Douglas A. Bayliss - US grants

The overall goal of the proposed work is to elucidate mechanisms underlying neurotransmission among respiratory-related brainstem neurons during postnatal development. A structure/function approach is taken in which neuroanatomical techniques are used to define neurotransmitter and receptor phenotype of rat brainstem neurons and in vitro electrophysiological approaches are used to determine cellular mechanisms that mediate effects of putative transmitters; developmental changes in neurotransmitter and receptor expression are correlated with concomitant changes in electrophysiological effects of relevant transmitters. This proposal focuses on the relationship between caudal raphe neurons and the hypoglossal motoneurons (HMs) they innervate, which may have important clinical relevance. The activity of HMs follows closely that of raphe cells, being lowest during sleep. Because HMs regulate upper airway patency and decreased activity of HMs during sleep can lead to airway obstruction, clarifying transmitter mechanisms within the raphe- hypoglossal system may be important in understanding certain respiratory pathologies of sleep (e.g., obstructive sleep apnea, Sudden Infant Death Syndrome). To this end, the effects on HMs of three transmitters synthesized by raphe cells [i.e., serotonin (5-HT), thyrotropin-releasing hormone (TRH), and substance P (SP)] are determined during postnatal development and correlated with levels of receptor and transmitter expression. Mechanisms underlying autoinhibition of raphe cells by 5-HT are also studied. The first specific aim is to test the hypothesis that differential effects of 5-HT in neonatal and adult HMs reflect differences in receptor expression. This is done pharmacologically using intracellular recording techniques in a thick-slice preparation; those results are correlated with postnatal changes in expression of 5-HT receptors by HMs determined anatomically by in situ hybridization and receptor autoradiography. The same approaches are used in the second specific aim to determine the pharmacological and ionic bases for effects of SP on HMs and to test the hypothesis that developmental changes in SP expression by raphe neurons are matched by changes in SP receptor expression in HMs. The third specific aim is to determine neuroanatomically if raphe neurons influence developmental patterns of receptor (5-HT, TRH and SP) expression in HMs using a specific neurotoxin (5,7-dihydroxytryptamine) to lesion serotonergic raphe neurons. The fourth specific aim is to test the hypothesis that TRH inhibits resting K+ channels in HMs via a soluble second messenger using single channel recording techniques in a medullary thin-slice preparation. The fifth specific aim is to test the hypothesis that 5-HT modulates Ca2+ and inwardly-rectifying K+ currents in caudal raphe neurons as a mechanism of autoinhibition; for these experiments whole-cell recordings are made from directly visualized ra he neurons in the thin-slice preparation.

Membrane depolarization resulting from inhibition of background K+ channels is a widespread but poorly understood modulatory mechanism that increases activity of excitable cells. Metabotropic receptors that are capable of mediating such a slow depolarization have been identified for most major transmitters. This represents perhaps the predominant mechanism for slow synaptic excitation in the brain and may contribute to positive chronotropic effects in the heart. The molecular identity of the K+ channel(s) targeted for inhibition is unknown in most systems, but in a number of cases Kir3.x (GIRK) family channels are implicated. This is particularly interesting since activation of the same GIRK channels by receptors utilizing PTx-sensitive pathways can mediate a slow membrane hyperpolarization. This dual modulation of GIRKs provides a mechanism for dynamic control of membrane potential in the same cell by different classes of receptor targeting the same ion channel. The cellular and molecular mechanisms of GIRK activation are well-described. By contrast, there is little information regarding mechanisms that underlie receptor-mediated inhibition of GIRK channels. We have developed a model system, based on heterologous expression in mammalian cells of the thyrotropin-releasing hormone (TRH) receptor with GIRK-1 and -4, that recapitulates features of the dual modulatory mechanism. We use this system and the tools of molecular biology to manipulate individual components of the pathway in order to test the hypothesis that specific G protein subunits, distinct from those involved in GIRK activation, are critical to support receptor-mediated GIRK inhibition. The specific aims are: [1] Determine which G protein subunits are capable of causing inhibition of GIRKs. Specific Galpha subunits or Gbetagamma dimers are tested to determine if they: a) inhibit GIRK currents in whole cell assays; b) bind directly to purified GIRK protein domains; and c) inhibit GIRK channels in excised patches. [2] Determine which domains of Gbeta are important for GIRK inhibition and activation. Chimeric and site-directed mutants of Gbeta1 and Gbeta5 are used to identify regions that support GIRK channel modulation. [3] Determine G protein subunits that mediate GIRK inhibition by TRH receptors. Signaling by Galpha and/or Gbetagamma subunits is disrupted to determine which subunit supports receptor-mediated GIRK inhibition. [4] Determine second messenger pathway(s) that mediate GIRK inhibition by TRH receptors. Pharmacological tools are used to perturb second messenger pathways hypothesized to mediate GIRK channel inhibition by TRH receptors. These experiments will provide important information regarding not only mechanisms of K+ channel modulation that underlie slow synaptic excitation, but also regarding determinants of G protein-effector signaling specificity.

DESCRIPTION: Multiple ion channels influence neuronal excitability, and these are often subject to modulation by neurotransmitters. Prominent among these is a background or 'leak' K+ channel that is targeted for inhibition by neurotransmitters, leading to membrane depolarization and increased excitability. G protein-coupled receptors capable of mediating this effect have been identified for many transmitters (invariably those that couple via Gaq/l 1-family subunits), and whereas it represents a predominant mechanism for slow synaptic excitation throughout the brain, this phenomenon is particularly well described in motoneurons. Despite its widespread presence, the molecular identity of leak K+ channel(s) targeted for inhibition are unknown in most native systems, and the mechanisms of receptor-mediated channel inhibition remain obscure. A major goal of the current proposal is to identify the molecular substrate for a motoneuronal leak K about current. Evidence from our laboratory indicates that the two-pore domain K+ channel, TASK-1 (KCNK3), contributes to a pH- and neurotransmitter-sensitive leak K+ channel in hypoglossal motoneurons. New observations indicate that the closely related TASK-3 (KCNK9) subunit is also expressed in motoneurons. Moreover, preliminary data suggest that it may form heterodimers with TASK-1. We hypothesize that TASK-1 and TASK-3 form functional heterodimers that contribute to motoneuronal pH- and neurotransmitter-sensitive leak K+ currents. The second major goal is to characterize molecular mechanisms involved in receptor-mediated inhibition of these channels, focusing in turn on the molecules that represent the beginning (i.e., G proteins) and end points (TASK channels) of the receptor-activated signaling pathway. We hypothesize that Gag-family subunits provide the initial receptor-activated signal and that key determinants located in cytoplasmic domains of TASK channels are required for receptor-mediated TASK channel inhibition. For these studies, we utilize two experimental systems: a model system, based on heterologous expression of Gaq-coupled receptors and TASK channel subunits in mammalian cells, which recapitulates this modulatory mechanism; and a native neuronal system, in which heterologous gene expression is obtained in motoneurons using adenovirus vectors. The following Specific Aims are proposed: To determine if TASK channels can form functional heterodimers; To determine G protein subunits and channel domains involved in receptor-mediated TASK inhibition; and To determine contributions of TASK channels to motoneuronal currents and mechanisms of their modulation. These experiments will characterize molecular substrates underlying a native neurotransmitter-modulated leak K+ current and test key aspects of the mechanisms by which they are modulated.

Inhalation anesthetics are valuable agents in widespread clinical use. However, the cellular and molecular mechanisms by which these compounds elicit clinically important actions, such as loss of consciousness and immobility, are still incompletely understood. Accumulating evidence implicates neuronal membrane ion channels as direct targets for anesthetic effects, with much emphasis historically on GABAA and glycine receptors. Our laboratory has recently demonstrated that anesthetics decrease excitability in somatic motoneurons via modulation of two distinct ion channels: activation of background or 'leak' K+ channels and inhibition of hyperpolarization-activated cationic channels (Ih). This channel modulation occurs at clinically relevant concentrations and the motoneuronal inhibition that results could account, at least in part, for the immobilizing effects of anesthetics. The relatively recent cloning of KCNK and HCN channel families, the substrates for neuronal leak K+ and Ih, channels, provides an opportunity to determine molecular mechanisms underlying anesthetic effects on these channels. Our published and preliminary data indicate that the anesthetic- activated K+ current in motoneurons involves the pH- and neurotransmitter-sensitive TASK-1 (KCNK3) and TASK-3 (KCNK9) channel subunits, either in homo- or heteromeric configurations; anesthetic effects on these leak K+ channels appear to be modulated by neurotransmitter action. Likewise, the cyclic- nucleotide-gated HCN1 and HCN2 subunits are co-expressed in motoneurons, where they also may associate into homo- or heteromeric channels; our preliminary data indicate that volatile anesthetics affect homomeric HCN subunits differentially and that cAMP modulates effects of anesthetic. We hypothesize that the effects of volatile anesthetics on neuronal leak K+ currents and Ih, and their modulation by neurotransmitters, are fully recapitulated in cloned TASK and HCN channels, and that these channels include determinants critical for anesthetic effects within their primary structure. The Specific Aims are: [1] Elucidate molecular mechanisms underlying volatile anesthetic effects on 'leak' K+ (TASK) channels; [2] Elucidate molecular mechanisms underlying volatile anesthetic effects on hyperpolarization-activated cationic (HCN) channels. For these studies, we record cloned TASK and HCN channel currents in a mammalian heterologous expression system and native leak K+ currents and Ih in motoneurons. We characterize anesthetic effects and their modulation by neurotransmitters (or cAMP) on cloned and native channels, and use site-directed mutagenesis in order to identify channel domains that are necessary for these actions. These experiments will determine molecular mechanisms by which volatile anesthetics modulate TASK and HCN channels native to motoneurons, with implications for their immobilizing actions. Widespread expression of these channels in the CNS suggests that their modulation by anesthetics in other brain regions may contribute to additional anesthetic actions.

[unreadable] DESCRIPTION (provided by applicant): The discovery of a means to anesthetize patients for surgical procedures, some 150 years ago, represented a major advance in clinical medicine. Since that time, despite numerous important developments in the anesthetic pharmacopoeia, the mechanisms by which most general anesthetic drugs mediate clinically important actions have remained enigmatic. In this regard, cellular and molecular studies have led to a prevailing current view that membrane ion channels represent the most relevant anesthetic targets, and recent behavioral assays from genetic mouse models implicate GABAA receptor channels as preeminent for some, though not all, actions of intravenous anesthetics. The GABAA receptors may not be as critical for actions of inhalational anesthetics, for which other ion channel targets are sought. In this application, we propose molecular, cellular and behavioral experiments with mouse knockout models to examine a role for two types of alternative anesthetic-sensitive ion channels - TASK background potassium channels and HCN pacemaker cation channels - in immobilizing and hypnotic anesthetic actions. Our working model is that anesthetic modulation of either or both these channels contributes: to decreased excitability in motoneurons that is associated with immobilization; to induction of sleep-like hypnotic states through actions in thalamocortical.circuits; and to inhibition of brainstem aminergic neurons that is associated with both atonia and sleep. In Specific Aim 1, we use conventional and conditional mouse knockouts of TASK-1 and TASK-3 that are developed in our laboratory; and in Specific Aim 2, we use previously described conventional HCN1 and HCN2 knockout mice. For both aims, the knockout mouse models are first validated by molecular, immunochemical and behavioral approaches. We use patch clamp recordings from brain slices to determine effects of channel knockout on electrophysiological properties of the key neural elements identified in our working model (i.e., motoneurons, thalamocortical relay neurons, cortical pyramidal neurons, and brainstem aminergic neurons). Finally, we test if knockout animals have altered sensitivity to actions of inhaled and intravenous anesthetic agents using established behavioral assays of immobilization and hypnosis. The proposed studies provide a critical test of TASK and HCN channel subunit contributions to cellular actions of anesthetics, and of their role in behaviorally relevant actions of these clinically important drugs. Identification of molecular and neural substrates for anesthesia may lead to discovery of safer, more effective anesthetic compounds, a fundamental goal of anesthesia research. In addition, the studies may provide new insights into neural mechanisms of arousal. [unreadable] [unreadable] [unreadable]

Multiple ion channels influence neuronal excitability, and these are often subject to modulation by neurotransmitters. Prominent among these are background K+ channels that are targeted for inhibition by neurotransmitters, leading to membrane depolarization and increased excitability. G protein-coupled receptors capable of mediating this effect have been identified for many transmitters (invariably those that couple via Gaq/n-family subunits), and it represents a predominant mechanism for slow synaptic excitation throughout the brain. The molecular identity of background K+ channels targeted for inhibition are unknown in most native systems, and the mechanisms of receptor-mediated channel inhibition remain obscure. Proposed research explores novel mechanisms and molecular substrates underlying Gaq-linked inhibition of background K+ channels. Our studies of cloned two-pore-domain background K+(K2P)channels - TASK-i (K2Ps) andTASK-3 (K2Pg) - has revealed a novel mechanismfor Gaq-mediated ion channel modulation. We find that TASK channel inhibition is independent of phospholipase C (PLC) activation and PI(4,5)P2 depletion, but instead requires Gaq interaction with the channels or with a closely-associated intermediary. Wepropose studies designed to identify molecular determinants that accountfor Gaq association and TASK channel inhibition, and to examine if this PLC- independent mechanism contributes to inhibition of other types of background K+ channels and their neuronal correlates by Gaq. Our published and preliminary work has identified TASK channels as substrates for background K+ currents in cholinergic neurons, specifically motoneurons and striatal interneurons, based on a constellation ofvoltage-dependent and pharmacological properties. This tentative identification requires verification. We propose to use newly available knockout mice to test definitively the TASKsubunit contributions to these native neuronal backgroundK+currents. Interesting preliminary data indicates that TASK currents are not targets for Gaq-mediated inhibition in striatal cholinergic interneurons. Rather, a novel Ch-activated background K+ channel is inhibited by Gaq-linked metabotropic receptors (mGluRs). Wepropose experiments to determine if the recently identified Slo2 channels account for this mGluR-sensitive channel, and to identify the relevant Gaq-mediated inhibitory mechanism. The following Specific Aims are proposed: [i] Establish mechanisms underlying PLC-independent modulation ofTASK and GIRK channels by Gaq subunits;[2] Identify background K+ channels in striatal cholinergic interneurons and elucidate mechanisms that contribute to Gaq-mediated activation. These experiments will characterize molecular substrates underlying native neuronal neurotransmitter-modulated background K+ currents and examine molecular mechanisms by which they are modulated.

DESCRIPTION (provided by applicant): Anesthesiologists routinely select from a host of chemically diverse compounds to render patients unconscious and insentient, allowing painless performance of surgical procedures. Remarkably, however, the molecular and neuronal mechanisms by which most anesthetic drugs mediate their clinically important actions still remain uncertain. The development of new mouse genetic models in which candidate anesthetic targets can be disabled, either globally or in specific cell types, is beginning to reveal relative contributions of different molecular targets and cell groups to particular anesthetic actions. In this application, we employ such models to examine contributions of two subthreshold anesthetic-sensitive ion channels - HCN hyperpolarization- activated cation channels and TASK background potassium channels - in mediating clinically important anesthetic actions. The hypothesis guiding Specific Aim 1, which builds on recently published results from our laboratory, is that selective inhibition of dendritic HCN1 channels in cortical pyramidal neurons contributes to anesthetic-induced hypnosis. Proposed experiments focus primarily on HCN1-mediated actions of ketamine, a dissociative anesthetic for which a robust decrease in hypnotic sensitivity was obtained in conventional HCN1 knockout mice. Our aims are to: examine the role of pyramidal neurons in hypnotic anesthetic actions by using a forebrain-selective HCN1 knockout model;characterize effects of other dissociative anesthetics on HCN channels and anesthetic-induced hypnosis;identify molecular determinants for subunit-selective effects of ketamine on HCN1 channels;and develop/test a knock-in mouse model in which HCN1 channels are intact, but rendered insensitive to ketamine. The working model underlying Specific Aim 2, which derives from new preliminary data, is that inhibition of TASK channels by local anesthetics contributes to their deleterious effects. Our aims are to: evaluate pro-convulsive CNS effects of systemically administered local anesthetics in conventional TASK-1-/-:TASK-3-/- knockout mice;characterize relative sensitivity of TASK channels to different local anesthetics that vary in systemic toxicity;determine TASK channel contributions to local anesthetic action on excitatory thalamocortical circuit neurons;and examine specifically the role of those neurons in TASK channel-mediated CNS effects of local anesthetics by using cell- specific conditional TASK knockout mice. For both aims, we use a variety of approaches, including channel mutagenesis and patch clamp recordings from transfected cells;molecular and immunochemical validation of novel conditional mouse models;somatic and dendritic recordings from key neurons in wild type and mutant mice;and behavioral assessments of anesthetic sensitivity in those mice. The proposed studies provide a test of HCN and TASK channel subunit contributions to specific neuronal and behavioral actions of general and local anesthetics. Identification of these molecular and neural mechanisms may lead to discovery of safer, more effective anesthetic compounds, an important goal of anesthesia research. PUBLIC HEALTH RELEVANCE: General and local anesthetics remain among the most widely used and clinically useful drugs. Despite prevalent use and extensive clinical experience with these compounds, the molecular and neural mechanisms by which they mediate their desirable and untoward actions remain uncertain. The research undertaken in this proposal seeks to clarify those mechanisms, and thus to provide insights that may lead to development of more effective and safer anesthetic agents.

A group of excitatory neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b, integrate sensory inputs and information regarding brain state for transmission on to respiratory rhythm/pattern-generating circuits. In addition, RTN neurons adjust their firing in response to changes in CO2 (or H+) and adjust breathing to maintain physiologically appropriate levels of pH and PCO2, a homeostatic process called central respiratory chemoreception. Dysfunction of central chemoreception is implicated in various central disorders of breathing that often occur during sleep (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). In the last project period, we showed that Phox2b-expressing RTN neurons are intrinsically chemosensitive, and identified two independent molecular proton sensors in RTN neurons - TASK-2, a proton-inhibited background K+ channel and GPR4, a proton-activated G protein-coupled receptor - that are required for stimulation of breathing by CO2. Important questions remain, however, regarding: the ionic basis for baseline firing properties and modulation by arousal state-dependent factors; the effector systems engaged downstream of GPR4 in RTN neurons; and mechanisms by proposed astrocytic modulation can be integrated with the requirement for GPR4 and TASK-2 in RTN-mediated respiratory chemosensitivity. This proposal addresses these issues using: novel conditional knockout mouse lines; viral-mediated shRNA knockdown and/or rescue; single cell electrophysiology and molecular biology; and whole animal assays of respiratory function and vigilance states. The hypothesis underpinning Specific Aim 1 is that TTX- resistant subthreshold Na+ channels, NALCN and NaV1.9, contribute to baseline excitability of RTN neurons and mediate facilitatory effects of neuropeptides associated with arousal state-dependent brain nuclei. We disrupt expression of these channels in RTN neurons and determine effects on subthreshold Na+ currents, basal and neuropeptide-modulated firing in vitro, and arousal state-dependent respiratory CO2 sensitivity in vivo. The hypothesis driving Specific Aim 2 is that GPR4 engages a cAMP-transduction pathway and background K+ channel (independent of TASK-2) for cellular pH sensing in RTN neurons, and that astrocytic amplification of respiratory chemoreflexes involves boosting local pH changes around RTN neurons. We use pharmacological and transcriptomic approaches in single RTN neurons to characterize the GPR4 signaling pathway and effector channel, and we disrupt a pH-modulating Na+ -HCO3 transporter, NBCe1, in medullary astrocytes to determine effects on the respiratory chemoreflex in vivo. This latter may support a convergent theory for astrocyte-neuron contributions for this highly sensitive chemoreflex, bridging a major current divide in the field. Collectively, the proposed studies provide critical information regarding molecular and cellular mechanisms that control activity of RTN neurons, and regulate this important homeostatic respiratory system. Identification of novel molecular mechanisms may provide new therapeutic targets for disorders of breathing.

DESCRIPTION (provided by applicant): Anesthesiologists routinely select from a host of chemically diverse compounds to render patients unconscious and insentient, allowing painless performance of surgical procedures. Remarkably, however, the molecular and neuronal mechanisms by which most anesthetic drugs mediate their clinically important actions still remain uncertain. The development of new mouse genetic models in which candidate anesthetic targets can be disabled, either globally or in specific cell types, is beginning to reveal relative contributions of different molecular targets and cell groups to particular anesthetic actions. In this application, we employ such models to examine contributions of two subthreshold anesthetic-sensitive ion channels - HCN hyperpolarization- activated cation channels and TASK background potassium channels - in mediating clinically important anesthetic actions. The hypothesis guiding Specific Aim 1, which builds on recently published results from our laboratory, is that selective inhibition of dendritic HCN1 channels in cortical pyramidal neurons contributes to anesthetic-induced hypnosis. Proposed experiments focus primarily on HCN1-mediated actions of ketamine, a dissociative anesthetic for which a robust decrease in hypnotic sensitivity was obtained in conventional HCN1 knockout mice. Our aims are to: examine the role of pyramidal neurons in hypnotic anesthetic actions by using a forebrain-selective HCN1 knockout model; characterize effects of other dissociative anesthetics on HCN channels and anesthetic-induced hypnosis; identify molecular determinants for subunit-selective effects of ketamine on HCN1 channels; and develop/test a knock-in mouse model in which HCN1 channels are intact, but rendered insensitive to ketamine. The working model underlying Specific Aim 2, which derives from new preliminary data, is that inhibition of TASK channels by local anesthetics contributes to their deleterious effects. Our aims are to: evaluate pro-convulsive CNS effects of systemically administered local anesthetics in conventional TASK-1-/-:TASK-3-/- knockout mice; characterize relative sensitivity of TASK channels to different local anesthetics that vary in systemic toxicity; determine TASK channel contributions to local anesthetic action on excitatory thalamocortical circuit neurons; and examine specifically the role of those neurons in TASK channel-mediated CNS effects of local anesthetics by using cell- specific conditional TASK knockout mice. For both aims, we use a variety of approaches, including channel mutagenesis and patch clamp recordings from transfected cells; molecular and immunochemical validation of novel conditional mouse models; somatic and dendritic recordings from key neurons in wild type and mutant mice; and behavioral assessments of anesthetic sensitivity in those mice. The proposed studies provide a test of HCN and TASK channel subunit contributions to specific neuronal and behavioral actions of general and local anesthetics. Identification of these molecular and neural mechanisms may lead to discovery of safer, more effective anesthetic compounds, an important goal of anesthesia research. PUBLIC HEALTH RELEVANCE: General and local anesthetics remain among the most widely used and clinically useful drugs. Despite prevalent use and extensive clinical experience with these compounds, the molecular and neural mechanisms by which they mediate their desirable and untoward actions remain uncertain. The research undertaken in this proposal seeks to clarify those mechanisms, and thus to provide insights that may lead to development of more effective and safer anesthetic agents.

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. In nearly all tissues, there is a continual turnover of cells, usually by the process of apoptosis. In healthy tissues, the dying cells are quickly recognized and cleared by phagocytes. However, failure to promptly clear apoptotic cells leads to their secondary necrosis, release of toxic cytoplasmic contents, and inflammation within tissues. Current evidence suggests that the apoptotic cells `advertise' their presence early on in the death process, via soluble factors termed `find-me signals' to attract phagocytes, and thereby promote their prompt clearance. Initial studies from the Principal Investigators of this proposal have identified the nucleotides ATP and UTP as one type of `find-me signal' that is critical for apoptotic cell clearance in vitro and in vivo; subsequent studies led to a key discovery that the membrane protein pannexin 1 (Panx1) is the channel mediating nucleotide release from apoptotic cells. The overall hypothesis tested in this proposal is that pannexin channel-dependent release of nucleotide find-me signal from apoptotic cells, and subsequent sensing by phagocytes is important for proper cell clearance in vivo, and that disruption of the Panx1-mediated find-me signal pathway would contribute to diseases. Based on our preliminary studies, in Aim 1, we study the molecular basis of a novel Panx1 activation mechanism, seeking new understanding that will allow manipulation of `find- me` signal release via these channels. In Aim 2, we use conditional cell-specific Panx1 knockout mice and conditional transgenic mice to determine if manipulation of Panx1 channels affects cell clearance in vivo. For this, we take advantage of a model where evidence suggests cell clearance is important in normal tissue homeostasis and in disease ? i.e., thymic development. Collectively, we expect these studies to yield new mechanistic understanding on how the regulated release of find-me signals influence cell clearance, and better define this mode of inter-cellular communication between dying cells and phagocytes, with implications for autoimmunity. These studies can also provide a rationale for considering Panx1 channels as a suitable target for therapeutic development.

Overall Program Project - Project Summary Inter-cellular communication between cells within a tissue environment is fundamentally important for many physiological processes. Channels and transmembrane transporters that conduct ions and other molecules across the plasma membrane in healthy living cells are also linked to pathologies of the cardiovascular and respiratory systems. Extracellular nucleltides (such as ATP) and their derivatires critically influence many aspects of vascular physiology such as vasoconstriction and blood pressure regulation, as well disease states such as metabolic syndromes. Recent exciting series of observations suggest that the pannexin proteins form channels on the plasma membrane, and by permeating ions and/or the release of nucleotides in a very regulated manner, these pannexin channels allow cells to communicate with other cells. Consistent with this, altered expression of pannexin channels have been linked to cardovascular and metabolic disorders. On an independent and inter-related set of observations, the pannexin channels also play a role in releasing nucleotides from early stage apoptotic cells that appear critical for communicating with phagocytes and in turn promoting prompt corpse removal. Since, failed clearance of dying cells is linked to atherosclerosis and airway inflammation, pannexin channels likely also play a role in regulating inflammation within tissues. The central hypothesis tested via this P01 application is that pannexin channels sit at a critical interphase between normal homeostasis within the cardiovascular system, and the disease states leading inflammation, atherosclerosis, and hypertension. The four projects that comprise this proposal address the role of pannexin channels as follows. Project 1 (Ravichandran) addresses the role of pannexin channels in cell death and recruitment of monocytes during atherosclerosis, cholesterol efflux, and in tissue inflammation; Project 2 (Isakson) addresses how pannexin channels in smooth muscle cells contribute to vasoconstriction in resistance vessels to regulate blood pressure and how this is altered in obesity; Project 3 (Leitinger) addresses how pannexin channels regulate adipocyte functions and the inflammation induced by dying adipocytes in obesity, insulin resistance and hypertension; Project 4 (Bayliss) addresses molecular mechanisms of pannexin channel activation in physiological and diseased states. With the combination of mouse models and ex vivo studies, and mechanistic approaches, and the preliminary identification of new compounds capable of altering Panx1 function, we expect to provide exciting new insights on pannexin channels and purinergic signaling in vascular physiology and hypertension, and provide the basis for novel treatment strategies targeting the regulated opening and closing of these channels in specific disease states. We expect this would have a broad impact to cardiovascular, metabolic, and respiratory diseases.

7. Project Summary / Abstract Low-renin hypertension (LREH) and idiopathic primary hyperaldosteronism (IHA) occur commonly, and predispose to the development of cardiovascular and renal disease. Within the disease spectrum of low renin hypertension (LRH), hyperaldosteronism ranges from mild to marked, but it always remains inappropriate for the level of plasma renin. The primary causes for LRH remain ill-defined. Here, we propose that excess aldosterone production may not be the sole causative factor contributing to low-renin hypertension in LREH or IHA. Our general hypothesis is that the low renin-hypertensive state in LRH is a consequence of an increased sensitivity to Angiotensin II (Ang II) manifest at multiple sites: the adrenal gland (hyperaldosteronism) the vasculature (hypertension) and/or the juxtaglomerular apparatus (feed-back inhibition of renin secretion, low- renin). We previously demonstrated that global disruption of genes encoding TASK two-pore domain potassium channels produces cardinal features of LREH and IHA (low renin hypertension with high aldosterone:renin ratios, hypersensitivity to Ang II and variable degrees of autonomous aldosterone production). Therefore, we further hypothesize that disrupting TASK channel activity, as well as the removal of TASK protein itself, is required to produce hyper-reactivity to Ang II. To provide human disease relevance to our proposed work, we use genomics to test for novel associations of human TASK channel gene variants with measures of hypertension, aldosterone, renin activity and ARR in MESA (Multi-Ethnic Study of Atherosclerosis) We propose to use a combination of molecular/cell biological and electrophysiological recording techniques, along with genomic approaches, to test our hypotheses in two Specific Aims. In Aim 1, we generate and validate new mouse models in which TASK channels are deleted specifically in aldosterone producing zona glomerulosa cells (ZG) and in which TASK KO ZG cells are marked by green fluorescent protein. We use these unique mouse models of LRH to determine which phenotypic features of LRH are produced by hyperaldosteronism, per se. We use these findings to inform a genetic analysis in humans. In Aim 2, we determine the cellular basis for hypersensitivity to Ang II testing contributions of: i) TASK channel activity; ii) AT1 receptor activity-state; iii) cellular electrical excitability; and iv) altered Ca channel activity. Our 2+ proposed studies will provide new information about the cell biology of ZG cells, the cellular mechanisms that underlie exaggerated responses in LRH, and the contribution of genetic differences in TASK channels to human hypertension. If our hypotheses are correct, they also will provide a rational basis for development or evaluation of new medical treatments for LRH, for which there remains a high prevalence of resistance to currently available therapies.

PROJECT 4 PROJECT SUMMARY Pannexin 1 (Panx1) is a widely-expressed membrane ion channel that, when activated, leads to transmembrane flux of large molecules (i.e., nucleotides, other metabolites) that can mediate intercellular signaling in multiple (patho)physiological contexts (e.g., see Projects 1-3). Thus, understanding the different cellular and molecular mechanisms for channel activation, and the determinants for large molecule permeation, are of paramount importance to reveal novel potential therapeutic strategies for pathway-specific pharmacological intervention that could selectively modulate permeation of specific signaling metabolites in different contexts. Among well-established Panx1 activation mechanisms, that mediated by G?q protein-coupled receptors (G?qPCRs) is widespread, but the essential cellular, molecular and biophysical mechanisms that mediate this prevalent form of channel activation have not been elucidated. Our preliminary data implicate the salt-inducible kinase, SIK1, a serine-threonine kinase that physically associates with Panx1, and is both necessary and sufficient for channel activation. In Aim 1, supported by additional preliminary observations, we test the hypothesis that G?qPCRs signal via non-canonical pathways involving LKB1 and RhoA-mDia-HDAC6, which converge to activate SIK1 to mediate phosphorylation and activation of Panx1. For this, we use genetic and pharmacological tools, in heterologous and native systems, to determine the relevant signaling pathways and identify critical channel phosphosites by mutational, mass spectrometric and in vitro kinase approaches. In addition, we use single channel recordings to characterize properties of partially and fully receptor-activated wild type and concatenated Panx1 constructs, examining whether channels activate in the novel stepwise fashion that we recently discovered for C-terminally cleavage-activated channels. Panx1 channels are renowned for their association with nucleotide release and dye uptake. Nonetheless, it has not been established whether these large molecules actually permeate via the channel itself, and even the ionic selectivity of Panx1 has not been established. In addition, channels activated by different mechanisms display distinct single channel properties, suggesting that they may also yield distinct permeation properties that support release of specific signaling molecules. In Aim 2, we implement a proteoliposome system incorporating purified Panx1 to test the hypothesis that activated Panx1 provides a permeation pathway that supports release of various cellular constituents, and that flux of different molecules is influenced by distinct modes of channel activation. By directly measuring permeation of specific signaling metabolites through these purified Panx1 channels, we will identify the range of metabolites that can transit the channel when activated by either caspase- mediated C-terminal cleavage or SIK1-mediated phosphorylation.!! This work defines molecular mechanisms underlying physiologically relevant forms of Panx1 regulation, and identifies permeation properties and signaling metabolites supported by specific activation mechanisms.