H Criss Hartzell, Ph.D. - US grants

The vertebrate heart contracts spontaneously, but the force and frequency of contraction are increased by norepinephrine (NE) released from sympathetic nerves and decreased by acetylcholine (ACh) released from parasympathetic nerves. At the molecular level, these transmiters act upon several different effector systems. The effects on beat frequency are produced by modulation of several kinds of ionic channels in the plasma membrane and consequent alteration of pacemaker currents. Effects on contractile force are mediated by changes in myosin cross-bridge activity which is regulated by (1) the influx of Ca during the action potential, (2) Ca sequestration and release by the sarcoplasmic reticulum (SR), and (3) the functioning of proteins in the contractile apparatus. The molecular mechanisms underlying these effects, however, are poorly understood. The overall goal of this research is to understand the molecular mechanisms which underly neural (particularly parasympathetic) control of the heart. We would like to understand how the binding of ACh to receptors is transmitted to different effector systems (ion channels, contractile apparatus, etc.), how the different effectors contribute quantitatively to changes in contractile force and frequency, and whether the opposing effects of sympathetic and parasympathetic activity are mediated through opposite effects on the same effector systems. This research will address three specific questions. (1) What are the molecular mechanisms underlying the increase in K+ conductance (hyperpolarization) produced by ACh in the heart. Electrophysiological techniques will be used to determine the mechanisms which are responsible for the long duration of this response. (2) Does phosphorylation of the myofibrillar protein, C-protein, play a role in regulating the force of cardiac contraction? Biochemical experiments will correlate phosphorylation of this protein with contractile activity of the heart. (3) What are the molecular mechanisms by which ACh stimulates dephosphorylation of C-protein? These studies hopefully will increase our understanding of the mechanisms by which ACh regulates heartbeat.

The vertebrate heart contracts spontaneously, but the force and frequency of contration are increased by norepinephrine (NE) release from sympathetic nerves and acetylcholine (ACh) released from parasympathetic nerves. These transmitters act upon several different effector systems including several different kinds of ion channels in the plasma membrane, the sarcoplasmic reticulum, and proteins in the contractile apparatus. The mechanism of action of NE is relatively well understood: NE increases contractility by stimulating adenylate cyclase which in turn activates the cAMP-dependent protein kinase that phosphorylates the appropriate effector proteins. The molecular mechanisms of action of ACh, on the other hand, are less well understood. The overall goal of this research will continue to be to elucidate the molecular mechanisms which underlie neural (particularly parasympathetic) and hormonal control of the heart. We would like to understand how the binding of ACh to receptors is transmitted to different effector systems, the nature of the "second messenger" systems invovled, and the role of each of the effector systems in regulating contraction. In this next 5-year period, we plan to focus heavily upon regulation of the trans-sarcolemmal calcium current (ICa), because this current plays a central role in determining the force of cardiac contraction. In addition, we will pursue experiments on phosphorylation of C-protein. The research will address 4 specific questions. (1) What is the mechanisms of cGMP action on ICa? We have previously shown that intracellular perfusion with cGMP decreases I Ca under certain conditions, and we have hypothesized that this decrease is mediated by a cGMP-stimulated phosphodiesterase. This hypothesis wil be tested extensively. (2) What are the mechanisms of ACh action on ICa? Does ACh act only by inhibiting adenylate cyclase, or are there other modes of ACh action? (3) Does ACh produce its positive inotropic effects on the heart by stimulating phosphoinositide metabolism and increasing ICa? (4) Are ICa and C-protein and troponin-1 phosphorylation controlled coordinately? It is hoped that these studies will provide new insights into neural control of cardiac function.

Endothelium-derived relaxing factors are known to play a key role in vasorelaxation. At least two different factors have been identified: endothelium-derived relaxing factor (EDRF, thought to be nitric oxide or a closely related compound) and endothelium-derived hyperpolarizing factor (EDHF). The mechanisms by which these substances produce relaxation remains incompletely understood. The goal of this research is to understand the effects of endothelium-derived factors on ion channels in arterial smooth muscle. Recent studies have suggested that EDHF, acting via arterial smooth muscle potassium channels, may be responsible for as much as 50% of endothelium-derived vasorelaxation. Furthermore, there are reports that EDRF may also affect ion channels in arterial smooth muscle, but the species of channels affected remains poorly defined. Previous studies on endothelium-dependent relaxation have provided an incomplete picture of the ionic mechanisms involved for two reasons: Intact segments of artery are very difficult to voltage clamp; and, only pharmacological vasodilators rather than endothelium-derived substances have been studied in isolated patch clamped cells. We propose to patch clamp cells isolated from rabbit cerebral and systemic arteries and to test the effects of endothelial effluent from a column of cultured bovine aortic endothelial cells on ion channels. The properties and pharmacology of the channel(s) affected by endothelial-derived factors will be identified. There are four specific aims: 1) What are the effects of endothelial factors on ion channels in arterial smooth muscle? 2) What are the effects of vasoconstrictors on these ion currents? 3) What is the nature of the endothelial factors that produce these effects? 4) What are the signal transduction pathways involved? By investigating the effects of endothelial-derived factors on isolated vascular smooth muscle ion channels with patch clamp techniques, we will determine new aspects of this important facet of endothelial control of vascular tone. These studies will lead to better understanding of vascular control in health and in disease states.

9317359 Quarmby The goal of this proposal is to analyze the signaling pathways in Chlamydomonas, a unicellular, biflagellate alga. Chlamydomonas exhibits three behaviors that all involve increases in Ca++: these are phototaxis, mating and flagellar excision. The ultimate objective is to understand the mechanisms by which different stress stimuli are transduced to flagellar excision, while other stimuli, such as light, result in the change of swimming behavior and not in flagellar excision, even though all these pathways appear to involve Ca++ in signaling. The proposal has two objectives. The first is to isolate and characterize excision mutants of C. reinhardtii to test the hypothesis that low pH activates a signaling pathway that is different from the signaling pathways activated by the chemical inducers of flagellar excision. The hypothesis for such a dual pathway is supported by physiological and pharmacological data, but a genetic analysis will verify the existence of independent signaling pathways leading to a common response. Mutants will be generated by insertion of exogenous DNA and selected for signaling defects. Heterocaryon analysis will be used to study the relationship of selected mutants. The second objective is to use Ca++ flux and electrophysiology to test the hypothesis that activation of a Ca++ channel is a key event in low pH induced flagellar excision. Dr. Quarmby has strong circumstantial evidence that influx of Ca++ that is stimulated by decreasing pH is necessary for acid-induced flagellar excision. %%% The research objectives chosen by the PI provide a powerful experimental system to study how different signals elicit the same response, and how the different signals that use Ca++ elicit distinctly different physiological responses. Because it is presently thought that the fundamental mechanisms of such signaling pathways have been conserved throughout evolution, results of these studies will be instrumental in our understa nding of how intracellular communication takes place in living organisms. ***

In many cells, release of Ca from intracellular stores is followed by Ca influx from the extracellular space ("capacitative Ca entry") through store-operated Ca channels (SOCCs). Ca influx through SOCCs is important for many cellular activities including activation of T lymphocytes by antigen, secretion of insulin from pancreatic acinar cells in response to secretagogues, and stimulation of mast cell degranulation. Despite the importance of SOCCs, very little is known about their molecular structure or control mechanisms, but an ion channel (TRP) cloned from Drosophila photoreceptors may provide insights into SOCC structure and function. Recently, highly conserved homologs of TRP have been identified in several species and it appears that TRP and these homologs are SOCCs. The purpose of this proposal is to test the hypothesis that TRP and its homologs are SOCCs and to investigate the structure, function, and regulation of TRPs using biophysical and molecular genetic approaches. Specific aims will be to study the cellular distribution of TRP homologs in human tissues and cell lines, to determine the topology of TRP homologs in the membrane, to characterize the properties of TRP ion channel homologs expressed in Xenopus oocytes and mammalian cell lines, to identify accessory subunits of the channel, and to develop C. elegans as a genetic system for studying TRP ion channels. These studies are expected to provide valuable knowledge about the mechanisms by which Ca is regulated in cells. Because intracellular Ca is a key element in signal transduction cascades in virtually every cell type, we anticipate that a better understanding of this fundamental cell biological process will provide new insights into a variety of human diseases.

Ca-activated Cl (CaC) channels play fundamental roles in many physiological processes, including secretion in airway epithelium, repolarization of the cardiac action potential, regulation of vascular tone, and neuronal excitability. These channels are important in several human diseases, including cystic fibrosis and cardiac arrhythmias. Our understanding of the biophysics of CaC channels, however, remains rudimentary, and the mechanisms of CaC channel activation by Ca are enigmatic. This proposal aims to elucidate the mechanism of regulation and gating of CaC channels in Xenopus oocytes and rabbit cardiac myocytes at the single channel level using the patch clamp technique in the cell-attached and excised-patch configurations. These studies will provide important insights into the control and function of this important class of ion channels and will likely have an impact on the understanding of how other types of ion channels are regulated by Ca. Aim 1 addresses whether the apparent diversity in CaC channels and currents is due to molecular diversity in channel types or to complexity of CaC channel regulation. Xenopus oocytes are an ideal system for studying this question because they express three different types of CaC currents. We present evidence that CaC channels exhibit strong voltage-dependent Ca sensitivity and we suggest that at least some of the diversity in CaC currents can be explained by the complexities in channel function that result from this voltage-dependence of Ca regulation. Aim 2. Addresses how this complex regulation comes about, by examining the mechanisms of channel regulation by Ca binding, by CaM binding, and by phosphorylation. Aim 2 then extends these studies to cardiac myocytes. CaC channels play a normal role in repolarization of the cardiac action potential and can also play a pathological role in initiating arrhythmias during Ca overload. The goal of this aim is to characterize CaC channels in cardiac myocytes to understand whether the same CaC channels are responsible for action potential repolarization and the after depolarizations that trigger arrhythmias and to explore the features of channel regulation that permit the CaC channel to become arrhythmogenic.

DESCRIPTION (provided by applicant): The aim of this proposal is to help support an international symposium on "Chloride Signaling" to be held September 3-7, 2003, at the Marine Biological Laboratory in Woods Hole, Massachusetts. This meeting will be the 57th Annual Symposium of the Society of General Physiologists (SGP). The major, if not sole, purpose of the SGP is to host a symposium each year on a different subject. The topic of this meeting is especially timely and compelling. Anion channels in general have received less attention than cation channels, partly because their functions and pathophysiology was not as evident and because the available methodology was not powerful enough to crack them. Recently it has become clear that CI channels play very important roles in fundamental physiological and cell biological processes in ways that were not anticipated. Diseases due to CI channel defects are rapidly being discovered. This year the crystal structure of the first CI channel was published, which is certain to provide new and exciting insights into CI channel permeation. The symposium is titled "Chloride Signaling" rather than "Chloride Channels" because of the recent realization that cytosolic CI is dynamic and may have direct actions on the function of some proteins and because the regulation of intracellular CI depends on a variety of other active transport proteins. The hope is that this symposium will be formative in identifying important directions for research in the biology of chloride. The speakers have been selected to represent the leaders in chloride signaling around the world. The high-profile character of the subject makes us confident of high attendance (approximately 300 participants). Our publicity for the meeting will encourage participation from a wide sweep of scientists at all career levels-from structural biologists exploring the mechanisms of anion permeation and anion binding to proteins, to cell biologists interested in the roles of chloride in membrane trafficking, neuroscientists interested in the signaling of chloride channels, transport physiologists involved in ion homeostasis, and geneticists interested in channelopathies

A grant has been awarded to Dr. H. Criss Hartzell of Emory University School of Medicine to support a symposium on the Biology of Chloride. The symposium will be held at the Marine Biological Laboratory from September 3 -7, 2003. Presentations will be given by approximately 30 leaders in the field from around the world. There will also be sessions featuring young investigators. The purpose of the meeting is to define new directions in the physiology of chloride. Only recently have we begun to appreciate that chloride ions play fundamental roles in cell have recently been identified at a molecular level and the structures of a few are now known at atomic resolution. The impact of their disruption has been, in several cases, completely surprising. Disruption of chloride channels produces a wide range of diseases affecting muscle, bone, kidney, and the nervous system. This meeting will bring together the leaders in chloride physiology to formulate the directions this field will take in the immediate future.
With scientific progress progressing so rapidly, a meeting such as this is invaluable in identifying the areas that require the most attention and are most likely to provide high payoff in terms of new insights. Poster sessions where people present their newest, as yet unpublished, data will serve as a forum for discussion of general directions and identification of the highest priority issues. Certainly it is now clear that chloride ions are important in human disease, but chloride has other functions that we have only glimpsed. Proteins that regulate chloride are present in other organisms, including worms, flies, and plants, but their functions remain poorly elucidated. These proteins may have potential as targets that can be manipulated genetically or chemically to enhance food production or control pests. This meeting of experts in the field and their students will provide a fertile ground for exploring these ideas.

DESCRIPTION (provided by applicant): The ability to read this page without magnification depends upon the integrity of the macula, a small region of the retina including the fovea. Macular degeneration is the leading cause of blindness in developed countries. Age-related macular degeneration (AMD) is a progressive degeneration of the macula that affects approximately 20% of individuals over the age of 65, but its causes remain unknown. The hypothesis driving this proposal is that CI currents play a role in phagocytosis of shed photoreceptor discs by the retinal pigment epithelium (RPE). Defects in this process can lead to macular degeneration as the result of accumulation of retinoids and lipofuscin pigment in the subretinal space. We propose that CI channels are important in normal phagocytosis because they are involved in the regulation of cell volume during ingestion of large quantities of outer segments. A variety of well-known CI channels including CFTR, CIC-2, CIC-3, and CIC-5 are expressed in RPE cells and recently it has been suggested that bestrophin, an RPE protein that causes Best macular dystrophy, is the founding member of a new family of CI channels. The goal of this project is to characterize the CI currents, especially bestrophin-mediated currents, that are expressed in RPE cells and to understand their function. There are three specific aims. (1) To determine the properties of bestrophin CI channels. We will test the hypothesis that bestrophins are subunits of a chloride channel by patch clamp analysis of heterologously expressed bestrophins. (2) To characterize chloride channels in RPE cells. This aim tests the hypothesis that several types of CI channels are functionally specialized for specific RPE functions. The strategy is to use whole-cell and patch clamp recording to characterize CI channels in RPE cells and to compare them to the properties of known CI channels, including bestrophin. (3) To determine the role of CI channels in photoreceptor disc phagocytosis. This aim will test the hypothesis that CI channels are important in phagocytosis of rod outer segments by RPE cells. This hypothesis will be tested by determining the effects of pharmacological inhibitors and antisense knockdown of CI currents on the phagocytosis of rod outer segments by RPE.

? DESCRIPTION (provided by applicant): Ca2+-activated Cl- channels (CaCCs) are ion channels that are opened by increases in cytosolic Ca2+ and selectively conduct Cl- and other anions down their electrochemical gradient. Their best known function is epithelial fluid secretion. CaCCs are encoded by members of two different gene families, Bestrophins and Anoctamins (also called TMEM16). Mutations in BEST1 cause a spectrum of retinal degenerations called bestrophinopthies. ANO1 and ANO2, while not yet linked directly to retinal disease, are expressed in a variety of cells in the retina including photoreceptors and RPE. Since their discovery in 2008, it has since become increasingly apparent that anoctamins are amazingly versatile: ANO1 has a very broad range of functions that encompass sensory transduction and adaptation, regulation of myoepithelial cell and smooth muscle tone, control of neuronal and cardiac excitability, and nociception. In addition, evidence is accumulating that ANO1 plays fundamental roles in cell biological processes like regulation of cell motility, proliferation, and primary ciliogenesis. Recently, we observed that ANO1, while spread over the cell surface in non-confluent cells, becomes concentrated in a distinctive apical torus (the nimbus) as cells become confluent and polarize. The nimbus becomes the site where the primary cilium emerges from the cell. The primary cilium, a non-motile cellular antenna that is packed with sensory receptors, plays a pivotal role in tissue morphogenesis and development and is closely linked to cell proliferation and migration. Mutations in various genes involved in ciliogenesis produce a diverse spectrum of human disorders called ciliopathies that are very frequently characterized by retinal degeneration. We have found that disrupting ANO1 function or expression has profound effects on the development of the primary cilium. The goal of this research is to elucidate the role of ANO1 in the genesis and function of the primary cilium. We hypothesize that ANO1 plays a key role in organizing a subdomain of the apical membrane to make it competent for ciliogenesis to occur. We imagine two mechanisms by which ANO1 may function in this context. (i) ANO1 may operate as a scaffold to organize and coordinate ciliogenic proteins at the apical membrane. This mechanism is supported by our proteomic data that ANO1 interacts with proteins essential for ciliogenesis and that these ciliary proteins regulate ANO1 currents. (ii) Alternatively, Cl- transport through ANO1 may play a role in ciliogenesis. This idea is supported by data that ANO1 channel blockers disrupt ciliogenesis and that the species of anion in the extracellular solution has profound effects on ciliogenesis. Because the primary cilium is a fundamental property of both photoreceptors and retinal pigment epithelial cells, understanding the role of ANO1 in primary ciliogenesis has important implications for retinal biology and therapy of retinal diseases.

DESCRIPTION (provided by applicant): The function of the kidneys is to maintain normal ionic concentrations in the blood. Normally the kidneys maintain the blood ionic composition within very tight range and imbalance produces very serious health consequences including death. The goal of this grant is to understand the ion channels that function in the kidney to transport chloride and maintain chloride homeostasis in the body. This application will focus on an exciting new family of chloride channels that was just discovered, the anoctamins (also called TMEM16). We have preliminary data that several anoctamins are expressed in kidney and are likely to play important roles in chloride secretion and/or absorption. This application will investigate the expression of anotamins in normal mouse and human kidney and in kidney from human and from mouse models of polycystic kidney disease. Expression will be determined using quantitative RT-PCR, western blot, and immunofluorescent confocal microscopy. The functional structure and organization of the anoctamin channel will be investigated using electrophysiological (whole-cell and single channel patch clamp recording) and biochemical analysis of cells expressing Ano1. The location of Ca2+ binding sites and mechanisms of regulation of Ano1 by voltage and Ca2+ will be explored to develop a quantitative model of how anoctamin channels are gated.

? DESCRIPTION (provided by applicant): Recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E) cause Limb-Girdle Muscular Dystrophy 2L (LGMD2L), Miyoshi Muscular Dystrophy 3 (MMD3), and other generalized myopathies. ANO5 is a member of a 10-gene superfamily, the founding members of which (ANO1 and ANO2) are plasma membrane Ca2+-activated Cl- channels. Because ANO5 is 38% identical (54% similar) to ANO1, it is widely assumed that ANO5 is a Cl- channel and that ANO5 myopathies are explained by defects in ion transport. Recently, however, it has become apparent that some ANOs, notably ANO6 - which is 75% similar to ANO5, have an additional function: they stimulate phospholipid scrambling (PLS). PLS is the physiological loss of phospholipid asymmetry in the plasma membrane, typified by the translocation of phosphatidylserine (PtdSer) from its location in the cytoplasmic leaflet of the plasma membrane to the extracellular leaflet. The arrangement of PtdSer in the membrane is important for two reasons: PtdSer is known to serve as a platform for the assembly of membrane-associated protein complexes and is an important regulator of membrane fusion during endo- and exo- cytosis. This application tests the hypothesis that ANO5 is a phospholipid scramblase and an ion channel and then uses this information to explore the mechanisms of ANO5-associated skeletal muscle pathology. ANO5-myopathies, and related myopathies like ones caused by mutations in dysferlin, are explained by defects in mechanisms that repair membrane injury produced normally by exercise. Such injury is healed by two processes: (1) resealing of small lesions by assembly of new plasma membrane to fill the holes and (2) fusion of muscle progenitor stem cells (satellite cells) to regenerate new muscle fibers at sites of more severe damage. We propose that reorganization of membrane lipids mediated by ANO5 plays a fundamental role in these processes. There are three specific aims. (1) We will determine if ANO5 is a phospholipid scramblase, a regulator of a scramblase, and/or an ion channel. We will evaluate ion channel function by patch clamp and PLS by imaging fluorescent phospholipid probes in both HEK cells overexpressing ANO5 and in muscle cells endogenously expressing ANO5. (2) We will then investigate the cellular mechanisms of ANO5-mediated PLS in cultured myotubes and test whether ion transport plays a role. (3) We will elucidate the role of ANO5 in membrane repair using myotubes expressing wild type, disrupted, or mutant ANO5. Further, we will evaluate the function of pathogenic ANO5 variants to determine the functional consequences of human variations in ANO5 that are linked to myopathy. The effects of disease-associated ANO5 sequence variants on ion channel function, PLS, membrane repair, and myoblast fusion will be characterized in myotubes transfected with these variants. This study has the potential to open a completely novel line of investigation that may lead to new therapies for muscular dystrophies, especially those caused by ANO5 dysfunction, but potentially also other types of muscular dystrophies caused by muscle membrane fragility or defective repair.

Understanding the mechanisms by which small molecules are transported across cell membranes is a fundamental challenge in cell physiology. This application focuses on one family of transport proteins, the Anoctamins / TMEM16s, because they play diverse and indispensable roles in cellular physiology. The founding members of the Anoctamin (ANO) family are Ca2+-activated Cl- channels (ANO1 and ANO2). These channels are ubiquitously expressed and are intimately engaged in keeping our epithelia moist by driving the secretion of bodily fluids, controlling gut motility, facilitating the secretion of hormones, and regulating neuronal excitability and smooth muscle contractility, among other functions. Dysfunction of ANO1 has been implicated in a variety of human disease states including hypertension, colitis, asthma, and lung disease. Genetic disruption of the ANO1 gene in mice causes major developmental abnormalities, behavioral disorders, altered gastrointestinal motility, and ability to sense pain. Because ANO1 and ANO2 play such varied but essential roles in cell physiology, they represent novel targets for therapeutic drug development, but as yet ANOs as drug targets have received relatively little attention. Recently, 3-D structures of various ANOs including ANO1 have provided valuable insights into how these proteins work, but major questions remain. The long-range goal of our research is to understand the structure and function of ANO1 (TMEM16A) and ANO2 (TMEM16B). Specifically in this application, we focus on the regulation of ANO1 by the phospholipid phosphatidylinositol-(4,5)bisphosphate (PI(4,5)P2). While PI(4,5)P2 is a minor lipid in the cell membrane, it is clear that it plays a critical, but scantily understood, role in ANO1 and ANO2 function. We will use a combination of single-cell electrophysiology, directed mutagenesis, and computational molecular dynamics modeling to elucidate how the opening and closing of ANO1 is controlled by PI(4,5)P2 and calcium ions. There are 3 aims: (1) We will characterize the biophysical mechanisms of ANO1 and ANO2 regulation by PI(4,5)P2, the functional interactions between PI(4,5)P2 and calcium, and the structural requirements of phosphoinositides and inositol phosphates for channel regulation. (2) We will identify the amino acids involved in PI(4,5)P2 regulation and locate the PI(4,5)P2 binding sites in ANO1 and ANO2. Preliminary data provides strong support for the existence of 3 different PI(4,5)P2 binding sites in ANO1. (3) We will determine the functional roles for each of the 3 different PI(4,5)P2 binding sites in regulating ANO1 Ca2+ sensitivity, gating, and inactivation. A compelling reason for comparing ANO1 and ANO2 is that although these 2 proteins are 70% similar (57% identical) in sequence, ANO1 is stimulated by PI(4,5)P2 while ANO2 is inhibited. This difference provides a rich opportunity to understand how PI(4,5)P2 binding is coupled to channel function. These studies will answer pressing outstanding questions about the regulation of these channels that are crucial to human health and disease.