The long-term goal of our research is to understand the mechanisms underlying phototransduction in the fruitfly, Drosophila melanogaster. Drosophila phototransduction functions through a phospholipase C (PLC)-dependent signaling system, and culminates in Ca2+ and Na+ influx, via TRP channels. It is now clear that there exists a large family of mammalian TRP channels, many of which participate in a diversity of sensory signaling processes. The over-riding theme of this proposal is that phosphoinositide (PI) signaling is critical for regulatory events in Drosophila photoreceptor cells, which are more complex and varied than the established concept of gating the TRP channels through hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Experiments in this proposal are designed to challenge the current view that the only effector pathway for light-activated rhodopsin is a heterotrimeric G-protein/PLC pathway. Rather, we propose to test the idea that light activated rhodopsin also couples to a small GTPase, which in turn stimulates a phospholipase D (PLD), and the small GTPase and PLD are required for the light-dependent translocation of the rhodopsin regulatory protein, arrestin. We also propose to test the idea that PIs have an additional role in vivo, namely to regulate the TRP channel by preventing its interaction with inhibitory calcium/calmodulin. As part of a long-term goal to define the proteins that participate in PI signaling in photoreceptor cells, we propose to address the functional requirement for a type of PI-transfer protein that is conserved from flies to humans, but has not been characterized in any organism. To address these questions, we propose to employ a combination of genetics, electrophysiology, cell biology, germline transformation and biochemical approaches. Finally, to generalize from our work on fly vision to mammalian phototransduction, experiments are proposed to provide genetic evidence for a requirement for PI signaling in the intrinsically photosensitive retinal ganglion cells (ipRGCs) of the mouse, which function in the pupillary light reflex and several accessory light-driven behaviors. To study the regulation of TRP channels and phototransduction by PIs, we propose to test the hypotheses that: 1) light-dependent movement of Arrestin functions through a small GTPase and PLD-dependent pathway, 2) Drosophila TRP undergoes dual regulation by PIP3 and Ca2+/calmodulin in vivo, 3) a new PI-transfer protein is required for the Drosophila photoresponse, and 4) a mouse PI-transfer protein is required for function of the ipRGCs. During the last few years, four human diseases have been shown to be due to mutations in TRP channels, including the most common disease due to mutation in a single gene, autosomal dominant polycystic kidney disease, and mucolipidosis type IV, which causes severe neurodegeneration, mental retardation and retinal degeneration. Given that the molecular mechanisms underlying the activation of these human TRPs are poorly understood, Drosophila TRP provides an in vivo model for defining the mechanisms regulating TRP channels.

Public Health Relevance

TRP channels are a type of protein that allows calcium and sodium into cells, and have broad roles in many senses, including the sense of taste and the ability to feel noxious temperatures and mechanical stimuli. Furthermore, there are at least four diseases that are caused by mutations in TRP channels, including two kidney diseases, one of which afflicts 6 million people worldwide, and a neurodegenerative disease. The research on fly vision offers a uniquely useful model to characterize the means by which these channels open and close, and has potential as a model system for testing drugs that affect the activity of these channels.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY010852-17
Application #
8005508
Study Section
Biology and Diseases of the Posterior Eye Study Section (BDPE)
Program Officer
Neuhold, Lisa
Project Start
1995-01-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
17
Fiscal Year
2011
Total Cost
$389,664
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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
Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig (2015) TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon. Cell Rep 13:573-84
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
Akitake, Bradley; Ren, Qiuting; Boiko, Nina et al. (2015) Coordination and fine motor control depend on Drosophila TRPγ. Nat Commun 6:7288
Liu, Chao; Montell, Craig (2015) Forcing open TRP channels: Mechanical gating as a unifying activation mechanism. Biochem Biophys Res Commun 460:22-5
Liman, Emily R; Zhang, Yali V; Montell, Craig (2014) Peripheral coding of taste. Neuron 81:984-1000
Stefan, Christopher P; Zhang, Nannan; Sokabe, Takaaki et al. (2013) Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters. Eukaryot Cell 12:204-14
Zhang, Yali V; Raghuwanshi, Rakesh P; Shen, Wei L et al. (2013) Food experience-induced taste desensitization modulated by the Drosophila TRPL channel. Nat Neurosci 16:1468-76
Wong, Ching-On; Li, Ruoxia; Montell, Craig et al. (2012) Drosophila TRPML is required for TORC1 activation. Curr Biol 22:1616-21
Montell, Craig (2012) Drosophila visual transduction. Trends Neurosci 35:356-63

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