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 C? (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 ? subunits. Many ion channels, such as voltage-gated cation channels, intimately associate with ? 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 the framework for addressing similar questions relevant to the TRPC6 and TRPC7 channels in ipRGCs, which are essential for multiple behaviors important for human health, including normal circadian rhythms and sleep patterns.

Public Health Relevance

Mutations disrupting members of the TRP family of channels underlie multiple diseases, including those that cause complete stationary night blindness and retinal degeneration. However, our concepts as to how these proteins are opened and closed are quite incomplete. The goals of the proposed project are to use a combination of technical advantages available in the fruit fly to determine how these channels are regulated, thereby providing the framework for manipulating the activities of the related mammalian TRPs, and ameliorate human disease.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology of the Visual System Study Section (BVS)
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Neuhold, Lisa
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University of California Santa Barbara
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Santa Barbara
United States
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Liu, Jiangqu; Sokabe, Takaaki; Montell, Craig (2018) A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae. J Vis Exp :
Hofherr, Alexis; Seger, Claudia; Fitzpatrick, Fiona et al. (2018) The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism. PLoS Biol 16:e2005651
Luo, Junjie; Shen, Wei L; Montell, Craig (2017) TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae. Nat Neurosci 20:34-41
Xu, Chiwei; Luo, Junjie; He, Li et al. (2017) Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut. Elife 6:
Walker, Marquis T; Montell, Craig (2016) Suppression of the motor deficit in a mucolipidosis type IV mouse model by bone marrow transplantation. Hum Mol Genet 25:2752-2761
Walker, Marquis T; Rupp, Alan; Elsaesser, Rebecca et al. (2015) RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells. Mol Biol Cell 26:3671-8
Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig (2015) TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon. Cell Rep 13:573-584
Liu, Chao; Montell, Craig (2015) Forcing open TRP channels: Mechanical gating as a unifying activation mechanism. Biochem Biophys Res Commun 460:22-5
Akitake, Bradley; Ren, Qiuting; Boiko, Nina et al. (2015) Coordination and fine motor control depend on Drosophila TRP?. Nat Commun 6:7288
Liman, Emily R; Zhang, Yali V; Montell, Craig (2014) Peripheral coding of taste. Neuron 81:984-1000

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