Berridge, 1997) was originally postulated to be the second messenger that leads to TRP and TRPL activation (Hardie and Minke, 1993). However, several recent lines of 1373 The TRP family of ion channels mediates a wide range of calcium-influx phenomena in eukaryotic cells. Many members of this family are activated downstream of phosphoinositide hydrolysis but the subsequent steps that lead to TRP channel activation in vivo remain unclear. Recently, the lipid products of phosphoinositide hydrolysis (such as diacylglycerol and its metabolites) have been implicated in activating TRP channels in both Drosophila and mammals. In Drosophila photoreceptors, lack of diacylglycerol kinase (DGK) activity (encoded by rdgA) leads to both constitutive TRP-channel activity and retinal degeneration. In this study, using a novel forward-genetic screen, we identified InaD, a multivalent PDZ domain protein as a suppresser of retinal degeneration in rdgA mutants. We show that InaD suppresses rdgA and that the rescue is correlated with reduced levels of phospholipase Cβ (PLCβ), a key enzyme for TRP channel activation. Furthermore, we show that light, Gq and PLCβ all modulate retinal degeneration in rdgA. The results demonstrate a previously unknown requirement for a balance of PLCβ and DGK activity for retinal degeneration in rdgA. They also suggest a key role for the lipid products of phosphoinositide hydrolysis in the activation of TRP channels in vivo. Key words: Phosphoinositides, Diacylglycerol kinase, TRP channels, Screen Summary Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant Plamen Georgiev 1 , Isaac Garcia-Murillas 1 , Danny Ulahannan 2 , Roger C. Hardie 2 and Padinjat Raghu 1, * 1 The Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, UK 2 Department of Anatomy, University of Cambridge, Downing St, Cambridge, CB2 3DY, UK *Author for correspondence (e-mail: raghu.padinjat@bbsrc.ac.uk) Accepted 7 January 2005 Journal of Cell Science 118, 1373-1384 Published by The Company of Biologists 2005 doi:10.1242/jcs.01712 Research Article Journal฀of฀Cell฀Science