TRPV1 ion channels are multimodal receptors that can be activated by heat, high [H+]o, voltage, arachidonic acid metabolites, capsaicin (the pungent extract of hot chili peppers), and the signaling lipid PI(4,5)P2 (PIP2). Ca2+/Calmodulin (Ca2+/CaM) and ATP may modulate its activity as well. Our long-term goal is to understand the molecular mechanism by which TRPV1 integrates these multiple physiological stimuli. We and others have previously established that PIP2 directly activates TRPV1. Our recent work indicates that the proximal part of the intracellular C-terminal domain comprises at least part of the PIP2 binding site. However, the inability to control the lipid composition of native membranes, the presence of myriad enzymes and other proteins in cells and excised patches, and the difficulty of specifically labeling intracellular domains of channels within cells have proven serious experimental barriers to understanding regulation of TRPV1 by PIP2 and other activation modalities. We have developed a novel approach to reconstitute purified TRPV1 channels at high density in synthetic Giant Unilamellar Vesicles (GUVs). In this proposal we will apply standard patch-clamp methods, Patch-Clamp Fluorometry (PCF), and Transition Metal Ion FRET (tmFRET) to study purified TRPV1 channels in GUVs of defined lipid composition. Single cysteines engineered into our cysteineless TRPV1 background will be used to site-specifically label channels in the GUVs with fluorophore, completely eliminating the background fluorescence problem. The GUVs used for reconstitution will include synthetic lipids that bind transition metals which act as short- distance FRET quenchers in the novel short-range tmFRET approach we have developed. PCF allows us to simultaneously record the function of the channel with electrophysiology and the rearrangement of the channel with fluorescence. These new tools will allow us to measure dynamics of the intracellular N- and C-terminal domains associated with PIP2 activation as well as with activation by heat, Ca2+/CaM, and ATP.

Public Health Relevance

TRPV1 ion channels mediate the response to painful chemical and thermal stimuli in pain- receptor neurons. We will use a number of innovative new techniques which we have developed to probe the mechanisms by which painful stimuli regulate the activity of these ion channels. We particularly focus on the molecular events that underlie TRPV1 regulation by signaling lipids.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Nie, Zhongzhen
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University of Washington
Schools of Medicine
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Gordon, Sharona E; Munari, Mika; Zagotta, William N (2018) Visualizing conformational dynamics of proteins in solution and at the cell membrane. Elife 7:
Stratiievska, Anastasiia; Nelson, Sara; Senning, Eric N et al. (2018) Reciprocal regulation among TRPV1 channels and phosphoinositide 3-kinase in response to nerve growth factor. Elife 7:
Aman, Teresa K; Gordon, Sharona E; Zagotta, William N (2016) Regulation of CNGA1 Channel Gating by Interactions with the Membrane. J Biol Chem 291:9939-47
Zagotta, William N; Gordon, Moshe T; Senning, Eric N et al. (2016) Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET. J Gen Physiol 147:201-16
Gordon, Sharona E; Senning, Eric N; Aman, Teresa K et al. (2016) Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane. J Gen Physiol 147:189-200
Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M (2015) Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase. Biophys J 109:2480-2491
Senning, Eric N; Gordon, Sharona E (2015) Activity and Ca²? regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons. Elife 4:e03819
Ufret-Vincenty, Carmen A; Klein, Rebecca M; Collins, Marcus D et al. (2015) Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels. J Gen Physiol 145:431-42
Gordon, Sharona E (2014) Getting nowhere fast: the lack of gender equity in the physiology community. J Gen Physiol 144:1-3
Gordon, Sharona E (2014) Restoring integrity to the scientific literature: lowering the bar to raise our standards. J Gen Physiol 144:495-7

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