Barbara E. Ehrlich - US grants

In this project two different calcium (Ca) channels from paramecia will be studied after they have been incorporated into planar lipid bilayers. The long term goals of this project are to understand at the molecular level how Ca channels work, how cells regulate the entry of Ca across the cell membrane via Ca channels, and how channels and channel regulation control cell function. Paramecia are the most primitive organism known to have voltage-dependent Ca channels. While paramecia have neither a cardiovascular nor a nervous system, its Ca channels seem to be similar to one class of Ca channels found in mammalian heart and nerve cells. The other channel of paramecia to be studied in a mechanoreceptor-associated (mechano) channel which is permeable only to divalent cations. This channel may be related to other pressure- and stretch-activated channels found in variety of cells. Paramecia channels were selected for study because paramecia have many features which make it particularly suitable to electrophysiological, biochemical, and genetic manipulations that would be more difficult in a mammalian system. In all the proposed experiments, channel activity will be monitored electrically after they have been incorporated into planar lipid bilayers. The specific experiments planned for the voltage-dependent Ca channel are: 1) to continue the testing of pharmacological agents for this channel with the hopes of finding a biochemical probe for the channel protein and of finding an agent that cross reacts with mammalian channels, 2) to investigate the mechanism of Ca-dependent inactivation, and 3) to identify the functional consequences of Ca channel alterations in mutant paramecia. The specific experiments planned for the mechano channel are 1) to verify that this channel is mechanically activated and 2) to test the generality of an ion permeation model that was developed for a different type of Ca channel.

The overall goal of the conference is to bring together a diverse group of scientists working on all phases of calcium and its role in cell function. In addition to its role as an essential mineral for bone growth, regulation of calcium levels inside cells and the action of calcium on intracellular targets impinges on virtually every primary biological activity in cells from both prokaryotes and eukaryotes. The action of calcium in cells is critical for regulation of cardiac and skeletal muscle function, neuronal communication in the brain, the response of immune cells to infection and in most other critical cellular functions. The field provides the intersection of well established physiology and pathophysiology with new findings at the molecular level that provide insights into calcium and cell function. In addition to the formal sessions, informal workshops will be arranged during the meeting to discuss current questions in calcium research. Poster sessions will be held to allow all attendees to exchange new information and participate in the conference. Speakers will be from various fields including physiology, cell biology, biochemistry, pharmacology, molecular biology and genetics. This meeting is an important mechanism by which scientists funded by government grants exchange information that reduces redundancy in research and increases the speed by which new discoveries are incorporated by other scientists across the calcium field. The topics to be covered are: Structure and function of calcium-binding proteins--these are the calcium sensors that indicate to cells when they have been stimulated; Molecular properties of calcium channels--these proteins regulate entry of calcium into cells that trigger cell excitation; Calcium signaling in the cell and the nucleus--how changes in calcium levels in cells is used to provide information within the cell and how this information is transmitted throughout the cell to regulate cell division and the produc tion of protein products from specific genes; Calcium in human diseases--findings from the studies on cell biology above have enabled muscular dystrophy, stroke, Alzheimer's and other diseases to be better understood at a molecular and cellular level and allows for design of new therapeutics which will be discussed.

9505969 Clapham This is a grant for support of an international symposium on Organellar Ion Channels and Transporters to be held under the auspices of the Society of General Physiologists. The symposium will focus on the biochemistry, molecular biology, structure/function and physiology of ion channels and transporters in intracellular membranes of the endoplasmic reticulum and other organelles. The aim of the symposium is to integrate the physiological, biochemical and genetic approaches to the study of this class of membrane proteins and provide a focus for discussion of this new frontier of membrane biology. The meeting consists of five sessions : 1)endoplasmic reticulum ion channels and transporters, 2)mitochondrial channels and transporters, 3)nuclear channels and transporters, 4) secretory mechanisms and 5)organellar localization of calcium. There will also be poster sessions and a session entitled new ideas, new faces, consisting of shorter talks by young investigators selected from abstracts submitted for the poster session. These proceedings will be published. %%% This meeting covers an area which is relatively recent in the studies on membrane structure and function. Most of these studies have been concerned with the plasma membrane, however this meeting concerns the membranes of the intracellular organelles. Since these proceedings will be published, this information will be available to all interested scientists. ***

Intracellular Calcium (Ca) is a key cellular messenger that triggers a variety of cellular processes. This messenger Ca can come from internal stores, or can enter the cytosol from the extracellular medium. Ca can be released from intracellular stores via at least two types of Ca channel: the inositol 1,4,5-trisphosphate receptor (InsP3R) and the ryanodine receptor (RyR). This proposal addresses the integrative regulation of Ca release via the IsnP3R using biophysical, biochemical, and immunological techniques. We will begin these studies of cellular regulation using the richest known source of IsnP3R, the cerebellum. These brain channels will provide a model for subsequent studies of the regulation of less abundant smooth muscle and cardiac InsP3R. Specific long-term goals of this project are to understand how the InsP3-gated channel functions, how the cell regulates the channel to optimize cellular responses, and how regulation goes awry in pathophysiological situations. In the proposed experiments, we will monitor the activity of native and purified InsP3-gated channels after incorporation into planar lipid bilayers. In the first series of experiments in this proposal, the InsP3R will be studied using biophysical techniques to determine how the receptors are regulated, in particular by cytoplasmic Ca. In the second series of experiments, channel properties of the InsP3R will be studied with emphasis on an investigation into the role of luminal Ca in the Ca- dependent regulation of the channel. The third series of experiments will examine channel components necessary for Ca regulation of channel function by testing the role of associated proteins and utilizing antibodies targeted to portions of the InsP3R thought to be involved in regulation of receptor function by Ca. The many interacting levels of regulation of Ca release, which utilize several different intracellular Ca channels, provide for both positive feedback (amplification) and negative feedback (inhibition) of the intracellular Ca signal. Identification of the molecular mechanisms controlling these channels will assist in the understanding of processes as diverse as muscle contraction and motor learning, and may provide insights into conditions such as hypertension and heart failure, processes that involve abnormalities in the activity of these channels.

In the project, the Virtual Cell will be used to predict a cell's response of the association between FKBP and the rvanodine receptor in calcium signaling (Barbara E. Ehrlich, Yale University, NIH funded): In the project, the Virtual Cell will be used to predict a cell's response to changes in the interaction between FKBP and the ryanodine receptor (RyR). In single channel experiments it is possible to replace the FKBP with a different isoform and/or mutant FKBP, but the same experiment in an intact cell is quite difficult. The experimentally derived single channel behavior and cellular response predicted by the Virtual Cell will allow them to select the appropriate avenues for investigation. FKBP and its homologs have been shown to modulate the gating of RyR. The hypothesis to be studied here is that removal of FKBP from lipid bilayer preparations of RyR will allow the RyR to open at lower Ca2+. The basic data will be collected in the laboratory of the applicant by patch clamp record ing. The Resource will provide imaging data to show in intact cardiac and skeletal muscle cells, the distribution of SR and RyR. These data, in combination with patch clamp results, will be fed into the Virtual Cell to predict the role of FKBP on RyR activity in intact cells. 13816-01A1

This project examines the properties of a new class of intracellular Ca channel formed by polycystin-2. To date two major classes of Ca release channel have been identified: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (InsP3R). Most cells contain both types of channel, but the relative densities vary dramatically. The co-existence of a variety of intracellular channels is not surprising as cells need to respond to diverse stimuli with specific responses. The hypotheses to be tested are: 1) Polycystin-2 is a unique calcium- permeable channel in the endoplasmic reticular membrane. 2) The calcium channel formed by polycystin-2 is regulated by intracellular factors, including magnesium, eicosanoids, cAMP and polycystin-1. 3) specific regions of the protein can be divided into functional domains. These domains are based upon mutations found in polycystin-2 from subsets or individuals affected with autosomal dominant polycystic kidney disease (ADPKD). 4) Changes in channel activity of polycystin-2 will modify intracellular calcium signaling. The preliminary results presented here show for the first time that polycystin-2 makes a novel calcium permeable channel in the endoplasmic membrane. The experiments outlined in this project will investigate the functional properties of polycystin-2 at the single channel level and will correlate the channel properties with cell and organ function. The results to be obtained will identify regulatory factors that may determine the mechanism of action of polycystin-2 at a molecular level and may suggest useful tretments for individuals affected with polycystic kidney disease.

DESCRIPTION (applicant's description) Intracellular calcium (Ca) is a key cellular messenger that triggers a variety of cellular processes. It can enter the cytosol from the extracellular medium or it can be released from intracellular stores via at least two types of channel: the inositol 1,4,5-trisphosphate receptor (InsP3R) and the ryanodine receptor (RyR). The hypothesis of this proposal is that control of the lnsP3R by Ca and lnsP3 allows Ca release from the receptor to be regulated at the subcellular level. This hypothesis will be addressed using biophysical, biochemical and molecular biological techniques. Specific long-term goals of this project are to understand how the lnsP3-gated channel functions, how the cell regulates the channel to optimize cellular responses, and how regulation is altered in pathophysiological Situations. In the proposed experiments, we will investigate the interaction Of Ca and lnsP3 in the regulation of the type I lnsP3R. The Ca dependence of channel activity is a function of the lnsP3 concentration. At low concentrations of lnsP3 Ca can both activate and inhibit channel activity whereas at high concentrations of lnsP3 the inhibition by Ca is decreased. In this proposal the two models that have been put forward to explain this complex interaction between Ca and lnsP3 will be examined. The first model suggests that channel activity depends upon the kinetics and order of binding of lnsP3and Ca. The second model suggests that lnsP3.binding to a recently identified low affinity site of the type I lnsP3R is necessary for sustained channel activity. First, the models will be tested by characterizing the single channel properties of normal and mutant forms of the lnsP3R. Then, the functional consequences of the single channel properties will be investigated in intact cells. Our working hypothesis suggests that components of both models will be important to explain channel function. Identification of the molecular mechanisms controlling these channels will assist in the understanding of processes as diverse as secretion, smooth muscle contraction, and motor learning and may provide insights into conditions such as hypertension and ataxia, processes that involve abnormalities in the activity of this channel.

Bile secretion is one of the principal functions of the liver. In order to maintain bile flow, not only must hepatocytes secrete bile, but this must then be modified and conditioned further by bile duct epithelial cells, or cholangiocytes. Abnormal cholangiocytes function results in cholestasis, which is a cardinal manifestation of liver disease. Cholestatic liver diseases are responsible for 20% of liver transplants in the US, and are the most common cause of liver disease among pediatric transplant patients. In addition, abnormal cholangiocyte function is responsible for the hepatic manifestations of cystic fibrosis, one of the most common inherited diseases. Bile secretion in cholangiocytes is regulated in part by cytosolic Ca2+. In general, cells are regulated both by the pattern of Ca2+ signals over time and by the regions of the cells in which Ca2+ signals occur. However, little is known about temporal or spatial aspects of Ca2+ signaling in cholangiocytes, and nothing is known about how these Ca2+ signals are regulated. Inositol 1,4,5-trisphosphate receptors (InsP3R) mediate Ca2+ signaling in epithelia, and cholangiocytes express all three isoforms of this receptor. The hypothesis of this proposal is that Ca2+ signals in the cholangiocyte are regulated by the subcellular distribution of the InsP3R isoforms. This hypothesis will be investigated through the following specific aims: 1. The function and regulation of the InsP3Rs will be compared at the single channel level. 2. The contribution that each of the receptors plays to Ca2+ signaling in cholangiocytes will be examined in a bile duct cell line modified to express either one or a combination of these receptors. 3. These findings will be related to the organization of Ca2+ signals and secretory function in native cholangiocytes, as determined in isolated microperfused bile duct segments. This work should not only identify the molecular mechanisms responsible for Ca2+ signaling in cholangiocytes, but serve as a model for how the molecular organization of signaling pathways is responsible for regulation of ductular secretion.

This project examines the properties of polycystin-2 (PC2) as an intracellular calcium channel. In this project two classes of potential regulators will be tested where each regulator is part of the complex or cascade. The consequences of disrupting the regulation on the intracellular calcium signaling and on the subsequent downstream signaling will be tested. The results obtained from these experiments will identify regulatory factors that modulate the activity of PC2, will outline the molecular basis for these interactions and how they are regulated, and will suggest downstream targets for the siganling cascade. The hypotheses to be tested are: 1) The interactions between PC1 and PC2 are functional and can be predicted from the molecular properties of PC2. 2) PC2 and the ryanodine receptor (RyR) make a channel complex where PC2 regulates the activity of the RyR which then can modulate global intracellular calcium signaling. 3) Changes in the regulation of the channel complex will modify intracellular calcium signaling, and downstream signaling in intact cell. These changes will have consequences on organs in the intact animal. The preliminary results presented here show that PC2 has several protein partners and that these associated proteins are important for regulating the channel complex. The experiments outlined in this project will investigate the functional properties of PC2 at the single channel level and will correlate the channel properties with cell and organ function. The results to be obtained may determine the mechanism of action of PC2 at a molecular level and may suggest useful treatments for individuals affected with polycystic kidney disease.

This project will focus on how growth factors and the activation of the MARK pathway control intracellular[unreadable] Ca2+ in intact cells and will suggest a molecular basis for the specific roles for nuclear and cytosolic Ca2+[unreadable] signaling and the importance of the regulation of these effects on the growth and function of hepatocytes.[unreadable] Three isoforms of the InsPSR have been identified. The cellular localization of each receptor isoform may be[unreadable] correlated with its functional properties. In hepatocytes the InsPSR type 1 isoform (lnsP3R-1) is cytosolic[unreadable] whereas the type 2 isoform (lnsP3R-2) is found throughout the cell, but is predominantly found in the nucleus[unreadable] and the canalicular region. We hypothesize that in hepatocytes MKP-1 differentially regulates the function[unreadable] and distribution of the InsPSR. In this project the interplay between the InsPSR and MAP kinases will be[unreadable] tested on the activity of single InsPSR channels, the ability of isolated cells to generate transient changes in[unreadable] intracellular Ca2+, and the dynamics of InsPSR distribution. An understanding of these complex interactions[unreadable] is necessary to explain the molecular mechanisms of Ca2+ signaling and thus the regeneraton of liver.[unreadable] The hypotheses to be tested include 1) Does activation of MAPK alter the function of the InsPSR and[unreadable] intracellular Ca2+ signaling? 2) Does MKP-1 inactivation of MAPK alter the function of the InsPSR and[unreadable] intracellular Ca2+ signaling? and 3) Does MAPK phosphorylation modulate the InsPSR isoform distribution[unreadable] within the cell?[unreadable] The preliminary results presented here show for the first time that the InsPSR of hepatocytes are regulated[unreadable] by the MAPK pathway The experiments outlined in this project will investigate the functional of this[unreadable] regulation of the InsPSR at the single channel level and will correlate the channel properties with cell and[unreadable] organ function. The results to be obtained will identify regulatory factors that determine isoform-specific[unreadable] Ca2+ signaling responses, how the cell regulates the channel isoforms to optimize cellular responses, and[unreadable] how this regulation goes awry in pathophysiological situations, such as liver degeneration and regeneration.[unreadable] A long term goal will be to use the molecular information obtained in these studies to suggest useful[unreadable] treatments for individuals affected with liver disease.

DESCRIPTION (provided by applicant): Polycystic kidney disease (PKD) is a systemic hereditary disease characterized by renal and hepatic cysts. There is no known cure for this disease and it results in end-stage renal failure in approximately 50% of affected individuals. Mutations in either the PKD1 or the PKD2 gene, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, are seen in over 95% of PKD cases. An inability of PC1 or PC2 to function normally is highly correlated with cystogenesis in PKD. There are currently, however, serious deficiencies in the molecular-level descriptions of PC1 and PC2, and in understanding how alterations in these primary amino acid sequences affect three-dimensional structure, in vitro function and in vivo phenotypes. Our plan is to use a multi-disciplinary approach to significantly improve the description of PC2. We will conduct studies that fall along a structure-function-phenotype axis that will describe the consequences of PC2 mutation in PKD. In Aim 1 we will concentrate on describing the role Ca2+ binding to the EF-hand motif in the C-terminal cytoplasmic region of PC2. This region is critical for normal PC2 Ca2+ channel function and is often truncated in PKD. We will determine the structure of the C-terminal cytoplasmic region of PC2 in the presence of Ca2+ using NMR techniques. This structure will allow us to rationally design mutations we expect to alter Ca2+ binding and therefore PC2 channel function. We will then investigate the functional relevance of this region to PC2 Ca2+ signaling by conducting single channel measurements of PC2 channels in bilayers and by monitoring intracellular Ca2+ changes after agonist addition. In Aim 2 we will investigate the role of phosphorylation of PC2 at serine 812, a residue located between the EF-hand and coiled-coil domains of PC2 that has been shown to be important for channel regulation and is often disrupted in PKD. We will investigate the effect of phosphorylation mimics on Ca2+ binding, on the global structure of the PC2 C-terminal tail, and on the direct C- terminal tail mediated PC1-PC2 interaction. The functional consequences of phosphorylation will be studied using single channel techniques and imaging of fluorescent Ca2+-sensitive dyes in intact cells. In Aim 3 we will investigate the functional consequences of the interactions described in aims 1 and 2, the roles of Ca2+ binding and phosphorylation of PC2. For wild-type and mutant PC2 we will examine flow induced changes in Ca2+ signals, in cells obtained by transfection and in cells obtained by isolation from transgenic mice. We will generate transgenic mice that express wild-type or mutant PC2 and examine the hallmarks of PKD, the right- left axis and cyst formation. The studies proposed will allow us to correlate atomic-level molecular structures of PC2 with biophysical functional analyses and PKD phenotypes in mice. Or results will significantly enhance our molecular, functional and physiological understanding of PC2 in normal kidney function and the consequences of their dysregulation in PKD. PUBLIC HEALTH RELEVANCE: The project aims to enhance our molecular, functional and physiological understanding of the two proteins most often mutated in polycystic kidney disease, polycystin-1 and polycystin-2. We will determine the structure of a region important for regulation of this complex, will conduct functional studies and will describe the effects of mutations in mice for both polycystin-2. The proposed studies will help us understand the role of polycystin-1 and polycystin-2 in normal kidneys and in those affected by polycystic kidney disease.

Calcium (Ca2+) regulates a wide range of functions in the liver in response to hormonal and nutritional signals. The broad goals of this project are to (1) define how nutrients regulate nuclear and cytosolic Ca2+ signaling through post-translational modifications of the inositol 1,4,5 trisphosphate receptor (lnsP3R), a Ca2+ release channel found in both the endoplasmic reticulum (ER) and nucleoplasmic reticulum (NR) and (2) how the dysregulation of this pathway contributes to metabolic liver disease. Nutrient flux leads to posttranslational modifications of cytoplasmic and nuclear proteins by O-linked P-N-acetylglucosamine (OGlcNAc). This dynamic and reversible modification is emerging as a key nutrient sensor and regulator of cell signaling and metabolic physiology. We recently discovered that the lnsP3R is modified by 0-GlcNAc and that this modification decreases lnsP3R single channel activity and Ca2+ release from ER. Preliminary data generated in this PPG suggest that fatty liver induces stress in the ER as well as the nuclear envelope and NR, which may result in impaired cytosolic and nuclear Ca2+ signaling. Based on these findings, we hypothesize that 0-GlcNAcylation of the lnsP3R is controlled by glucose and free fatty acids, which is translated into regulation of the lnsP3R in distinct subcellular compartments of hepatocytes. These regulatory events are, in turn, involved in the perturbation of Ca2+ signaling by ER/NR stress in nonalcoholic fatty liver disease. We will test this hypothesis through the following specific aims: (1) We will identify the effects of glucose and free fatty acids on 0-GlcNAcylation of the lnsP3R isofomns in the nucleus and cytosol; (2) we will examine whether 0-GlcNAcylation of the lnsP3R alters lnsP3-gated channel activity and subsequent intracellular Ca2+ signaling with nuclear and cytoplasmic specificity; and (3) we will detemnine whether metabolic stress promotes hepatic steatosis by perturbing lnsP3R 0-GlcNAcylation and nuclear Ca2+ signaling. Collectively, these studies will synergize with Projects 1 and 3 to define the role of 0-GlcNAcylation of the lnsP3R in nutrient sensing and the regulation of nuclear Ca2+ signaling in the development of hepatic steatosis.