Chemical Biology Probes to Decipher Regulation of TRPV1 in Living Cells

In this proposal, we apply new chemical biology tools to probe the molecular and cellular mechanisms by which the inflammatory mediator nerve growth factor (NGF) regulates the trafficking of TRPV1 ion channels. Although genetically encoded fluorescent proteins have facilitated major breakthroughs, they can perturb protein trafficking and do not clearly distinguish protein in the plasma membrane from that in intracellular compartments. To overcome these limitations, we developed novel tetrazine-based noncanonical amino acids and evolved aminoacyl tRNA synthetases to incorporate them into proteins in mammalian cells via amber codon suppression. We also developed membrane-impermeant, fast-reacting strained trans- cyclooctene-conjugated fluorophores that react with the tetrazine noncanonical amino acids on the extracellular side of proteins within seconds, even at sub-micromolar concentrations compatible with cell imaging. We utilize these new chemical biology probes to probe the cellular and molecular mechanisms by which NGF signaling increases TRPV1 trafficking to the plasma membrane. NGF binds to a receptor tyrosine kinase, which then activates multiple signaling pathways including phosphoinositide 3-kinase (PI3K). We combine genetic code expansion and bio-orthogonal labeling with our recently published system for activating PI3K. This “OptoPI3K” system bypasses the NGF receptor to activate PI3K directly, without activating other signaling pathways. We will use genetic code expansion and our new, rapid click chemistry labels to determine the cellular mechanisms by which NGF promotes trafficking of TRPV1 channels to the PM (Aim 1). We will activate OptoPI3K with light to determine the role of the positive feedback of TRPV1 on PI3K and whether other pathways contribute to the NGF response (Aim 2). We will use multi-color single-molecule imaging with our bright, photostable click chemistry labels to determine how NGF regulates the complex that includes TrkA, PI3K, and TRPV1 (Aim 3). Finally, we will generate mutations in TRPV1 to disrupt the PI3K interaction and determine the role of the interaction in NGF-induced sensitization (Aim 4). The approaches we apply to this system will be broadly useful for studying signaling via G-protein coupled receptors and receptor tyrosine kinases in a variety of cell contexts.