1988 — 1992 |
Parker, Ian |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Intracellular Messenger Functions of Inositol Phosphates @ University of California Irvine
Inositol phosphates are used as intracellular messengers within a signalling pathway which serves to control many diverse cellular functions, such as neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signalling pathway have been implicated in diseases including tumorigenesis and manic depressive illness, and the relevance of this system to clinical studies will almost certainly grow as we come to understand it more. Great progress has recently been made in the biochemical elucidation of the pathways leading to the formation of several different inositol phosphate compounds. However, the physiological study of the actions of these compounds is less well advanced, and although it is thought that they act primarily by raising intracellular free calcium levels, little is known about the specific actions of many of them, or about the mechanisms by which they regulate calcium fluxes across intracellular and cell surface membranes. The main object of this proposal is to study the messenger functions of inositol phosphates, by using electrophysiological and optical techniques to measure membrane currents and intracellular calcium. Xenopus oocytes will be used as a convenient model cell system, as these cells have a well characterized phosphoinositide signalling system, and their large size facilitates many procedures including voltage-clamp recording and micro-injection. The initial aim is to characterize the abilities of different inositol phosphates to liberate intracellular calcium, to activate influx of calcium across the plasma membrane, and to activate membrane conductances independent of calcium. Subsequently, the properties of inositol phosphateactivated calcium channels will be examined in detail by voltage- and patch-clamp recording of currents across the plasma membrane, and by reconstituting channels from internal membranes into lipid bilayers. This will give information about the kinetics and conductances of single channels, their ionic specificity, modulation by inositol phosphates and other second messengers, and blocking by calcium antagonists and other pharmacological agents. Similar studies will also be made of the calcium-activated chloride channels which mediate the final electrical response to phosphoinositide activation in the oocyte, and of any calcium-independent currents which are found to be modulated by inositol phosphates. Recordings of intracellular calcium will be made to determine whether the oscillatory membrane current response to inositol phosphates arises from an oscillatory liberation of calcium, and the feedback mechanism responsible for this process will be studied. The oocyte will also be used as a translation system for exogenous mRNA, to see whether calcium channels, or other components of the phosphoinositide signalling system, can be functionally expressed by mRNA from brain or salivary gland.
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1 |
1992 — 1994 |
Parker, Ian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Spatial and Temporal Aspects of Insp3 Signalling @ University of California Irvine
Inositol 1,4,5-trisphosphate (InsP3) is used as an intracellular messenger within a signalling pathway that serves to control many diverse cellular functions, including neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signaling have been implicated in disorders including manic depressive illness, tumorigenesis and teratogenesis, and the relevance of this system to clinical studies will certainly grow as we come to understand it more. It is now well established that InsP3 functions principally by causing the liberation of Ca2+ ions sequestered within intracellular stores. However, recent improvements in techniques for monitoring intracellular Ca2+ have revealed great complexities in the patterns of InsP3-mediated Ca2+ liberation. Ca2+ is released as repetitive spikes or oscillations, and waves of Ca2+ release propagate through the cell. Furthermore, individual cells appear to contain many functionally independent Ca2+ stores, that each release their contents in a 'quantal', all-or-none manner. The spatial and temporal aspects of InsP3 signalling are undoubtedly important for the encoding of information by the InsP3-mediated transduction pathway. To study them we will employ Xenopus oocytes as a model system, as these cells have a well characterized InsP3 pathway, and their large size to obtain good spatial and temporal control of intracellular InsP3, and resolution of the resulting elevations in intracellular free Ca2 InsP3 will be formed in the cell by photolysis of a 'caged' precursor, and Ca2+ will be imaged by confocal video microscopy using long-wavelength indicator dyes. 'quantal' subcellular Ca2+ release units, to determine how their properties lead to the generation of repetitive Ca2+ spikes and propagating Ca2+ waves, and to see how information in the spatial and temporal patterns of Ca2+ liberation is encoded as changes in Ca2+-dependent membrane currents. High resolution imaging of Ca2+ evoked by photoreleased InsP3 will allow mapping of the distribution and morphology of subcellular InsP3-sensitive Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and endoplasmic reticulum marker proteins. At the whole cell level, we will also study the polarization of Ca2+ signalling between the two hemispheres of the oocyte. Functional studies of InsP3-evoked Ca2+ release at individual sites will elucidate the nature of the positive feedback that gives rise to regenerative Ca2+ release, and the roles of positive and negative feedback by Ca2+ will then be investigated in the generation of repetitive Ca2+ spikes, and propagating Ca2+ waves. Finally, the complex dependence of Ca2+-activated membrane C1- conductance on intracellular Ca2+ will be determined by simultaneous Ca2+ imaging and voltage-clamp recording.
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1995 — 1998 |
Parker, Ian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Spatial and Temporal Aspects of Insp3 Signaling @ University of California Irvine
Inositol 1,4,5-trisphosphate (InsP3) is utilized by virtually all cells as an intracellular messenger within a signaling pathway controlling many diverse functions including neurotransmitter, hormone and growth factor responses, secretion and muscle contraction. Disruptions of this pathway have been implicated in disorders including manic depressive illness, tumorigenesis and teratogenesis, and the relevance of this system to clinical studies will certainly grow as we come to understand it better. It is well established that InsP3 functions principally by liberating Ca2+ ions sequestered within intracellular stores. Furthermore, advances in techniques for monitoring cytosolic Ca2+ have revealed great complexities in the patterns of its liberation; Ca2+ may be released as local 'puffs', or as repetitive circular and spiral waves. These spatial and temporal aspects of InsP3 signaling are undoubtedly important for determining whether Ca2+ signals remain localized to sub-cellular regions or act globally, for the 'digital' encoding of information as frequency of repetitive waves, and for propagation of signals within and between coupled cells as Ca2+ waves. In the presence of InsP3 the cell cytoplasm acts as an excitable medium, formed from multiple discrete and autonomous sites that release 'quanta' of Ca2 in a regenerative manner in response to dual positive and negative feedback by Ca2+. Ca2+ waves are thus akin to a chemical action potential, and their characteristics will be determined by three factors; the functional properties of release sites, the ability of Ca2+ ions to act as diffusible messengers within and between sites, and the spatial organization of release sites. Our overall goals are to study each of these aspects, with the aim of elucidating how they contribute to the final Ca2+ dynamics evoked by InsP3 signaling. We will use Xenopus oocytes as a convenient and well characterized model cell system, utilizing non- metabolizable InsP3 analogues and photolysis of caged InsP3 to evoke Ca2+ liberation that will then be monitored with high spatial and temporal resolution by video-rate confocal microscopy. Functional studies of release sites will include the roles of Ca2+ feedback in evoking graded and regenerative release, the stochastic triggering of regenerative responses, processes underlying activation and inactivation of release, and variability between release sites. The mobilities and range of action of InsP3 and Ca2+ will be determined in intact cells, and we will study the effects of endogenous and exogenous buffers on Ca2+ diffusion and consequent changes in dose-dependence and dynamics of puffs and waves. High resolution imaging of spontaneous Ca2+ puffs and 'hot spots' evoked by photoreleased InsP3 will allow mapping of the three-dimensional distribution and morphology of Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and e.r. structure. Finally, in collaboration with theoretical groups, these quantitative data will be used to model the effects of Ca2+ mobility and the spatial organization of discrete release sites on the cellular dynamics of InsP3-mediated Ca2+ signaling.
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1999 — 2020 |
Parker, Ian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Elementary Events of Intracellular Calcium Signaling @ University of California-Irvine
DESCRIPTION (provided by applicant): The entry of Ca2+ ions into the cytosol from the extracellular fluid and from endoplasmic reticulum (ER) stores is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as electrical excitability, secretion, proliferation and cell death. Improved optical technology now enables visualization of a hierarchy of Ca2+ signaling events, ranging from openings of single-channel Ca2+-permeable channels ('fundamental' events), concerted openings of clustered channels ('elementary' events) and propagating Ca2+ waves. The localized free [Ca2+] elevations arising through individual and clustered channels serve autonomous signaling functions, and their activity may further be coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release to propagate global cellular Ca2+ waves. Fundamental and elementary events thus form hierarchical building blocks underlying the complex spatiotemporal Ca2+ signals that permit graded and selective regulation of cell functions. Elucidation of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological functioning of the ubiquitous Ca2+ messenger pathway and its involvement in disease. Our overall goal is to elucidate, at the single-channel level, how cells generate the hierarchy of Ca2+ signals and how disruptions in Ca2+ signaling may be involved in disease pathogenesis. We focus on physiological Ca2+ signals generated by the ubiquitous inositol trisphosphate second messenger pathway, and on the pathogenic Ca2+-permeable pores formed by amyloid oligomers implicated in Alzheimer's disease. By utilizing novel biophotonic tools that now enable the optical imaging of calcium flux through individual channels and the localization and tracking of channel proteins with nanometer precision we aim to: (i) Further refine optical and analytical techniques for simultaneously monitoring Ca2+ flux through hundreds of individual channels in the plasma membrane and ER of intact cells. (ii) Elucidate how the activity of individual IP3 receptors (IP3R) at a release site is orchestrated to generate elementary Ca2+ puffs. (iii) Employ superresolution imaging techniques to determine the nanoscopic spatial distribution of IP3R, and how this impacts their functioning. (iv) Simultaneously monitor Ca2+ flux and peptide stoichiometry of individual amyloid pores to study fundamental mechanisms of membrane incorporation, channel gating and ion permeation.
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2009 — 2017 |
Foskett, J. Kevin Mak, Don-On Daniel Parker, Ian Pearson, John E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Multi-Scale Observation and Modeling of Ip3/Ca Signaling @ Los Alamos Nat Secty-Los Alamos Nat Lab
DESCRIPTION (provided by applicant): The overall goal of this project involves a synergistic approach of multi-scale modeling and experimental observation to elucidate the fundamental mechanisms underlying cellular calcium signaling. Cytosolic Ca2+ transients serve as a ubiquitous signaling mechanism that regulates cellular functions as diverse as secretion, contraction and proliferation. Information is encoded by spatio-temporal patterns of cytosolic Ca2+ signals at scales ranging from nanometers and microseconds to millimeters and minutes, involving `phonemes'of Ca2+ constructed hierarchically through the activity of individual channels;multiple channels within clusters;and interactions between clusters. These levels cannot simultaneously be observed by any single experimental technique and the shorter scales are below experimental resolution. We therefore employ a dual, tightly integrated and iterative approach of data-driven mathematical modeling together with experimental measurements involving electrophysiological single-channel recording and high-resolution cellular Ca2+ imaging to elucidate how 'elementary'Ca2+ events involving individual channels and clusters are triggered and coupled to produce global cellular calcium signals. Specific aims are to: (i) characterize the gating and Ca2+ permeation properties of IP3R, and develop a predictive mathematical model to account for its complex regulation by IP3 and Ca2+;(ii) observe and model the stochastic, Ca2+-mediated functional coupling between individual channels within a cluster, and;(iii) determine the mechanisms underlying cluster-cluster interactions that allow for propagation of global signals and the powerful differential modulation of this process by Ca2+ buffers of differing kinetics. We focus on IP3 signaling in a single experimentally-tractable system (human SH- SY5Y neuroblastoma cells), but the experimental and theoretical tools we develop will be widely applicable, and the emergent principles will illuminate fundamental mechanisms of Ca2+ signaling in many cell types. Our group involves five Lead Investigators, with expertise and responsibilities as follows: John Pearson. Los Alamos. Theoretician - provides overall direction and synthesis of data;construction of low-dimensional IP3 receptor model and comprehensive multi-scale cellular models. Kevin Foskett and Daniel Mak U. Penn. Experimentalists - electrophysiological single-channel recording and IP3 receptor/channel modeling. Ian Parker. U.C. Irvine. Experimentalist - cytosolic Ca2+ imaging and modeling. Jianwei Shuai. Xiamen University. Theoretician. Computer modeling of Ca2+ signals. Our results will help elucidate the mechanisms underlying complex calcium signals that regulate the normal functioning of almost all cells in the body, and whose disruption is implicated in diseases as diverse as Alzheimers, bipolar disorder, and heart failure. PUBLIC HEALTH RELEVANCE: This unique integrative approach to discover the fundamental mechanisms by which intracellular Ca2+ signals are generated will fundamentally enhance our understanding of their normal functioning and provide insights into how their disruption affects numerous diseases as varied as pancreatitis and Alzheimer's.
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