Emily R. Liman - US grants

DESCRIPTION: The application is concerned with the mechanisms of sensory - transduction by chemosensory neurons of the vomeronasal organ (VNO). A series of patch-clamp recording experiments are proposed to characterize the voltage-gated ionic currents in dissociated mouse vomeronasal neurons, and to determine if second messenger-gated ion channels are present in these cells. Retrograde labeling of VNO neurons in vivo prior to dissociation, or antibody labeling for cell specific markers in fixed, dissociated cells, will be used to confirm visual identification of dissociated chemosensory VNO neurons from which recordings will be obtained. Whole cell patch recordings in voltage clamp mode will be used to analyze Na+, Ca++, and K+ currents in terms of which ion carries them, their I/V functions, their inactivation kinetics and their pharmacology. In other experiments, three approaches (excised patch recordings, intracellular dialysis, and application of membrane permanent analogs) will be used to test whether cAMP, cGMP, or IP3-gated channels are present in these cells.

DESCRIPTION: (adapted from applicant's abstract)The mammalian olfactory system consists of at least two functionally distinct organs, the main olfactory epithelium and the vomeronasal organ (VNO). The VNO, a pair of tubular structures at the rostral end of the nasal cavity, is thought to mediate detection of pheromones, chemical signals that regulate reproductive and social behavior and neuroendocrine status. Sensory transduction in the VNO is distinct from that in the main olfactory epithelium, and remains poorly understood. The experiments proposed in this application will test the hypothesis that sensory transduction in the VNO is mediated by a signaling pathway similar to that which mediates phototransduction in invertebrates. In support of this hypothesis, the PI has recently found a VNO-specific homolog (TRP2) of the Drosophila light-activated channel (dTRP) that is highly localized to sensory microvilli of rat vomeronasal sensory neurons. The proposed research is aimed at elucidating the role of the TRP2 channel in vomeronasal sensory transduction, and in VNO-mediated behaviors. A central goal of this work is to identify second messenger pathways that activate TRP2. Extensive studies in Drosophila suggest that activation of dTRP is downstream of phospholipase C, but the direct stimulus for opening dTRP channels is not known. The PI will use techniques of patch-clamp recording and ratiometric calcium imaging in heterologous cell types to study gating of TRP2 in response to receptor-mediated stimulation of phospholipase C and direct application of putative second messengers. A second goal is to identify molecules that interact with TRP2 in a yeast two-hybrid screen of a VNO-cDNA library. By analogy to Drosophila phototransduction, the PI expects TRP2 to be part of a signaling complex that includes other components of the sensory transduction cascade. It has recently been recognized that air-borne pheromones play a role in regulating human reproduction as seen in the menstrual synchrony of women who are in close contact. The sensory reception of human pheromones is not well understood, but may be mediated by the VNO, a structure previously thought to be vestigial in humans. The proposed experiments to elucidate basic mechanisms of sensory transduction in the VNO may, thus, provide a better understanding of the control of human reproductive function.

DESCRIPTION (provided by applicant): The mammalian olfactory system consists of at least two functionally distinct organs, the main olfactory epithelium and the vomeronasal organ (VNO). The VNO, a pair of tubular structures at the rostral end of the nasal cavity, is thought to mediate detection of pheromones, chemical signals that regulate reproductive and social behavior and neuroendocrine status. Sensory transduction in the VNO is distinct from that in the main olfactory epithelium, and remains poorly understood. The experiments described in this grant will test the hypothesis that sensory transduction in the VNO is mediated by a signaling pathway similar to that which mediates phototransduction in invertebrates. In support of this hypothesis, we have recently found a VNO- specific homolog (TRP2) of the Drosophila light-activated channel (dTRP) that is highly localized to sensory microvilli of rat vomeronasal sensory neurons. The proposed research is aimed at elucidating the role of the TRP2 channel in vomeronasal sensory transduction, and in VNOmediated behaviors. A central goal of this work is to identify second messenger pathways that activate TRP2. Extensive studies in Drosophila, suggest that activation of dTRP is downstream of phospholipase C, but the direct stimulus for opening dTRP channels is not known. We will use techniques of patch-clamp recording and ratiometric Ca++ imaging in heterologous cell types to study gating of TRP2 in response to receptor-mediated stimulation of phospholipase C and direct application of putative second messengers. A second goal is to identify additional components of the sensory transduction cascade. The candidate, Dr. Liman, has recently joined the faculty at the University of Southern California, where she is an assistant professor. This ISA will greatly enhance the candidate's career development by allowing her much needed time to set up a strong research program, involving a variety of sophisticated techniques. The candidate is an experienced electrophysiologist as well as a molecular biologist, and the research proposed will take advantage of her expertise in these disparate techniques to tackle basic questions in the biology of the vomeronasal system.

DESCRIPTION (provided by applicant): Recent work indicates that vomeronasal and taste transduction in vertebrates share similar transduction pathways: G protein-coupled receptors signal through phospholipase C (PLC) to open a member of the TRP family of ion channels, TRPC2 for the vomeronasal organ (VNO) and TRPM5 for taste. The importance of these ion channels for chemosensation is highlighted by the lack of response to pheromones in TRPC2 knockout mice and to bitter, sweet and amino acid tastes in TRPM5 knockout animals. Understanding the mechanisms by which TRP ion channels are gated, has proven difficult in any system, and remains an essential goal for understanding taste and VNO transduction. In the last grant period, we discovered that TRPM5 is activated by intracellular Ca +, suggesting that Ca + may be the second messenger for some forms of taste transduction. In the next grant period we aim to understand the molecular mechanisms that underlie regulation of TRPM5, and how molecular properties of TRPM5 influence taste sensation. We will specifically address the following questions: (1) What are the mechanisms by which TRPM5 channels are activation and inactivated? In these studies we will use patch-clamp recording of expressed TRPM5, together with pharmacological and structural manipulations of the channel. (2) Do blockers of TRPM5 interfere with taste detection? Blockers will be identified by patch-clamp recording from cells expressing TRPM5 and the effects of identified blockers on taste thresholds in mice will be determined (3) Do similar channels to TRPM5 play a role in VNO transduction? TRPM5 and related channels are widely expresed in the body, and mutations in these channels may underlie certain pathological states. By understanding the regulation of these channels, we can understand their contribution to signaling in health and disease.

DESCRIPTION (provided by applicant): Studies conducted under this grant are directed at understanding the second messenger-gated ion channels that mediate the transduction of bitter, sweet and umami taste. These tastes are detected by G-protein-coupled taste receptors that initiate a signaling cascade for which the enzyme PLC22 and the ion channel TRPM5 are essential elements. It is hypothesized that TRPM5 is activated downstream of hydrolysis of PI(4,5)P2 to IP3 and DAG, by the release of Ca2+ from intracellular stores. Our recent results show that the TRPM5-dependent current in mouse taste receptors cells is activated by, and desensitizes in response to, intracellular Ca2+. These results raise the following questions which will be addressed in this grant application (1) Is Ca2+ the second messenger for taste transduction, coupling receptor signaling to an electrical response and transmitter release? (2) Is there a functional association of TRPM5 with elements of the Ca2+ signaling pathway? and (3) What are the biochemical or structural events that underlie desensitization of TRPM5? Together these experiments will allow us to develop a model for the contribution of TRPM5 channels to taste signaling. Taste plays an important role in determining the acceptance of foods and drugs and the ability to selectively modulate taste can therefore lead to significant health benefits. PUBLIC HEALTH RELEVANCE Taste plays an important role in determining the acceptance of foods and drugs. By understanding the signaling pathways that underlie taste sensation, what the key molecular events are, how they are organized in space, and how they change over time, we can better develop ways to modify taste to increase or decrease sensation. Moreover, information about the signaling pathways in taste cells can form the basis for understanding how taste sensation changes with development and aging or in response to disease.

DESCRIPTION (provided by applicant): Most vertebrate species are responsive to five basic tastes: sweet, bitter, umami, sour and salty, each of which provides unique information on the nutritional content and safety of ingested food. Each of the five taste qualities is detected by a distinct subset of taste cells, which express distinct receptors and signaling molecules. While great progress has been made in understanding the molecules and pathways that mediate bitter, sweet, umami and salty tastes, relatively little is known about sour taste. The cells that detect sour taste have been shown to be defined by expression of the TRP ion channel PKD2L1, but the Pkd2l1 gene itself is dispensable for sour taste and the ionic mechanisms that underlie sensory responses to sour remain poorly understood. In recent work, using a mouse in which yellow fluorescent protein (YFP) was driven by the promoter of Pkd2l1, we showed that sour taste cells express a novel proton conductance which contributes to the response to acids. The specific goal of the current proposal is to identify the gene that encodes this proton conductance. To do this, we propose two specific aims. In aim 1, we will use deep sequencing to perform transcription profiling of YFP-tagged sour cells and we will identify transcripts that ae enriched in Pkd2l1 cells and are likely to encode novel transmembrane proteins. Under Aim 2, we will express the top candidates in a heterologous cell type and determine with patch clamp recording whether they generate a proton channel. The identification of this proton channel will represent an important step in understanding the taste system, and may help define a new class of ion channels. Taste is an essential way in which humans and other organisms regulate their ingestive behavior and the identification of key molecular components of taste signaling can therefore have a direct impact on human health and well-being. PUBLIC HEALTH RELEVANCE: The proposed experiments use next generation sequencing of mRNA transcripts from genetically labeled taste cells to identify a novel ion channel that mediates sour taste transduction. Taste is an essential way in which humans and other organisms regulate their ingestive behavior and the identification of key molecular components of taste signaling can therefore have a direct impact on human health and well-being.

Project Summary The broad goal of the proposed experiments is to identify key molecules that allow mammals to detect basic tastes and generate electrical responses that are conducted to brain regions. Molecular mechanisms of taste reception have been a subject of intense investigation over the last 30 years, with great strides made in identifying receptors for bitter, sweet and umami. Much less is known about the nature and function of receptors for sour, the taste that allows us to detect acids in spoiled foods or citrus fruits. In this proposal, we will begin to unravel this problem as we test the contribution of the newly discovered otopetrin proton channels in the transduction of acidic and ionic tastes. These experiments build on the progress made in the last grant application, where we used a combination of cellular, molecular and functional approaches to identify the pH sensitive ion channels in Type III taste receptor cells (TRCs) that mediate sour taste. Notably, we described a novel proton-selective ionic current that is likely to be a key component of sour taste transduction. In the last funding period, we successfully identified the gene that encodes the proton channel, through functional screening of genes enriched in Type III TRCs. Among 41 genes tested, we identified one, encoding the transmembrane protein Otop1 that upon expression induced a proton current in both Xenopus oocytes and HEK-293 cells. Interestingly, Otop1 was first identified as a gene mutated in mice with vestibular defects (?tilted? or tlt) but its function in the vestibular system and elsewhere in the body was not understood. Building on these new results, we propose three specific aims to test the role of the Otop channels in taste signaling. The first aim will examine the functional distribution of Otop1 across the tongue and palate epithelium, allowing us to answer the question of whether Otop1 is the sole ion channel mediating proton influx in the gustatory system. In the second aim, we will measure cellular responses to acids in wildtype and Otop1 KO mice in order to determine the degree to which Otop1 contributes to sensory responses, ex vivo. In the third aim, we will measure responses from gustatory nerves and assess behavioral thresholds for acid detection in wildtype and Otop1 KO mice to determine the extent to which Otop1 mediates responses to sour taste stimuli in vivo. Together our experiments will allow us to determine if Otop1 functions as a sour taste receptor. Our efforts to identify mechanisms of taste transduction may allow the development of taste modifiers that can be used to enhance palatability of food, reducing the need to add sweeteners that contribute to the development of diabetes or salts that contribute to hypertension. Moreover, the proposed experiments will provide basic information regarding the functional properties of this new family of proton channels that will help us understand their contributions to diverse physiological processes, including brown fat metabolism and the development and maintenance of the vestibular system.

DESCRIPTION (provided by applicant): This proposal is aimed at generating reagents with which to visualize the distribution of the transient receptor potential ion channel (TRPA1) protein in living animals. TRPA1 is expressed in sensory neurons of the trigeminal and dorsal root ganglia where it serves as the main sensor for environmental irritants or chemicals that cause tissue damage. It also plays a critical role in the development of hypersensitivity under inflammatory pain conditions, and is therefore considered an important target for the development of pain therapeutics. However, a major obstacle in further establishing the contribution of TRPA1 to cellular physiology, and in understanding how the channel is regulated under normal and inflammatory conditions, has been the lack of reagents with which to visualize the channel protein, either in fixed or in living tissue. Although several groups have reported the production of antibodies against TRPA1, these have generally exhibited low specificity or sensitivity. Moreover, antibodies can only be used in fixed tissues or in cell culture, precluding the study of the protein in intact living tissues. To address this shortcoming, we propose to generate novel genetic probes to visualize TRPA1 channels in fixed tissue and living cells. These probes, termed FingRs (fibronectin intrabodies generated with mRNA display), will be identified through a selection for proteins that bind TRPA1 from a library built on the highly stable fibronectin backbone. We anticipate that a TRPA1 FingR will be an extremely powerful tool, which could be used to examine the distribution of TRPA1 throughout the body, in a variety of cell types, in healthy and disease conditions. Moreover, the genetic nature of the material, wil allow it to be easily propagated and introduced into living cells.

DESCRIPTION (provided by applicant): Our senses provide us with the information that we need to perform all the essential functions of life: navigate the world, avoid danger, find food, choose mates and nurture our offspring. Each sensory modality is tuned to a specific set of stimuli; this tuning sets the limits of our sensory experience. Thus understanding how sensory cells detect and transduce sensory stimuli is critical to understanding how the nervous system responds to the environment to generate appropriate behaviors and perceptions. Moreover, disruption of sensory signaling in diseases ranging from blindness to deafness to insensitivity to pain can be devastating for humans. Despite ample evidence of shared molecular components and shared principles for transforming sensory input to neural signals across the senses, researchers investigating distinct senses rarely have opportunities to interact directly. With new technologies emerging at an ever- increasing rate, the timely sharing of information is needed to greatly accelerate research in sensory biology. The goal of this conference proposal is to assemble leading researchers across all sensory modalities (vision, smell, taste, touch, temperature, pain) and create an event designed to spark new collaborations among established and emerging investigators as well as graduate students and postdoctoral fellows. The conference, entitled Sensory Transduction, will be the 68th Annual Symposium of the Society of General Physiologists (SGP), a conference that has long been recognized as a pioneering and high- impact meeting for physiologists, cell biologists, and biophysicists. The venue, the campus of the Marine Biological Laboratory in Woods Hole, MA, is a magical place that affords participants with an intimate environment and ample opportunities to share scientific insight in both formal and informal settings.

? DESCRIPTION (provided by applicant): This proposal is for partial support for an international meeting on Ion Channels, as part of the Gordon Research Conference series, to be held at Mount Holyoke College in South Hadley, Massachusetts, July 10 - 15, 2016. The long-term goal of this conference is to increase understanding of the fundamental structure, function and physiological roles of ion channels and how these functions are disrupted in human disease. The specific aim of this meeting will be to convene 40 speakers and discussion leaders who represent critical areas of ion channel research together with 160 participants-at-large for a maximum of 200 participants for a five-day meeting in a relatively secluded setting. The program will consist of nine sessions in which invited speakers and speakers chosen from the participants-at-large will present their latest, unpublished, research, with ample time for discussion of the results. In addition, four poster sessions will permit all participants to presen their own work. The significance of this meeting is that is has been for many years, and remains, the premier meeting for scientists interested in fundamental mechanisms of ion channel structure and function. The small size and intensive discussions engendered by the Ion Channels GRC make it uniquely important for the catalysis of new ideas, directions and collaboration among the participants. Ion channels play a critical role in establishing the electrical excitability of membranes and their dysfunction underlies a large variety of disorders, from congenital hearing loss, to epilepsy and heart disease. Thus information disseminated through this meeting has the potential to be of wide relevance to all areas of human health.

PROJECT SUMMARY/ABSTRACT The proposed experiments are aimed at elucidating the structural and functional properties of a newly discovered family of ion channels, the Otopetrins (Otops). The Otops are just the second class, after Hv1, of proton-selective ion channels described in eukaryotic cells. Otop1 was first discovered in the vestibular system where it is required for the development of calcium carbonate-based otoconia that allow hair cells to detect changes in gravitational forces and acceleration. It was subsequently identified in an unbiased screen for proton channels based on functional analysis of transcripts enriched in taste cells that detect sour. When expressed in Xenopus oocytes and HEK- 293 cells, Otop1 generates a proton-selective ion current that is blocked by extracellular Zn[2+]. The mammalian genome encodes two homologs of Otop1 (Otop2 and Otop3), which also form proton permeable ion channels with distinct functional properties. Here we propose a series of experiments to define mechanisms of gating and permeation of Otop channels, and to identify the underlying structural elements. The CryoEm structure of Otop1 and Otop3 have led us to identify several possible proton permeation pathways; the function of specific residues in permeation or Zn[2+] inhibition will be interrogated using site directed mutagenesis and patch clamp electrophysiology. Other experiments will address the outstanding question of whether Otop channels are gated and identify structural determinants for gating. Members of the otopetrin gene family are widely expressed throughout the body, including in the taste, vestibular and immune systems, in the gastrointestinal tract, and in brown adipose tissue. By understanding the structural and functional properties of each of the Otop isoforms, we will be better able to understand how they contributes to cellular physiology. Moreover, ion channels are the preferred targets for pharmaceuticals and mutations that disrupt their function underlie a growing number of inherited or acquired disorders (channelopathies).