Kevin Strange - US grants

The collecting duct is the final site in the kidney for regulation of urine volume and composition and is the primary target for aldosterone and vasopressin, two of the most important hormones known to control whole animal salt and water homeostasis. Under normal conditions the cortical collecting tubule (CCT) actively reabsorbs NaC1 and secretes K+, H+ and HCO3-. Cortical collecting tubules of the rabbit are composed of principal and intercalated cells present in a ratio of ca. 2:1. A large body of evidence suggests that the principal cell is primarily involved in NaC1 reabsorption and K+ secretion. The function of the intercalated cell has been inferred almost entirely from indirect studies, but this cell is thought to be responsible for H+ and HCO3- secretion. The proposed study will determine the transport functions of identified cell types in isolated perfused rabbit CCT. Specifically, solute entry and exit steps will be characterized in principal and intercalated cells by studying the mechanisms of ouabain-induced cell swelling using quantitative differential interference contrast microscopy. Techniques to impale identified cell types with ion and voltage-sensitive microelectrodes will be developed to characterize membrane conductive properties and electro-chemical gradients for transported ions. Electrophysiological studies are important for corroborating and complementing results of the ouabain experiments. Mechanisms of volume regulatory decrease behavior in principal and intercalated cells will be studied using quantitative microscopy and microelectrode techniques. These studies are important not only for determining the transport properties of principal and intercalated cells, but for characterizing intrinsic cellular homeostatic regulatory mechanisms. The technical and experimental approach described in this proposal will characterize normal CCT cell function and will provide a crucially important foundation for future attempts to elucidate the role played by hormones, acute and chronic physiological disturbances and disease states in altering principal and intercalated cell physiology.

This proposal request direct costs for the purchase of instrumentation necessary to construct a multiuser, real-time, combined transmitted light and epifluorescence microscopy digital imaging facility in the Renal Division at Children's Hospital. Th main instrumentation requested includes a Zeiss Axiovert 35 microscope equipped with fluorescence and DIC optics, a Photon Technology International dual wavelength epifluorescence illumination system, a Panasonic laser disc recorder and an Image-1/AT image processing and analysis system integrated with a Dell Model 325 80386 computer. A group of seven major users consisting of faculty members at the Children's and Brigham and Women's hospitals have been identified. Each major user has NIH grant support. The imaging facility will enable the major users to greatly extend their NIH-supported studies and initiate new lines of investigation not currently possible using existing instrumentation. For example, the imaging system will allow Drs. Harris, Strange and Zeidel to use fluorescent antibodies and fluid phase markers to characterize for the first time the dynamics and control of ADH-induced water channel insertion and retrieval in tight epithelial cells. Drs. Strange and Hebert will be able to address fundamental problems of cell volume regulation in renal epithelial cells by directly correlating changes in cell pH and Ca2+ with changes in cell volume. Dr. Neutra, who has extensively utilized ultrastructural methods to characterize apical endocytosis in intestinal epithelial cells, will be able for the first time to study the dynamics of this process and will be able to begin to elucidate cellular sorting mechanisms. The capability of the system for rapid sequential DIC and fluorescence and multiple fluorophore imaging will allow Dr. Lux to map ankyrin distribution in single living cells and to localize ankyrin with respect to other membrane an cytoskeletal components such as vimentin, tubulin, the Na+/K+ ATPase, etc. Finally, Dr. Simmons will be able to quantify the cellular and subcellular distribution of Na+/K+ ATPase subunits in native tissues and cell lines, detect transient transfection of mutant Na+/K+ ATPase subunits in cell cultures and characterize the role of the Na+ pump in membrane protein recycling and endocytic vesicle acidification.

Maintenance of the solute composition and volume of intra- and extracellular fluid compartments in the CNS is crucial for normal brain functioning. Small changes in solute composition can dramatically alter neuronal signaling and information processing. Because of the rigid confines of the skull and complex brain architecture changes in total brain volume can cause devastating neurological damage. It is not surprising to find, therefor, that the composition and volume of brain fluid compartments are controlled tightly under both normal and pathological condiitons. CNS osmotic and ionic balance are regulated by solute and water transport across the blood-brain barrier, choroid plexus an plasma membrane of glial cells and neurons. Despite its clinical and physiological significance, the underlying mechanisms of brain osmotic homeostasis are incompletely understood. Studies carried out in the first 2 years of NS30591 funding, however, have provided the first detailed cellular and molecular description of how cultured CNS glial cells adapt to acute and chronic hypertonicity and their correction. Several of our findings have important clinical implications and suggest new strategies for correcting plasma osmolality disorders and treating brain volume disturbances. Glial cells exposed chronically to hypertonicity accumulate the organic osmolyte myo-inositol via enhanced Na+/myo-inositol cotransporter gene expression. When returned to normotonic conditions, the cells swell and remain swollen for prolonged periods of time due in part to slow downregulation of the cotransporter. Myo-inositol is lost from the swollen cells via activation of a volume-sensitive anion channel we have described and termed VSOAC (Volume-Sensitive Organic osmolyte /Anion Channel). Studies outlined in the current proposal will complete the cloning and characterization of the brain cotransporter and will quantify osmotically regulated cotransporter gene transcription. Cotransporter protein downregulation will be characterized by western analysis. Pulse-chase labeling of cellular mRNA and in vitro mRNA degradation assays will characterize postulated post-transcriptional regulation of cotransporter message levels. Key components of our cell culture model will be tested in the whole animal to integrate cellular and molecular data into a description of brain osmoregulatory behavior. Using a hypernatremic rat model, we will quantify cotransporter mRNA levels in the brain by Northern analysis and in situ hybridization and we will measure myo-inositol transport in brain plasma membrane vesicles. The properties and regulation of VSOAC will be characterized by patch clamp, video microscopy and Xenopus oocyte expression techniques. Studies outlined in this grant provide a central foundation for the comprehensive understanding of brain volume homeostasis. Such an understanding is essential for treating a variety of disease status.

Patients with ESRD are exposed to a constantly increasing plasma osmolality as urea accumulates in extracellular fluids. ESRD, particularly in children, can also commonly be associated with a variety of serious and usually episodic electrolyte and osmolality disorders such as hypernatremia. Plasma hyperosmolality and its correction can lead to serious central nervous system (CNS) complications including brain damage, coma, brain developmental disorders and even death. The underlying causes of these complications are not well understood, but changes In brain osmoregulatory metabolism are likely to play an important role. Effective treatments for the CNS complications of hyperosmolality are lacking. The brain adapts to plasma hyperosmolality by activating volume regulatory mechanisms. For example, hypernatremia results in brain shrinkage which is followed by return of the brain to its original volume. This behavior, termed regulatory volume increase (RVI), is mediated initially by uptake of electrolytes from the extracellular fluids. With chronic exposure to hypernatremia, electrolytes are replaced by organic solutes ("organic osmolytes") such as inositol and amino acids. Correction of hypernatremia results in brain swelling and volume regulatory loss of electrolytes and organic solutes. Remarkably, the cellular and molecular mechanisms by which the brain adapts to hyperosmolality and its correction are largely unknown. Recently, however, we developed the first CNS cell culture models that exhibit a pattern of hyperosmolar volume regulation similar to that seen in vivo. We will utilize these models to 1) elucidate the mechanisms and control of volume regulatory electrolyte uptake and organic osmolyte loss and accumulation pathways in glial and neuronal cells 2) determine the mechanisms by which Na-dependent inositol transport is upregulated by hypernatremia, 3) characterize the osmoregulatory role of methylamine compounds in uremic brain cells, and 4) determine how volume regulatory electrolyte and organic osmolyte accumulation pathways are temporally coordinated. Studies outlined in this grant will provide the first detailed understanding of the cellular and molecular mechanisms by which cells of the CNS adapt to acute and chronic hyperosmolar disturbances and to the clinical correction of the hyperosmotic state. Investigations such as these are essential for. understanding the CNS complications of osmolality disturbances and in the development of more effective therapies to treat plasma hyperosmolality and ESRD. As such, our proposed investigations address directly several of the major objectives of the RFA.

Patients with ESRD are exposed to a constantly increasing plasma osmolality as urea accumulates in extracellular fluids. ESRD, particularly in children, can also commonly be associated with a variety of serious and usually episodic electrolyte and osmolality disorders such as hypernatremia. Plasma hyperosmolality and its correction can lead to serious central nervous system (CNS) complications including brain damage, coma, brain developmental disorders and even death. The underlying causes of these complications are not well understood, but changes In brain osmoregulatory metabolism are likely to play an important role. Effective treatments for the CNS complications of hyperosmolality are lacking. The brain adapts to plasma hyperosmolality by activating volume regulatory mechanisms. For example, hypernatremia results in brain shrinkage which is followed by return of the brain to its original volume. This behavior, termed regulatory volume increase (RVI), is mediated initially by uptake of electrolytes from the extracellular fluids. With chronic exposure to hypernatremia, electrolytes are replaced by organic solutes ("organic osmolytes") such as inositol and amino acids. Correction of hypernatremia results in brain swelling and volume regulatory loss of electrolytes and organic solutes. Remarkably, the cellular and molecular mechanisms by which the brain adapts to hyperosmolality and its correction are largely unknown. Recently, however, we developed the first CNS cell culture models that exhibit a pattern of hyperosmolar volume regulation similar to that seen in vivo. We will utilize these models to 1) elucidate the mechanisms and control of volume regulatory electrolyte uptake and organic osmolyte loss and accumulation pathways in glial and neuronal cells 2) determine the mechanisms by which Na-dependent inositol transport is upregulated by hypernatremia, 3) characterize the osmoregulatory role of methylamine compounds in uremic brain cells, and 4) determine how volume regulatory electrolyte and organic osmolyte accumulation pathways are temporally coordinated. Studies outlined in this grant will provide the first detailed understanding of the cellular and molecular mechanisms by which cells of the CNS adapt to acute and chronic hyperosmolar disturbances and to the clinical correction of the hyperosmotic state. Investigations such as these are essential for. understanding the CNS complications of osmolality disturbances and in the development of more effective therapies to treat plasma hyperosmolality and ESRD. As such, our proposed investigations address directly several of the major objectives of the RFA.

DESCRIPTION (Adapted from the Applicant's Abstract): The ability to sense and respond to mechanical force and changes in cell volume is a universal property of all cells. Mechanical signaling via mechanosensitive ion channels plays a key role in many cellular and organismal processes. Cell volume regulation is an essential housekeeping function and is mediated in part by ion channels that sense cell size. With few exceptions, the genes that encode these channels have eluded molecular identification. C. elegans offers unique experimental advantage for defining the physiological roles and regulation of ion channels. However, a major disadvantage is the relative inaccessibility of its cells for detailed electrophysiological characterization. To circumvent this limitation, the investigator's group developed techniques to routinely isolate and patch clamp C. elegans embryo cells and oocytes. They discovered a novel, abundantly expressed outwardly rectifying mechanosensitive anion current, ICl, mec, and a robust inwardly rectifying swelling-activated anion current, IClir, swell. The proposed studies will characterize the functional properties and regulation of the ICl, mec and IClir, swell channels, and will test the hypothesis that they are encoded by one or more of the 6 C. elegans ClC anion channel genes. The embryonic and oocyte expression patterns of the 20 identified DEG/EnaC cation channel genes will be also characterized as these channels play key roles in C. elegans mechanosensory behavior. Knowledge of expression patterns will allow the investigator to study channel function by patch clamp in native cells and to determine whether DEG/ENaCs are mechanically gated. The combination of genomic analysis, cellular and molecular biology methods, and electrophysiological measurement of channel activity will be used.

Ion channels and solute transporters are essential players in a diverse array of cellular, organ and whole organism functions. Defining the molecular structure, regulation and physiological roles of these proteins represents the leading edge of the field of membrane transport biology. In the wake of genome sequencing, two new questions in this field have emerged: What are the physiological functions of an identified ion channel or transporter gene? What are the genes in an organism's genetic blueprint responsible for a given ion channel or transporter mediated physiological process. Answering these questions presents enormous intellectual and technical challenges that have resulted in a paradigm shift in investigation strategies. It has become essential to utilize simpler, genetically and experimentally more manipulatable organisms to understand fully the genetic and molecular basis of complex physiological process and human pathophysiology. The remarkable conservation of structure and function demonstrated by genome sequencing underscores the necessity of using "model organisms" to address basic biological questions. The nematode Caenorhabditis elegans offers tremendous experimental advantages for studies of membrane transport physiology. These advantages include a fully sequenced genome, molecular and cellular manipulability a rapid lifecycle, and the ability to carry out powerful genetic analysis of physiological processes. This Program, exploit the experimental power of C. elegans to define fundamental aspects of CIC anion channel, acetylcholine receptor and neurotransmitter transporter function, regulation, and structure. The scientific questions posed by Program investigators are relevant to the biology of all organisms and are not tractable using other eukaryotic experimental systems.

DESCRIPTION (provided by applicant): C1C anion channels are found in virtually all organisms. While the functions of most identified CICs are obscure, the presence of CIC genes in widely divergent organisms and the existence of disease-causing CIC mutations in humans and other mammals indicate that the channels play important physiological roles. C. elegans offers significant experimental advantages for characterizing CIC anion channel biology. We have demonstrated that C. elegans oocytes express a mammalian CIC-2 channel ortholog encoded by clh-3, one of six nematode C1C genes. CLH-3 is activated by swelling, but plays no role in oocyte volume control. Volume sensitivity appears to link channel activity to oocyte growth and development. In full-grown oocytes undergoing rneiotic maturation, CLH-3 is constitutively activated. Oocyte maturation induces ovulatory contractions of electrically-coupled sheath cells. RNA interference of clh-3 expression disrupts the timing of sheath contractions indicating that the channel modulates ovulation via oocyte-sheath cell intercellular signaling pathways. CLH-3 thus functions as a cell cycle sensor to ensure synchronization of maturation with ovulation and fertilization. The central focus of this proposal is to identify CLH-3 regulatory mechanisms and define the role of the channel in cell-to-cell signaling pathways. Specifically, we will characterize the roles of oocyte growth, oocyte cell cycle progression and fertilization in regulating CLH-3 activity, test the hypothesis that cell cycle-dependent kinases regulate CLH-3, and test the hypothesis that CLR-3 modulates sheath cell Ca2+ signaling pathways via depolarization of oocyte and sheath cell membrane potential. Results of these studies have significant implications for understanding human physiology and pathophysiology. Proposed investigations will continue to broaden our understanding of C1C-2 specifically, and of C1C anion channels in general. Such understanding is essential in order to identify the functions of C1C channels, their regulatory mechanisms, and their potential as therapeutic targets for diseases such as cystic fibrosis. In addition, our studies will likely provide new insights into the fundamental problems of oocyte development, oocyte cell cycle control, cell-to-cell communication mechanisms, and agonist-induced smooth muscle contraction and regulation.

DESCRIPTION (provided by applicant): The nematode C. elegans offers substantial experimental advantages for investigations of the molecular/genetic basis of ion channel- and transporter-mediated cellular and whole animal integrated physiological processes. These advantages include a fully sequenced genome, a short life cycle, genetic tractability, and molecular manipulability. However, a significant limitation of C. elegans for studies of membrane transport phenomena is the relative inaccessibility for direct physiological measurements of differentiated somatic cells. The absence of robust methods for either primary or continuous culture of differentiated nematode cell types poses an additional limitation for studies of a host of important physiological and cell biological processes. Recently, we defined conditions in which C. elegans embryonic cells develop and survive in culture for many days to weeks. Cultured embryonic cells undergo striking differentiation to form neurons, muscle cells and epithelial-type cells. Cells in culture can be readily patch clamped and loaded with ion-sensitive fluorescent dyes for study by quantitative imaging methods. Targeted gene function in cultured cells is disrupted by adding double strand RNA to the culture medium. Combining direct physiological measurements with a straightforward, potent and highly selective method for disrupting gene expression offers extraordinary opportunities for defining complex cellular processes at the molecular level. The central goals of this proposal are focused on further development and characterization of C. elegans primary cell cultures. Specifically, we will develop FACS methods for purifying specific GFP-labeled cell types. FACS methods will allow cellular biochemical analysis, genomic and proteomic profiling, and co-culture of interacting cell types. We will also further develop in vitro RNA-mediated gene interference strategies and carry out an initial functional characterization of cultured nematode epithelial cells. Given the relative ease and economy of manipulating C. elegans gene function in vivo, primary cell culture methods now make it possible to readily define the genetic basis of cellular physiological processes and integrate them into the context of the whole animal. The speed and economy with which this can be done in C. elegans cannot be duplicated in other metazoan animals.

DESCRIPTION (provided by applicant): The ability to tightly control solute and water balance during osmotic challenge is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by regulated accumulation and loss of inorganic ions and organic osmolytes. Organic osmolytes play essential roles in protecting renal medullary cells from extreme hypertonic stress and fluctuating extracellular osmolality associated with the urinary concentrating mechanism. While cellular osmoregulation has been studied extensively in a variety of cell types, including kidney cells, major gaps exist in our molecular understanding of this essential process. The nematode C. elegans provides powerful experimental advantages for defining the genetic bases of fundamental biological processes. These advantages include a fully sequenced genome, genetic tractability, and ease and economy of manipulating gene function. Nematodes normally live in soil where environmental variables such as water availability and solute levels change constantly and dramatically. Recently, we demonstrated that C. elegans readily adapts to and survives extreme hypertonic stress. Given its many experimental advantages, C. elegans thus provides an outstanding model system in which to define the genes and genetic pathways responsible for cellular osmoregulation. This R21 grant application addresses stated objectives of the NIDDK PA entitled, "Pilot and feasibility program related to the kidney". We propose to characterize organic osmolyte homeostasis in C. elegans during adaptation to and recovery from hypertonic stress, perform whole genome microarray analyses to identify genes transcriptionally upregulated by hypertonicity, and assess the role of MAPK signaling pathways in cellular osmoregulation using mutant worm strains and RNAi. We will also perform mutagenesis screens to identify genes required for cellular osmoregulation in C. elegans. Powerful forward genetic screening methods have been used with great success to define molecular aspects of osmoregulation in bacteria and yeast, but have not been utilized previously to characterize this process in animal cells. Our proposed investigations represent a novel approach to the molecular study of animal cell osmotic homeostasis. Given the evolutionarily conserved nature of this process, studies in C. elegans will likely provide unique insights into osmoregulation, signaling mechanisms, and cellular stress and damage repair )rocesses in kidney cells as well as other mammalian cell types and tissues.

DESCRIPTION (provided by applicant): The ability to tightly control solute and water balance during osmotic challenge is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by regulated accumulation and loss of inorganic ions and organic osmolytes. Organic osmolytes play essential roles in protecting renal medullary cells from extreme hypertonic stress and fluctuating extracellular osmolality associated with the urinary concentrating mechanism. While cellular osmoregulation has been studied extensively in a variety of cell types, including kidney cells, major gaps exist in our molecular understanding of this essential process. The nematode C. elegans provides powerful experimental advantages for defining the genetic bases of fundamental biological processes. These advantages include a fully sequenced genome, genetic tractability, and ease and economy of manipulating gene function. Nematodes normally live in soil where environmental variables such as water availability and solute levels change constantly and dramatically. Recently, we demonstrated that C. elegans readily adapts to and survives extreme hypertonic stress. Given its many experimental advantages, C. elegans thus provides an outstanding model system in which to define the genes and genetic pathways responsible for cellular osmoregulation. This R21 grant application addresses stated objectives of the NIDDK PA entitled, "Pilot and feasibility program related to the kidney". We propose to characterize organic osmolyte homeostasis in C. elegans during adaptation to and recovery from hypertonic stress, perform whole genome microarray analyses to identify genes transcriptionally upregulated by hypertonicity, and assess the role of MAPK signaling pathways in cellular osmoregulation using mutant worm strains and RNAi. We will also perform mutagenesis screens to identify genes required for cellular osmoregulation in C. elegans. Powerful forward genetic screening methods have been used with great success to define molecular aspects of osmoregulation in bacteria and yeast, but have not been utilized previously to characterize this process in animal cells. Our proposed investigations represent a novel approach to the molecular study of animal cell osmotic homeostasis. Given the evolutionarily conserved nature of this process, studies in C. elegans will likely provide unique insights into osmoregulation, signaling mechanisms, and cellular stress and damage repair )rocesses in kidney cells as well as other mammalian cell types and tissues.

DESCRIPTION (provided by applicant): CIC voltage-gated anion channels are ubiquitous and function in plants, yeast, eubacteria, archaebacteria and various invertebrate and vertebrate animals. Nine CIC genes have been identified in mammals. Mutations in five of these genes give rise to inherited human muscle, bone, neurological and kidney disorders. The presence of CIC genes in evolutionarily divergent organisms and the existence of disease causing mutations indicate that these channels play fundamental physiological roles. The precise functions of most identified ClCs are unclear and almost nothing is known about how these important channels are regulated. The nematode C. elegans offers significant experimental advantages for characterizing CtC biology. However, a drawback of C. e/egans for ion channel studies is its small size and limited physiological access. We developed a number of novel methods during the previous funding period of this grant that allowed us to circumvent these problems. Our approach provided us with the unique opportunity to characterize the regulation and physiological roles of a CIC channel that is assembled and operational in its native cellular environment. Using an isolated worm oocyte preparation and reverse genetics, we identified a CIC channel encoded by c/h-3. Two splice variants of the channel, CLH-3a and CLH-3b, have been identified. CLH-3b is responsible for the oocyte current, is activated by oocyte swelling and meiotic cell cycle progression, and functions to couple cell cycle events to ovulation. The present application builds on our recent successes and will provide further detailed characterization of CLH-3. We will address two broad questions that have important implications for understanding basic aspects of cell physiology and of CIC function: 1) How are CIC channels regulated? and 2) What role does splice variation play in controlling CIC function and gating? Specifically, we will characterize the role of a STE-20-related kinase in regulating CLH-3b activity, we will test the hypothesis that splice variation of the channel C-terminus modulates gating, and we will define the physiological roles of CLH-3 splice variants. Results of our proposed studies will continue to broaden our understanding of CIC anion channel biology. Such understanding is essential in order to identify the functions of ClC channels, their regulatory mechanisms, their role in disease processes, and their potential as therapeutic targets.

[unreadable] DESCRIPTION (provided by applicant): Cytoplasmic Ca2+ levels control numerous, diverse cellular processes including gene expression, exocytosis and secretion, motility and contraction, cell proliferation, programmed cell death, and differentiation. While physiologists have gained an impressive understanding of Ca2+ signaling events, many fundamental questions remain unanswered. The nematode C. elegans provides numerous experimental advantages for defining molecular mechanisms of Ca2+ signaling. These advantages include relative ease and economy of manipulating gene expression by RNA interference, knockout and transgenesis; ready availability of numerous molecular reagents and mutant worm strains; a fully sequenced and well-annotated genome; and the ability to perform mutagenesis and forward genetic analysis. Posterior body wall muscle contraction (pBoc) in C. elegans drives defecation behavior and occurs in rhythmic fashion every 45-50 seconds. Genetic analyses have identified numerous genes that, when mutated or knocked down, disrupt pBoc rhythm. These include genes encoding the IP3 receptor, PLC, K+ channels and TRPM cation channels. Physiological and molecular studies have demonstrated that pBoc is driven by rhythmic, IPs-dependent intracellular Ca2+ oscillations in the intestinal epithelium. Recently, we developed primary C. elegans cell culture methods that allow for the first time patch clamp characterization of intestinal cell Ca2+ conductances. In addition, we have developed a novel isolated intestine preparation that allows physiological characterization of intracellular Ca2+ oscillations. We will use a combination of Ca2+ imaging, electrophysiology, reverse genetics and immunofluorescence to test the hypothesis that PLC-B and PLC-y, the KCNQ channels KQT-2 and KQT-3, and the TRPM channels GON-2 and GTL-1 function together to regulate intracellular Ca2+ release. We will also use patch clamp electrophysiology and gene knockout to determine if the TRPM-like Ca2+ channel ORCa is encoded by gon-2 and/or gtl-1. The combination of experimental approaches we will use in our studies is substantially more costly and time-consuming, or not realistically possible in vertebrate experimental systems. By defining basic aspects of intestinal Ca2+ signaling, this proposal forms an essential foundation of a long-term effort that will exploit the considerable experimental advantages of C. elegans to develop an integrated molecular understanding of a non-excitable cell oscillatory Ca2+ signaling pathway. Given the fundamental and highly conserved nature of Ca2+ signaling, insights gained from C. elegans will clearly provide new and important insights into vertebrate Ca2+ signaling mechanisms. Detailed molecular understanding of Ca2+ signaling is essential for understanding and treating numerous disease processes including cancer, heart disease and diabetes. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Chloride is the most abundant anion in extracellular fluids and plays critical roles in numerous essential physiological processes including epithelial fluid transport, cell volume control, acid-base homeostasis and regulation of cell excitability. Anion channels mediate CI- transport across cell and organelle membranes in all organisms. Electrophysiological studies have identified a diverse array of anion channel types, but relatively little is known about anion channel molecular biology and regulation. The nematode C. elegans provides numerous experimental advantages for defining anion channel and CI- transport molecular physiology. A drawback of C. elegans, however, is its small size and limited physiological access. We recently developed a number of innovative methods that allow us to circumvent these problems. Using electrophysiology and reverse genetics, we identified a CIC CI- channel, CLH-3b, that is expressed in the C. elegans oocyte. CICs function in organisms from bacteria to animals and their physiological importance is underscored by the identification of mutations in five human CIC genes that give rise to kidney, muscle, bone and neurological disorders. The biophysical properties of CLH-3b resemble those of mammalian CIC-2. CLH-3b is activated by dephosphorylation during oocyte meiotic maturation and swelling, and functions to couple cell cycle progression to ovulation. The type 1 phosphatases CeGLC-7a/p and a newly identified Ste20 kinase, GCK-3, regulate CLH-3b. GCK-3 binds to CLH-3b, inactivates the channel in a phosphorylation dependent manner, and is a homolog of mammalian PASK. PASK regulates Na-K-2CI cotransporters involved in fluid secretion, osmoregulation, and cell volume and CI- homeostasis. This renewal application builds on the considerable progress and successes of the previous funding cycle of DK61168. During the next funding period we will use a combination of phosphopeptide analysis, forward and reverse genetics, electrophysiology, molecular biology and microscopy to 1) identify CLH-3b regulatory phosphorylation sites, 2) define the physiological roles of the CLH-3b regulatory kinase GCK-3, 3) identify components of the GCK-3 signaling cascade that regulates CLH-3b and whole animal fluid balance, and 4) begin characterizing the mechanisms and genes involved in regulating CLH-3b plasma membrane retrieval. Our proposed studies will provide significant new insights into CIC regulation, the function of GCK- 3 and its mammalian homolog PASK, and fundamental processes such as cell volume sensing and the coordinated regulation of ion channels and transporters that control cellular CI- content, epithelial fluid transport and cell volume. Detailed understanding of CIC regulation and GCK-3/PASK signaling is essential in order to define the role of anion channels in disease processes and their potential as therapeutic targets as well as to fully understand and treat fluid secretory diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cytoplasmic Ca2+ levels control numerous, diverse cellular processes including gene expression, exocytosis and secretion, motility and contraction, cell proliferation, programmed cell death, and differentiation. While physiologists have gained an impressive understanding of Ca2+ signaling events, many fundamental questions remain unanswered. The nematode C. elegans provides numerous experimental advantages for defining molecular mechanisms of Ca2+ signaling. These advantages include relative ease and economy of manipulating gene expression by RNA interference, knockout and transgenesis; ready availability of numerous molecular reagents and mutant worm strains; a fully sequenced and well-annotated genome; and the ability to perform mutagenesis and forward genetic analysis. Posterior body wall muscle contraction (pBoc) in C. elegans drives defecation behavior and occurs in rhythmic fashion every 45-50 sec. Genetic analyses have identified numerous genes that, when mutated or knocked down, disrupt pBoc rhythm. These include genes encoding the IP3 receptor, PLC, K+ channels and TRPM cation channels. Physiological and molecular studies have demonstrated that pBoc is driven by rhythmic, IPs-dependent intracellular Ca2+ oscillations in the intestinal epithelium. Recently, we developed primary C. elegans cell culture methods that allow for the first time patch clamp characterization of intestinal cell Ca2+ conductances. In addition, we have developed a novel isolated intestine preparation that allows physiological characterization of intracellular Ca2+ oscillations. We will use a combination of Ca2+ imaging, electrophysiology, reverse genetics and immunofluorescence to test the hypothesis that PLC-p and PLC-y, the KCNQ channels KQT-2 and KQT-3, and the TRPM channels GON-2 and GTL-1 function together to regulate intracellular Ca2+ release. We will also use patch clamp electrophysiology and gene knockout to determine if the TRPM-like Ca2+ channel ORCa is encoded by gon-2 and/or gtl-1. The combination of experimental approaches we will use in our studies is substantially more costly and time-consuming, or not realistically possible in vertebrate experimental systems. By defining basic aspects of intestinal Ca2+ signaling, this proposal forms an essential foundation of a long-term effort that will exploit the considerable experimental advantages of C. elegans to develop an integrated molecular understanding of a non-excitable cell oscillatory Ca2+ signaling pathway. Given the fundamental and highly conserved nature of Ca2+ signaling, insights gained from C. elegans will clearly provide new and important insights into vertebrate Ca2+ signaling mechanisms. Detailed molecular understanding of Ca2+ signaling is essential for understanding and treating numerous disease processes including cancer, heart disease and diabetes. [unreadable] [unreadable]

Chloride is the most abundant anion in extracellular fluids and plays critical roles in numerous essential physiological processes including epithelial fluid transport, cell volume control, acid-base homeostasis and regulation of cell excitability. Anion channels mediate CI" transport across cell and organelle membranes in all organisms. Electrophysiological studies have identified a diverse array of anion channel types, but relatively little is known about anion channel molecular biology and regulation. The nematode C. elegans provides numerous experimental advantages for defining anion channel and CI" transport molecular physiology. A drawback of C. elegans, however, is its small size and limited physiological access. We recently developed a number of innovative methods that allow us to circumvent these problems. Using electrophysiology and reverse genetics, we identified a CIC CI"channel, CLH-3b, that is expressed in the C. elegans oocyte. CICs function in organisms from bacteria to animals and their physiological importance is underscored by the identification of mutations in five human CIC genes that give rise to kidney, muscle, bone and neurological disorders. The biophysical properties of CLH-3b resemble those of mammalian CIC-2. CLH-3b is activated by dephosphorylation during oocyte meiotic maturation and swelling, and functions to couple cell cycle progression to ovulation. The type 1 phosphatases CeGLC-7a/p and a newly identified Ste20 kinase, GCK-3, regulate CLH-3b. GCK-3 binds to CLH-3b, inactivates the channel in a phosphorylation dependent manner, and is a homolog of mammalian PASK. PASK regulates Na-K-2CI cotransporters involved in fluid secretion, osmoregulation, and cell volume and CI" homeostasis. This renewal application builds on the considerable progress and successes of the previous funding cycle of DK61168. During the next funding period we will use a combination of phosphopeptide analysis, forward and reverse genetics, electrophysiology, molecular biology and microscopy to 1) identify CLH-3b regulatory phosphorylation sites, 2) define the physiological roles of the CLH-3b regulatory kinase GCK-3, 3) identify components of the GCK-3 signaling cascade that regulates CLH-3b and whole animal fluid balance, and 4) begin characterizing the mechanisms and genes involved in regulating CLH-3b plasma membrane retrieval. Our proposed studies will provide significant new insights into CIC regulation, the function of GCK- 3 and its mammalian homolog PASK, and fundamental processes such as cell volume sensing and the coordinated regulation of ion channels and transporters that control cellular CI" content, epithelial fluid transport and cell volume. Detailed understanding of CIC regulation and GCK-3/PASK signaling is essential in order to define the role of anion channels in disease processes and their potential as therapeutic targets as well as to fully understand and treat fluid secretory diseases.

DESCRIPTION (provided by applicant): Nematodes parasitize ~25% of the human population. Helminth targeting drugs, or anthelmintics, have been used to control parasitic nematodes for decades and many species are evolving multidrug resistance. In diverse organisms, multidrug resistance is mediated by increased expression and activity of enzymes that detoxify xenobiotics. Pharmacological compounds that target xenobiotic detoxification pathways would provide much needed tools for studying multidrug resistance and could greatly increase the useful life of current and future anthelmintics. Few pharmacological compounds are available for studying and targeting multidrug resistance and those that are available are not specific for nematodes and only target a single enzyme or class of enzymes. The transcription factor SKN-1 activates the expression of xenobiotic detoxification genes in the nematode Caenorhabditis elegans. Genetic inhibition of SKN-1 sensitizes C. elegans to diverse xenobiotics, and C. elegans can acquire resistance to anthelmintics by increasing the expression of genes that are regulated by SKN-1. SKN-1 is also essential for the development of embryos. Our recent studies have identified a principal pathway regulating SKN-1 that is highly divergent from pathways that regulate xenobiotic detoxification in mammals. Therefore, SKN-1 is a promising target for the development of drugs that disrupt embryonic development, decrease stress resistance, and inhibit xenobiotic detoxification in nematodes without affecting analogous pathways in humans. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal system to screen for inhibitors of xenobiotic detoxification genes. The first goal of this project is to develop and optimize a fluorescence-based assay of SKN-1 activity in C. elegans. The second goal of this project is to develop four secondary and counter screens to rapidly prioritize hit compounds and to test the assay in a pilot screen of 2000 compounds. After completion of these goals, a detailed description of the optimized assay will be submitted for entry into the Molecular Libraries Probe Production Centers Network (MLPCN). PUBLIC HEALTH RELEVANCE: Nematodes parasitize ~25% of humans and many strains are resistant to all currently used antiparasitic drugs. In many organisms, drug resistance is caused by over active drug detoxification pathways. The studies proposed in this application will develop, optimize, and validate a screen for pharmacological inhibitors of drug detoxification in nematodes.

DESCRIPTION (provided by applicant): ClC anion transport proteins are expressed in evolutionarily diverse organisms ranging from archaebacteria to mammals where they play essential roles in diverse processes such as systemic osmoregulation and regulation of cell and organelle Cl- and pH. Mutations in five of the nine human ClC genes give rise to or are associated with inherited muscle, bone, neurological and kidney disorders. Despite intensive study and their physiological importance, very little is known about how ClCs are regulated. We have exploited the genetic and molecular tractability of the nematode Caenorhabditis elegans to characterize the regulation and physiological roles of ClC channels that are assembled and operational in their native cellular environments. CLH-3b is a member of the mammalian ClC-1, 2, Ka and Kb anion channel subfamily, is expressed in the C. elegans oocyte and is activated by swelling and meiotic maturation via type 1 phosphatase mediated serine dephosphorylation. The Ste20 kinase GCK-3 binds to a 101 amino acid regulatory domain on the CLH-3b cytoplasmic C-terminus and functions to inhibit channel activity. Channel inhibition requires concomitant phosphorylation of two serine residues in the regulatory domain. Our studies of CLH-3b have provided the most detailed description of ClC channel regulation in the field. GCK-3 is a homolog of mammalian SPAK and OSR1. These kinases have emerged as critical regulators of diverse transport processes that play essential roles in cellular and systemic osmotic homeostasis. The SPAK/OSR1 signaling pathway is an important target for the development of new anti- hypertension drugs. We have shown that the role of GCK-3/SPAK/OSR1 signaling in osmosensing and systemic osmotic homeostasis is conserved from C. elegans to humans. The functional conservation of this signaling mechanism over hundreds of millions of years of evolution underscores its physiological significance. This renewal application builds on past successes of DK51610 and our unique understanding of CLH- 3b regulation to address two fundamental and unresolved questions: How do signaling events and conformational changes in the cytoplasmic C-terminus modulate and regulate ClC channel properties? How do cells and organisms detect osmotic perturbations and transduce those changes into specific responses? Our studies will use a variety of molecular, electrophysiological and biophysical approaches to characterize the signaling mechanisms by which GCK-3 and dephosphorylation control CLH-3b activity and to characterize conformational changes in the cytoplasmic C-terminus and outer pore that are induced by phosphorylation events. Detailed understanding of ClC biology is essential in order to fully define the role of these proteins in physiology and pathophysiology and their potential as therapeutic targets. Molecular understanding of cellular osmosensing represents a cornerstone for understanding and treating disturbances of salt and water balance that have a major impact on human health. PUBLIC HEALTH RELEVANCE: ClC anion transport proteins carry out essential physiological functions and are associated with inherited muscle, bone, neurological and kidney disorders in humans. Studies described in this application will provide the first detailed description of how phosphorylation regulates ClC channel function and will provide new insights into mechanisms of cellular osmosensing. Detailed understanding of ClC regulation and cellular osmosensing is essential for understanding and treating disturbances of salt and water balance that have a major impact on human health.

DESCRIPTION (provided by applicant): Cellular osmotic homeostasis is a fundamental requirement for life. All cells are exposed to osmotic challenges brought about by changes in intracellular solute flux and/or perturbations in extracellular osmolality. Most mammalian cells are protected from extracellular osmotic challenges by the kidney, which tightly regulates blood ionic and osmotic concentrations. Renal medullary cells are an important exception to this generalization and are subjected normally to extreme osmotic stress by the renal concentrating mechanism. Cells maintain osmotic homeostasis by the tightly regulated gain and loss of salt and organic solutes termed organic osmolytes, and by detecting and repairing osmotic stress induced damage. The transport and metabolic pathways that mediate animal cell osmoregulatory solute fluxes are well described. However, little is known about the signaling mechanisms by which animal cells detect osmotic perturbations, about the types of cellular and molecular damage induced by osmotic stress, and about how this damage is detected, repaired and prevented. DK61168 supported studies developed the nematode C. elegans as a novel genetically tractable model system for defining fundamental mechanisms of animal cell osmosensing and osmotic homeostasis. During the previous funding period, we made the novel observation that disruption of protein synthesis activates expression of genes required for organic osmolyte accumulation. We also demonstrated for the first time that hypertonicity causes rapid and extensive protein damage in vivo and that genes required for protein degradation are essential for survival during hypertonic stress. The current proposal builds on these new findings and addresses three questions with broad biological and pathophysiological significance. How does disruption of protein synthesis activate osmosensitive gene expression? What are the quality control mechanisms utilized by cells to detect, degrade and repair proteins damaged by hypertonic stress? What are the mechanisms by which acclimation to hypertonic stress suppresses hypertonicity induced protein damage? We will utilize a combination of cell biological, molecular and biochemical approaches to provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. We will also exploit the genetic tractability of C. elegans and begin to define the signals and signaling pathways that regulate expression of genes required for survival in hypertonic environments. Our work will provide novel insights into cellular osmosensing and signal transduction and into the mechanisms that protect hypertonically stressed cells from protein damage and associated injury and death. Detailed understanding of hypertonicity induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases. Public Health Relevance: Cellular osmotic homeostasis is a fundamental requirement for life. Studies described in this application will define the signals and signaling pathways that regulate expression of genes required for survival of cells in hypertonic environments and will provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. Detailed understanding of osmotic stress induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases.

DESCRIPTION (provided by applicant): This application proposes to create an innovative Center of Biomedical Research Excellence (COBRE) focused on the Comparative Biology of Tissue Repair, Regeneration and Aging. Tissue regeneration in mammals is extremely limited. However, robust regeneration is the norm for countless lower vertebrates and invertebrates. In addition, nature has endowed diverse animals with remarkable longevity and robust tissue repair mechanisms that slow aging-induced degenerative processes. The COBRE research theme uniquely focuses on using diverse non-mammalian and mammalian model organisms together with comparative biology approaches to address several overarching and fundamental questions in the field of regenerative biology that can only be addressed at the level of the whole organism. This research focus represents a cornerstone of the long term strategic scientific Vision of the Mount Desert Island Biological Laboratory (MDIBL), and builds on the MDIBL and Maine INBRE success stories and on MDIBL's longstanding and increasingly important expertise in comparative biomedical research. The four proposed COBRE projects have extensive points of intersection and interaction and focus on defining mechanisms of limb and sensory axon regeneration in zebrafish, the impact of aging and somatic mutation load on mouse hematopoietic stem cell tissue homeostasis, and the effects of aging and stress on zebrafish tissue regeneration. Proposed scientific cores will provide essential services and resources to Project Leaders and will be valuable to a larger scientific community. The Comparative Animal Models Core will provide COBRE investigators with resources necessary to utilize diverse non-mammalian animal models in regenerative and aging biology research. The Comparative Functional Genomics Core will provide data management and analysis expertise to COBRE investigators. It will also develop a novel web-based resource that integrates data generated by COBRE investigators with published data sets in order to provide a systems-level view of regeneration that will inform new hypotheses about how genes regulate regenerative responses in diverse organisms. The Administrative Core will provide administrative, scientific, and fiscal leadership; implement a career development plan for junior faculty that facilitates their transition to independence; and implement an evaluation strategy to assess the progress of the COBRE in accomplishing its goals. The proposed COBRE will greatly enhance MDIBL's growth and development, which in turn will contribute to the continued growth and enhancement of the biomedical research infrastructure in Maine.

Description and Justification of Renovation: Current Server Room Conditions The goal of the proposed COBRE funded alterations and renovations is to enlarge the Mount Desert Island Biological Laboratory's server room to support the data needs of COBRE Project Leaders and the COBRE program in the Comparative Biology of Tissue Repair, Regeneration, and Aging at the MDIBL. MDIBL requests support to expand and enhance an existing server room in order to provide additional server capacity and proper environmental conditions necessary to meet the objectives of the proposed COBRE program. The existing MDIBL server room (Figure 1, see also Line Drawings) occupies a 213 net square foot portion of a 776 net square foot building at the center of the MDIBL campus. This space was renovated in late 2006 to accommodate MDIBL servers and data storage devices. The building was selected as a server and data center because of its central location and proximity to the main data (internet) utilities entrance onto the MDIBL campus. The facility has met the needs of MDIBL and the Maine INBRE program including providing significant bioinformatics support and data storage of DNA sequence and confocal image data. The center has also supported the server and data needs of the NIEHS funded Comparative Toxicogenomics Database (ES014065

? DESCRIPTION (provided by applicant): CLC Cl- transport proteins are ubiquitous and perform essential and diverse physiological functions. Mutations in human CLC genes and genes encoding their accessory proteins underlie serious diseases including Dent's disease, Bartter syndrome, bone disease, neurodegeneration, deafness and myotonia. CLCs are regulated by phosphorylation, Ca2+, adenosine ligands and accessory proteins. However, little is known about the molecular mechanisms of regulation, which represents a major gap in the field and significant limitation in the development of pharmaceuticals that modulate CLC trafficking and function. Our laboratory, supported by DK51610, pioneered the use of the genetic model organism C. elegans to provide the first detailed molecular understanding of CLC regulatory mechanisms. CLH-3b is a C. elegans CLC-1/2/Ka/Kb anion channel subfamily member that is inactivated by GCK-3-mediated phosphorylation. GCK-3 is an ortholog/homolog of mammalian SPAK and OSR1 kinases, which play central roles in systemic fluid balance and are important targets for pharmaceutical development to treat blood pressure disorders. During the previous funding period, we began defining for the first time structure/function mechanisms of phosphorylation-dependent CLC regulation. The cytoplasmic C-terminus of CLCs includes two conserved CBS domains (CBS1 and CBS2) that dimerize to form a Bateman domain. An inter-CBS linker connects CBS1 and CBS2. The linker is an intrinsically disordered region (IDR) lacking rigid 3D structure. Much of the CLH-3b linker is dispensable for regulation. However, deletion of a 14 amino acid activation domain comprising two regulatory serine residues phosphorylated by GCK-3 inactivates CLH-3b to the same extent as phosphorylation. A newly identified CLC signal transduction domain comprising CBS2 and two membrane a-helices mediates phosphorylation-dependent intraprotein signaling that regulates channel activity. The overarching goal of this renewal application is to define for the first time the regulatory relationships between the CLC inter-CBS linker and the Bateman, signal transduction and membrane domains. Using NMR spectroscopy, patch clamp electrophysiology, biochemical and mutagenesis strategies; we will address three novel questions: Does the activation domain contain phosphorylation-sensitive secondary structure that modulates its regulatory interaction with the Bateman domain? Does activation domain phosphorylation induce regulatory conformational changes in the Bateman domain? Do CLC subunit interface conformational rearrangements mediate the effects of phosphorylation on channel activity? Proposed studies will yield new insights into regulatory structure/function relationships of CLC channels as well as those of CBS domains and IDRs, which are associated with numerous diseases and represent important targets for drug development. Results of our studies will provide an essential foundation for better understanding of CLC associated diseases and for development of pharmaceuticals that specifically target CLC intracellular regulatory domains.

ADMINISTRATIVE CORE PROJECT SUMMARY COBRE Phase I, Comparative Biology of Tissue Repair, Regeneration and Aging, supported the development and growth of the Kathryn W. Davis Center for Regenerative Biology and Medicine (Davis Center). The Davis Center is the major research focus of the MDI Biological Laboratory (MDIBL). COBRE Phase II will support three Project Leaders and two scientific cores, and will continue to support the growth of the Davis Center in order to establish a critical mass of investigators and a self-sustaining research program. The Administrative Core is critical to the management and success of COBRE Phase II. The COBRE PD/PI and Administrative Core Director, Dr. Kevin Strange, has primary responsibility for administering the program and overseeing the development of the COBRE, its faculty and its cores. He is assisted by the Administrative Core Co-Director, Dr. Nadia Rosenthal, and Program Coordinator, Ms. Amy Somes. Decisions regarding budgets, core usage and direction of the COBRE are made by the Director and Co-Director with advice from the External Advisory Committee (EAC) and Internal Advisory Committee (IAC). Leadership and oversight includes coordination and integration of Project Leader research programs with Center resources and activities; establishing and managing the allocation of Center resources; organizing Center activities; organizing EAC and IAC meetings; management of a rigorous faculty career development plan; faculty and program evaluation; faculty recruitment; and interactions with other groups to further COBRE goals. COBRE Phase I supported four early-career Project Leaders and one mid-career Project Leader. Phase I was highly successful both from formative and summative standpoints. All five Project Leaders graduated from Phase I with independent research program grant support and achieved multiple other successes including publication of significant peer-reviewed papers, creation of patented/patentable intellectual property, receipt of foundation and R21 grants, significant peer recognition and, for the mid-career Project Leader, a major and productive change in his laboratory research direction. The success of Phase I is directly attributable to the implementation of a rigorous career development plan that will be utilized in Phase II. Elements of this plan include a rigorous faculty recruitment process, biannual EAC reviews, biannual Project Leader career SWOT analyses and scientific advisor reviews, mandatory grant proposal development and review, peer-to-peer mentoring, and regular group meetings and meetings with the Administrative Core Director and Co-Director. MDIBL?s long-term strategic scientific goal is to build a world-class research program in regenerative biology and medicine. The Davis Center will remain MDIBL?s sole research focus for the foreseeable future and its sustainability is therefore of the highest priority to MDIBL leadership. Sustainability will be achieved by establishing a critical mass of investigators working at the forefront of regenerative and aging biology.