Abstract The goal of this project is to address the major unanswered questions concerning the mechanisms through which the TRP channels are activated, regulated and trafficked in Drosophila photoreceptor cells. These questions are of significance in part due to the striking similarities between the phototransduction cascades in Drosophila photoreceptor cells, and in mammalian intrinsically photosensitive retinal ganglion cells (ipRGCs). Both are initiated by similar visual pigments, which activate Gq and phospholipase Cβ (PLC), thereby leading to the opening of TRPC channels. It is long established that PLC activity is crucial for gating the TRP and TRPL channels in Drosophila photoreceptor cells. However, the link between stimulation of PLC and activation of these channels remains controversial. Aim 1 is devoted to addressing this question. We have identified a candidate agonist that increases in wild-type photoreceptor cells in a light-dependent manner, but not in a mutant devoid of PLC activity. We propose experiments to test whether this lipid represents the physiologically relevant agonist for the TRPC channels in photoreceptor cells. To provide further insights into the mechanisms activating and regulating TRP, we performed a proteomics analysis to identify the repertoire of proteins that associate with TRP in vivo. Aim 2 focuses on one such protein, which we propose has dual roles in phototransduction. In addition to its classical function in phototransduction, we outline experiments to discriminate between whether the direct interaction with TRP promotes activation, or serves to suppress dark noise in the photoreceptor cells. Aim 3 addresses the enigmatic mechanisms through which TRP traffics through the secretory pathway and inserts in the plasma membrane. Using a biochemical approach, we identified a candidate chaperone, which we propose is a critical component necessary for promoting TRP transport. While TRPL can be functionally expressed in heterologous cells, the classical TRP gets retained in the secretory pathway. As such, in vitro electrophysiological studies of TRP have been challenging. We propose that this new chaperone may solve this problem. Aim 4 is devoted to testing whether two small single transmembrane proteins that interact with TRP represent elusive TRP β subunits. Many ion channels, such as voltage-gated cation channels, intimately associate with β subunits. These proteins bear similar overall sizes and topologies with the candidate TRP regulatory subunits that are the focus here. We propose to test whether these proteins have two roles. The first is to enable TRP channels to traffic to the specialized portion of the photoreceptor cells where phototransduction takes place. The second is to shape the gating properties of the channel. To accomplish our goals, we propose a multidisciplinary approach, including electrophysiology, molecular genetics, biochemistry and cell biology. We propose that these studies will provide...