Ratneshwar Lal - US grants

DESCRIPTION: The experiments proposed by the investigator seek a better understanding of the quaternary structure of cardiac gap junction channels. To this end Atomic Force Microscopic (AFM) measurements will be made on hydrated cardiac gap junctions to determine the size, shape, subunit arrangement, subunit number, and general microstructure. These structures of fully formed gap junctions will be compared for reconstituted, purified and intact proteins. Additional measurements of connexon pairs will be compared to those obtained for isolated hemichannels to determine how the pairs are assembled during and prior to cell-cell contact. Finally, the investigator will utilize AFM measurement to begin unravelling the conformational changes which are associated with changes in the state of channel function. Open and closed states of the protein will be ascertained and compared to establish whether gross changes in morphology are associated with the transitions between the different functional states.

DESCRIPTION (provided by applicant): Ion channels and receptors are membrane interface macromolecules that mediate key physiological and pathological activity. Understanding their three-dimensional structure-activity relationship has been a central and yet elusive goal of biophysicists. The nanoscale size and structural complexity, such as an ion channel's subunit architecture and the central permeable pore, their membrane insertion and lipid-protein interactions put major constraints on their structural analysis. High resolution 3D structure of ion channels is being examined primarily with X-ray diffraction and electron microscopy. Ion channel activity is commonly analyzed by patch clamping and fluorescence microscopy. However, integration of these techniques into a combined system for the direct structure-function activity of ion channels is limited. Atomic force microscopy (AFM) provides high resolution structural information for many biological macromolecules, including ion channels and receptors. AFM images surfaces, the primary structural domains where ligands and other gating agents interact and alter channel activity. AFM allows online addition of pharmacologic or (patho-) physiologic stimuli. Its open architecture allows integration with other techniques permitting imaging of channel-stimuli complex, resulting 3D conformations and channel properties such as permeability, conductance, energetics, and mechanics. The main focus of this application is two-fold: a) to have a practical integrated double chamber AFM with the Support Chip, combine them with high sensitive electrical and fluorescence measuring tools and b) to use them to study hypothesis-driven mechanistic questions about the structure-activity relation for two different channels: i) Connexin hemichannels for high resolution and flexibility work, especially its cytoplasmic face, for which very little is known but much is speculated and for molecular permeability and ii) KcsA channels that form nice, high-conductance pH gated channels. Hemichannel will be used for structural and permeability study. KcsA channel will be used to test simultaneous measurement of conductance, the channel surface topography, and the opening of the bundle crossing implicated in channel opening and closing. Specific Aims of the application are: Aim 1: Design Support Chip with single or multiple nanopore(s) and integrate it with a high resolution AFM. Aim 2: Image 3D topography of reconstituted hemichannels and full-length or truncated KcsA channels. Aim 3: Examine the role of channel subunit structural changes in the open/closed functional states. This includes high resolution imaging of the structure while simultaneously measuring permeability, of dyes and large molecules specific to hemichannels, with integrated TIRF microscopy and examining role of various known gating agents, site-directed peptides and antibodies on the permeability. For KcsA channels, both truncated as well as full- length channels will be studied - truncated channel for 3D structure imaging and the full-length channel for imaging bundle crossing to correlate channel gating. While Aims 1 and 3 are interdependent to some extent, Aim 2 is independent and only relies on designing sharp AFM tips with low spring constant and high S/N ratio. PUBLIC HEALTH RELEVANCE: Ion channels and receptors are membrane structures that determine key physiological functions. Abnormality in their structure and activity underlie major human diseases, yet there is a limited understanding of their structure-activity relation. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D structure, yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying human diseases. The long term significance of our study is considerable. The techniques developed in our study, namely combining an equivalent of EM and single channel conductance (patch clamping) and permeability capability will provide a major breakthrough for 3D structure-function study of ion channels and receptors. Our results from study of hemichannels and potassium channel will provide new paradigms to examine the mechanism of cell-extracellular exchanges and their role in modulating tissue homeostasis. Mechanistic information gained from our study will then be used for designing effective prevention and treatment of diseases, including cardiac arrhythmias, neurodegenerative diseases, and cancer.

DESCRIPTION (provided by applicant): Abnormal (or mis-)folding alters protein's 3D conformation from native (soluble form) to non-native (insoluble from) polymorphic amyloid structures. Protein misfolding is linked to neurodegenerative (Alzheimer's, Huntington's, Parkinson's, familial dementia, prion encephalopathies), systemic (type II diabetes, light chain amyloidosis related cancer) and other (cystic fibrosis) diseases. Prevailing view suggests that protein misfolding-induced amyloids result into a gain-of-function and cause pathophysiologic cell response by destabilizing cell ionic homeostasis. Understanding protein misfolding and the resulting 3D conformations that induce pathophysiologic activity have been an important but challenging area of research. Mechanisms underlying amyloid fibril formation and its prevention are being studied extensively although amyloid fibers do not directly appear to cause neurodegenerative diseases; recent studies have shown that only globular amyloids are sufficient to cause pathophysiologic responses. The most direct mechanism of globular oligomer- mediated toxicity would involve their membrane poration as the key initial events. Our prevailing paradigm, therefore, is that protein misfolding diseases result from small globular amyloids forming ion channels to destabilize cell ionic homeostasis. Molecules and other interventions that modulate their channel structure and activity could thus be used for effective therapy. Indeed, amyloidogenic peptides induce ionic conductances in both native cell as well as artificial membranes. Structural study of membrane-bound amyloid complexes has been limited. Our studies have shown that amyloid peptides associated with several diseases form polymorphic ion channels. This continuing proposal primarily focuses on Alzheimer's disease (AD) linked amyloid beta (Ab) peptide that forms toxic channels. We intend to define the 3D structural polymorphism and identify amino acid (AA) epitopes in Ab peptide that can then be used as targets for designing effective therapeutics for AD. We will continue our multidimensional and complementary approaches of AFM imaging, ion conductance recording, molecular dynamics (MD) simulation, and cell Calcium uptake and degeneration to obtain a comprehensive understanding of amyloid ion channels. Our Specific Aims are 1) Image 3D structure of synthetic as well as tissue-derived amyloidogenic peptides and peptides with site-specific amino acid (AA) substitutions reconstituted in lipid membrane; 2) Image open-closed conformations, in response to pharmacologic agents, of channels made of peptides, normal as well as with site-specific amino acid (AA) substitutions, 3) Correlate open-close channel conformations with ion conductance using integrated ion conductance AFM and pharmacologic agents and site-specific AA substitutions, and 4) Examine the cellular effects (e.g., calcium uptake and degeneration) of amyloid peptides. Our study will define specific amyloid structures underlying Alzheimer's and other degenerative pathophysiology and will identify specific structural motif(s) in amyloid ion channels that can then be used for designing pharmacological intervention of therapeutic value.

The principal objective of the Biophysical Imaging Core is to provide Program investigators with a complete range of expertise, training, equipments, and data analysis tools to obtain nano-to-micro scale biophysical information pertaining to the cellular and molecular basis of endothelial barrier permeability. Core D personnel and equipment will allow PPG scientists to image 3D structures, evaluate physical and chemical properties and define perturbant-induced real-time changes in the structures and activity of cells and subcellular constituents, including membranes, cytoskeletal networks and cell-matrix and cell-cell junctions. This Biolmaging Core supports the PPG's five research projects with quantitative microscopy related to atomic force microscopy (AFM), light fluorescence microscopy and TIRF (total internal reflection fluorescence microscopy). It offers access to experienced use of the complete resources of the Center for Nanomedicine that includes state-of-the-art atomic force microscopes integrated with high resolution single photon microscopy and fluorescence microscopy systems for simultaneous multimodal correlative studies. In addition, this Core will also make use of the common resources available at the University of Chicago IBD (Institute for Biophysical Dynamics) Microscope Facility (which includes an excellent electron microscope facility) and the Department of Medicine's Multiphoton Laser Scanning Microscope Facility. Led by Core Leader Ratnesh Lai, PhD, the core has assembled the personnel and laboratory facilities to satisfy a wide range of experimental 3D imaging and mechanobiophysics needs. Core D personnel have professional experience spanning the fields of high resolution imaging and examining physical and chemical properties, including mechanobiophysics with various scanning probe microscopies. Despite relative recent arrival of Core personnel within the University of Chicago, these talented scientists have provided convincing preliminary data for each Project and all five research projects.

DESCRIPTION (provided by applicant): Overall goal of this application is to design and develop nanotechnology to study key nanoscale biostructures - Ion channels and receptors that are essential for all living functions. Changes in their three-dimensional (3D) structure and activity, in response to stimuli related to life style, including drug addiction, trigger severe health abnormalities. Understanding 3D structure-activity relationship of these nano-biostructures has been a central and yet elusive goal. 3D structure is currently examined with time and resource limiting X-ray diffraction and EM. Ion channel activity is analyzed by patch clamping and fluorescence microscopy. However, there is no integrated system for a direct 3D structure-activity study of these nano-biostructures in aqueous buffer. Atomic force microscopy (AFM) provides high resolution structural information, in aqueous medium, for many macromolecular complexes, including channels and receptors. AFM is ideally suited to image the surface topology - the primary structural domain where external stimuli, including drug molecules would normally interact. Open architecture of AFM permits integration of other techniques. An integrated multimodal AFM would allow real-time imaging of channel (or receptor)-stimuli (or perturbants) complex, their physicochemical properties and resulting channel conformations. We propose to design a state-of-the-art double chamber AFM integrated with high resolution imaging and permeability assay tools. As a test of its applications related to NIDA's mission, a potential supporter of this application, we will study two important ion channels: hemichannels and Acetyl choline receptor (AChR) that are intimately related to drug addiction. Their 3D structure and their permeability to ions and signaling molecules, in response to drug addiction-inducing stimuli will be examined. Hemichannels connect a cell to its extracellular milieu or its neighbor cells. They are linked to smoking-induced cell pathology and their presence is modulated by drug addiction-related cell receptors (e.g., dopamine receptor) and stimuli. Specific Aims of the application are: 1. Design a combined AFM, Support silicon Chip with a nanopore, TIRF, Single molecule FRET and voltage-sensitive dye imaging systems. As a test of this system, image 3D structure of hemichannels and AChR. 2a. Examine molecular permeability and ionic conductance, in response to physiological and drug addiction-related stimuli. This includes, a) measuring channel permeability to ions, sensor dyes and signaling molecules and b) examining role of defined gating agents and drug addiction-related perturbations, including smoking condensate, nicotine and ROS on the channel permeability, and 2b. Examine density, distribution and turnover of hemichannels and AChR (a drug addiction related receptor) in single cell plasma membrane in response to drugs (e.g., cocaine, nicotine) and pathological agents. The integrated imaging system developed in this study will be first of its kind and will have far reaching and broader role in defining our understanding of the molecular determinants of drug addiction, their pathological consequences as well as in development of therapeutics for drug addiction and treatment. PUBLIC HEALTH RELEVANCE: Consequences of drug addictions on human health and society at large are considerable, yet there is a limited understanding of the underlying mechanism(s) of the cause and/or deleterious effects of these addictions. Most of the drug addiction stimuli possibly induce their effects through them modulating the structure and activity of ion channels and receptors such as acetyl choline receptor (AChR) and gap junctional hemichannels. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D molecular structure, the 3D conformations;yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying drug addiction and/or their pathological consequences. Advances in nanoscience and technology provide perhaps, the best avenue to explore complex pathological processes that are mediated by nanoscale biostructures, such as ion channels and receptors. Here we intend to implement the most advanced integrated multimodal tools and test their applications on two major classes of ion channels, hemichannels and AChR. Our successful completion of the proposed undertaking will be like combining EM with patch clamping and single molecule imaging that fill the void as well as will provide viable avenues for development of therapeutics for drug addiction and treatment.

DESCRIPTION (provided by applicant): Ion channels and receptors are membrane interface macromolecules that mediate key physiological and pathological activity. Understanding their three-dimensional structure-activity relationship has been a central and yet elusive goal of biophysicists. The nanoscale size and structural complexity, such as an ion channel's subunit architecture and the central permeable pore, their membrane insertion and lipid-protein interactions put major constraints on their structural analysis. High resolution 3D structure of ion channels is being examined primarily with X-ray diffraction and electron microscopy. Ion channel activity is commonly analyzed by patch clamping and fluorescence microscopy. However, integration of these techniques into a combined system for the direct structure-function activity of ion channels is limited. Atomic force microscopy (AFM) provides high resolution structural information for many biological macromolecules, including ion channels and receptors. AFM images surfaces, the primary structural domains where ligands and other gating agents interact and alter channel activity. AFM allows online addition of pharmacologic or (patho-) physiologic stimuli. Its open architecture allows integration with other techniques permitting imaging of channel-stimuli complex, resulting 3D conformations and channel properties such as permeability, conductance, energetics, and mechanics. The main focus of this application is two-fold: a) to have a practical integrated double chamber AFM with the Support Chip, combine them with high sensitive electrical and fluorescence measuring tools and b) to use them to study hypothesis-driven mechanistic questions about the structure-activity relation for two different channels: i) Connexin hemichannels for high resolution and flexibility work, especially its cytoplasmic face, for which very little is known but much is speculated and for molecular permeability and ii) KcsA channels that form nice, high-conductance pH gated channels. Hemichannel will be used for structural and permeability study. KcsA channel will be used to test simultaneous measurement of conductance, the channel surface topography, and the opening of the bundle crossing implicated in channel opening and closing. Specific Aims of the application are: Aim 1: Design Support Chip with single or multiple nanopore(s) and integrate it with a high resolution AFM. Aim 2: Image 3D topography of reconstituted hemichannels and full-length or truncated KcsA channels. Aim 3: Examine the role of channel subunit structural changes in the open/closed functional states. This includes high resolution imaging of the structure while simultaneously measuring permeability, of dyes and large molecules specific to hemichannels, with integrated TIRF microscopy and examining role of various known gating agents, site-directed peptides and antibodies on the permeability. For KcsA channels, both truncated as well as full- length channels will be studied - truncated channel for 3D structure imaging and the full-length channel for imaging bundle crossing to correlate channel gating. While Aims 1 and 3 are interdependent to some extent, Aim 2 is independent and only relies on designing sharp AFM tips with low spring constant and high S/N ratio. PUBLIC HEALTH RELEVANCE: Ion channels and receptors are membrane structures that determine key physiological functions. Abnormality in their structure and activity underlie major human diseases, yet there is a limited understanding of their structure-activity relation. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D structure, yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying human diseases. The long term significance of our study is considerable. The techniques developed in our study, namely combining an equivalent of EM and single channel conductance (patch clamping) and permeability capability will provide a major breakthrough for 3D structure-function study of ion channels and receptors. Our results from study of hemichannels and potassium channel will provide new paradigms to examine the mechanism of cell-extracellular exchanges and their role in modulating tissue homeostasis. Mechanistic information gained from our study will then be used for designing effective prevention and treatment of diseases, including cardiac arrhythmias, neurodegenerative diseases, and cancer.

Project Summary Alzheimer?s disease (AD) causes a progressive loss of memory and cognition. In spite of sustained efforts over several decades, we lack the basic understanding of biophysical, physiological and pathophysiological mechanisms underlying AD pathology. Human cognition is controlled by a complex network of cells that are organized in a 3-dimensional architecture and the underlying neurological activity is heavily dependent upon the controlled and coordinated activity of precisely located membrane macromolecules, including channels and receptors. Indeed, cell membrane interactions of various amyloids, including amyloid beta, alpha-synuclein, FTD43 are primary drivers of AD pathophysiology. To obtain a complete understanding of the cellular behavior, in line with the goals of the AD initiative, technology enabling multi-modal and multi-scale structure-function imaging of live neuronal networks must be created to better understand the integrated neural activities. The structural connectivity and dynamic signal transmission within synaptic networks need to be understood in two fundamental ways: i) structural sub-components, including ion channels and receptors that propagate functional cellular signals and ii) their functional states. Our current understanding of the synaptic structure is limited to electron microscopy (EM) studies in fixed, dehydrated and metal-coated thin sections and which precludes real- time structural changes associated with the synaptic activity and brain function. The functional synaptic activity is currently examined by conventional electrophysiological setup. These studies have yet to elucidate the direct structure-function relationship at either individual synaptic level or at their interconnected clusters. Atomic force microscopy (AFM) allows imaging of native biological specimen in buffer at resolution equivalent to EM imaging and allows real-time introduction of agonists, including chemical, electrical, and mechanical while monitoring neuronal structures. However, current AFM technology is not developed to allow imaging of large areas and is limited to single point imaging and prohibits simultaneous high resolution imaging of connected networks. These connected networks coordinate the behavior of their ion channels to control membrane electrical potentials, producing one of the primary functional outputs of brain cells. The overall goal of this proposal is to develop a novel conducting atomic force microscopy (AFM)-array for simultaneous multi-point imaging with integrated electrical recording. As an initial application, we will study networks in cultured neurons. In order to accomplish our goals, we propose the following specific aims: Aim 1: Develop arrays of conducting AFM capable of imaging biological structures, Aim 2: Image live cultured neurons and synaptic networks, and Aim 3 Image structural and functional changes in response to various oligomeric amyloids, including amyloid beta, alpha-synuclein, FTD43 as well as our newly designed amyloid ion channel blockers which control memory loss in animals. Successful completion of this proposal will result in enabling technology that provides high-resolution imaging and increased understanding of synaptic networks linked with neurodegeneration and mental illness, especially AD.