Sharona E. Gordon - US grants

Ion channels activated directly by the binding of cyclic GMP (cGMP) mediate the response to light in vertebrate photoreceptors. Their closure during the sensory transduction event directly couples the chemical signal initiated by isomerization of rhodopsin into changes in membrane potential of the outer segment. Changes in membrane potential in turn direct changes in transmitter release from the synaptic terminal. A binding site for cGMP is present in the carboxyl-terminal domain, and a region that constitutes part of the pore is located between the fifth and sixth transmembrane domains of each subunit of the cGMP-activated ion channel. The molecular mechanism by which cGMP binding leads to opening of the pore, however, is completely unknown. Understanding the structural basis for the function of these channels is critical to understand the role of the channels in visual transduction, and the way its ability to respond to cGMP is tuned under various conditions. Our previous studies have identified two regions of sequence that influence the coupling of cGMP binding to opening of the pore: the amino-terminal region and the cytoplasmic loop that links the sixth (last) transmembrane domain to the cyclic nucleotide-binding domain (post-S6 region). The specific aims of this study are to examine the molecular mechanism by which these regions communicate binding with cGMP to other parts of the channel, including the pore. We will use electrophysiology, molecular biology, and biochemistry to take a multi-faceted approach to the question of the structural basis for channel function. The tools we have developed for studying the function of and interaction between these two important regions of channel sequence will also be used to study the stoichiometry and architecture of native photoreceptor channels.

binding sites; protein binding; intermolecular interaction; biomedical resource;

DESCRIPTION (provided by applicant): Chemical and thermal pain in the cornea is primarily transduced by a calcium- and sodium-permeable ion channel called TRPV1 expressed in nociceptors with cell bodies in the trigeminal ganglia. When injury (including surgery) or illness cause inflammation, the inflammatory process increases the sensitivity of TRPV1 ion channels to painful stimuli, a phenomenon known as inflammatory hyperalgesia. Our long-term goal is to understand the molecular mechanisms mediating inflammatory hyperalgesia, a critical first step in developing more effective pain therapies for corneal injury. In this study, we will focus on the molecular mechanisms of TRPV1 modulation by Nerve Growth Factor (NGF). Inflammation and injury lead to release of trophic factors such as NGF, insulin, and Insulin-like Growth Factor, which increase nociceptor excitability by activating receptor tyrosine kinases (RTKs). It has been proposed that RTK activation sensitizes TRPV1 through hydrolysis of phosphoinositide 4,5-bisphosphate (PIP2), relieving a tonic inhibition of TRPV1 by PIP2. The role of PIP2 is controversial, however, due to emerging evidence that phosphoinositide 3,4,5- trisphosphate (PIPS) may be involved. Based on our preliminary data, we propose that phosphorylation of PIP2 by phosphoinositide 3-kinase (PI3K) to form PIP3 may be an essential element of nociceptor sensitization by NGF. Our specific aims will address the molecular mechanism and functional significance of TRPV1 modulation by NGF. Understanding the regulation of TRPV1 by RTKs is critical to a complete understanding of how inflammation modulates corneal nociceptor excitability and to the development of improved therapies to treat inflammatory pain.

DESCRIPTION (provided by applicant): In the last two decades we have witnessed an explosion in knowledge of the normal and pathological function of the nervous system. Progress has been largely fueled by advances in two methodologies, fluorescence imaging and electrophysiology. We propose to begin the next wave of discovery, by combining optical and electrophysiological measurements of living cells, to probe their function in real time. The excitability of neurons, secretory cells, and muscle cells is influenced by a large number proteins and second messengers: transcription factors determine the number of ion channels and receptors that are produced;kinases, phosphatases, and ions determine the activity of channels and receptors;and complex recycling and degradation pathways determine the residence time of channels and receptors in the plasma membrane. As well, the phospholipids that compose the plasma membrane play an active role in determining channel activity and the rate of channel and receptor internalization. We will image the movement of proteins and signaling molecules within a cell with confocal and TIRF microscopy and read out the net effect of signaling on the cell's excitability using electrophysiological recording. This proposal requests funds for a Confocal-TIRF-Electrophysiological (CTE) workstation for ion channel biophysics. No such instrument exists in the Pacific Northwest. It would permit us to synchronize the movement of organelles, interactions between proteins, changes in intracellular calcium concentration, intra-molecular conformational changes, and electrical activity into a cohesive understanding of how cellular excitability is set, how it is perturbed by extracellular signals, and how it recovers after a signaling event. Using these methodologies, we will ask questions such as: Which calcium-activated signals regulate desensitization? What is the mechanism by which phosphoinositides regulate channels? How do growth factors regulate channel trafficking? How does phosphorylation regulate channel activity and expression? What are the molecular motions that underlie gating? These questions will be asked across a number of cell types, to define ion channel function quantitatively in several systems essential to human health. PUBLIC HEALTH RELEVANCE: Excitable cells depend on ion channel proteins to conduct electrical signals, regulate force, and mediate secretion. In fact, over half of all drug targets are ion channels or proteins that directly regulate ion channels. Our work is directed toward understanding the normal and pathological signaling by ion channels in the nervous system and vascular system.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms that control the excitability of pain-receptor neurons in the cornea. The cornea is innervated by nerve fibers expressing cold receptors (TRPA1 and TRPM8 ion channels), mechanoreceptors (molecular identity unknown), and polymodal receptors that respond to noxious chemical and thermal stimuli (TRPV1 ion channels). Sensory transduction of noxious stimuli protects the cornea from further damage by inducing protective blink reflexes and lacrimal secretions, whereas inflammatory hyperalgesia has a role in protecting injured tissue from repeated unintentional damage. Additionally, corneal nerves and their associated neurotrophins are important for corneal health and wound healing;dysfunction of the corneal nerves or damage during surgery may produce neurotrophic keratitis, characterized by corneal anesthesia and increased risk for corneal ulcers and perforation. TRPV1 is expressed in small-diameter corneal nerve fibers of the ophthalmic branch of the trigeminal ganglia. In response to tissue injury, TRPV1 undergoes sensitization in the presence of pro-inflammatory agents such as Nerve Growth Factor (NGF) and other neurotrophins. NGF induces neuritogenic and trophic signals when bound to the extracellular domain of its receptor, tropomyosin-receptor-kinase subtype A (TrkA). This catalytic receptor activates three enzymatic pathways inside the sensory nerve fiber: the phospholipase C (PLC) pathway, the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3K) pathway. The hyperalgesia induced by NGF and other trophic factors is due to an increase in the number of TRPV1 ion channels in the plasma membrane of sensory neurons. We and others have shown that the mechanism involves the PI3K signaling pathway downstream of TrkA. Furthermore, a direct interaction between a regulatory subunit of PI3K and TRPV1 channels appears to control the localization of the increase in pain receptors within sensory neurons. It is important to understand the sensitization of TRPV1 that occurs in response to NGF: interrupting this hyperalgesic sensory pathway would be of great utility in the clinical setting. In addition, understanding the converse pathway - desensitization of TRPV1 ion channels - may lead to new approaches for treating the pain of injury and disease and may open up therapeutic options that would otherwise be limited due to pain-inducing side effects. In this proposal we will study both hyperalgesia and desensitization. Combining new optical methods with electrophysiology, we will elucidate the role of the membrane lipid PIP2 in desensitization. We will also determine whether the product of PI3K activity, the membrane lipid PIP3, is sufficient for cellular hyperalgesia. By the end of the funding period, we will have gained a detailed understanding of how phosphoinositides (PIP2 and PIP3) tune the excitability of TRPV1-expressing pain receptor neurons. PUBLIC HEALTH RELEVANCE: In the cornea and periphery, pain-receptor neurons become hypersensitive to pain when tissue is injured or inflamed. We will determine how hypersensitivity to pain develops in pain-receptor neurons. Our goal is to identify targets for improved pain therapies.

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): The goal of our work is to elucidate the cellular and molecular mechanisms by which cell-surface receptors regulate the function, trafficking, and expression of ion channels. We are particularly interested in receptor regulation of ion channels in pain transduction, as sensitization to painful stimuli during inflammation (inflammatory hyperalgesia) profoundly influences our physical and mental health, as well as our economic and social well-being. We have chosen the Ca2+-permeable channel TRPV1 as our model both because its properties make it especially suitable and because of its importance in transducing painful stimuli and in tuning the excitability of pain-transducing neurons. Chronic pain is a significant public health and economic problem in the US. An analysis of the 2003 American Productivity Audit, a national survey of US workers, showed that, in a given two-week period, 13% of the workforce lost work time due to uncontrolled pain, with a mean loss of 4.6 hours per week. The 2003 National Center for Health Statistics Report found that 26% of adults report having a problem with pain lasting more than 24 hours. A 2006 study of chronic pain patients found that more than half felt they had little or no control over their pain. Headache, back pain, arthritis pain, tooth pain, cancer pain, and post-operative pain are just a few of the common conditions contributing to decreased quality of life and economic loss across the whole spectrum of the US population. Current treatments are clearly not sufficient to address the wide-spread need for pain relief, and have further problems related to specificity and addiction. Nerve Growth Factor (NGF) was discovered by Rita Levi-Montalcini and Stanley Cohen in the late 1950's. From the first, they understood its power to regulate the differentiation and growth of sensory neurons. NGF is involved in the guidance and survival of sensory neurons and is released onto TRPV1-expressing neurons during injury and inflammation. Our understanding of how NGF sensitizes TRPV1 in inflammatory hyperalgesia exploded in the last several years. In the previous funding period we showed that NGF increases TRPV1 currents by increasing the number of the TRPV1 channels in the plasma membrane. We further showed that a signal-transduction complex is present in nociceptors, composed of the NGF receptor (TrkA), TRPV1, and the enzyme PI3K, which phosphorylates phosphoinositide 4,5-bisphosphate (PIP2) to phosphoinositide 3,4,5-trisphosphate (PIP3). We and others further showed that PI3K activity is required for sensitization. For the remainder of this proposal use the term sensitization of TRPV1 to refer to the increase in the number of TRPV1 channels in the plasma membrane. Although cell surface receptor-stimulated trafficking of membrane lipids and membrane proteins is of broad significance to biology, the molecular mechanisms by which it occurs are poorly understood. One of the best-studied examples, trafficking of the Glut4 glucose transporter to the plasma membrane of adipocytes and muscle cells in response to insulin, has revealed a number of important players on which we based our model for NGF-induced trafficking of TRPV1 to the plasma membrane of pain-receptor neurons. Even in adipocytes and muscles, however, many critical steps in this process are not fully understood. Identification of the main players and their interactions in sensitization of TRPV1 may shed light on a signaling pathway essential to cell differentiation, metabolism, and survival in addition to leading to an understanding of TRPV1 regulation important for inflammatory pain.

Project summary The goal of this proposal is to determine the molecular mechanisms underlying multi-modal gating of the pain-receptor ion channel TRPV1 by interrogating the structural mechanisms for activation by capsaicin, heat, protons, savory compounds, and signaling lipids. We will use tmFRET and computational methods to determine the conformational change induced by each modality and the coupling between modalities.

Project Summary The goal of this equipment request is to acquire a Fluorescence Lifetime Imaging Microscope (FLIM) upgrade to an existing microscope body/optics. The FLIM upgrade will provide a previously unavailable ability to measure the energetics of activation of pain-transducing TRPV1 ion channels.