Min Li - US grants

DESCRIPTION (Adapted from the investigator's application): Organizing multimeric ion channel complexes through specific protein-protein interaction is a fundamental event in channel expression. Such protein-protein interaction is essential in determining the physiological function of the assembled channel complex, including kinetic properties, expression, and subcellular distribution. The proposed experiments in this application address questions critical for understanding subunit assembly: What molecular determinants mediate the specific subunit interactions? How the various forms of interactions may be coupled to channel assembly, expression, and properties? A large number of genes encoding potassium (K+) channels have been isolated. Vast diversity is created by homo- and hetero- multimeric assembly of the pore-forming alpha-subunits and modulatory beta-subunits. The proposed experiments in this application are focused on homo- and hetero- multimeric interactions among the Shaker -like alpha-subunits and two hydrophilic beta-subunits, Kvbeta1 and Kvbeta2. As indicated in our preliminary studies, the interactions to be studied are essential for alpha-alpha, alpha-beta, and beta-beta assembly, which are directly coupled to the K+ channel functions including kinetic properties and expression. Experiments in three areas will be performed: (1) to identify the regions and/or residues in the alpha-subunit that mediate the specific alpha-alpha, and alpha-beta interactions; (2) to identify regions in the beta-subunits that mediate the specific interaction of beta-beta and alpha-beta interactions; (3) to investigate role(s) of alpha-beta interaction in K+ channel expression. These experiments will employ a combination of molecular, biochemical, and electrophysiological methods that have been developed in the laboratory. The protein-protein interaction will be tested by biochemical binding, yeast two hybrid system, and coimmunoprecipitation. The expression of the channel protein will be assessed by immunoblot analysis and whole cell voltage clamp recording. K+ channels play critical roles in a variety of biological processes. Defects in their subunit interaction have been implicated in human diseases, such as cardiac tachyarrhythmia. The molecular understanding of the events central to their expression is essential for developing our knowledge in the area of nerve function both in health and in disease.

DESCRIPTION (Verbatim from applicant's abstract): The physiological significance of subcellularly localized G protein signaling is now increasingly appreciated in a number of systems. However, the molecular players and events involved in positioning and regulating the signaling complexes are poorly understood. The long-term objective of the proposed research is aimed at studying the molecular and biochemical mechanisms that govern the assembly and regulation of signaling complexes. The proposed experiments in this application focus on a Drosophila visual protein known as INAD (inactivation no afterpotential D). This protein is essentially composed of five PDZ domains, a class of protein interaction modules that possess diverse substrate specificity and usually bind to short linear peptides of target proteins. Genetic studies have shown that INAD acts as a scaffold to organize the phototransduction complex by interacting with multiple signaling proteins including rhodopsin, G proteins, phospholipases, post-translational enzymes, and ion channels. The fundament questions concerning scaffold proteins are their mechanistic roles both in assembly of the signal complexes and in activation and deactivation of G protein-coupled signal transmission. This project is aimed at understanding the molecular interaction between INAD and two signaling enzymes: phospholipase C (norpA) and protein phosphatases (rdgC). Both proteins are essential for phototransduction by mediating activation (PLC) and deactivation (RDGC, phosphatase). Interestingly, the spatially-localized activities of these two enzymatically as well as functionally distinct enzymes are coupled with INAD through specific protein-protein interactions. Results from the proposed experiments should provide insights into the mechanistic roles of the INAD-signaling enzyme interactions in Drosophila phototransduction. The G-coupled signaling is critical for phototransduction and many other biological processes. Our knowledge of the molecular principles of these interactions is, therefore, essential for the full understanding of their roles in both health and in disease.

DESCRIPTION(Adapted from applicant's abstract): Phosphorylation of ion channels and receptors is a key mechanism that contributes to the regualtion of neuronal excitability. At the molecular level, it is thought that the targeted protein kinase activity is achieved by specific protein-protein interactions that contribute to the biochemical and subcellular specificity of the posttranslational modification. Recent studies have identified proteins that may mediate specific recruitment of phosphorylation machinery into a channel complex, providing exciting possibilities to understand spatially localized biochemical reactions in neurons. Potassium channels are key players in electrical excitability. The native molecular composition and functional organization of this large family of channels are largely unknown. This application seeks to investigate the molecular and functional organization of Shaker-like potassium channels. A central hypothesis is that posttranslational machinery is recruited by a specific anchor protein into a channel complex. The formation of a macromolecular complex confers the specificity and effectiveness of posttranslational modification on targeted channel proteins. The proposed experiments will utilize a combination of biochemical, molecular and electrophysiological approaches. The proposed study will be centered on a macromolecular complex including channel subunits, adapter proteins and associated protein kinases. The resultant information should provide important insights into the mechanisms of molecular diversity and functional plasticity of ion channel proteins. Potassium channels have been thought to play critical roles in a variety of biological processes. Defects in their subunit assembly and macromolecular organization have been implicated in human diseases, such as cardiac tachyarrhythmia. The molecular understanding of the events that are central to their regulated expression is therefore essential for developing our knowledge in the area of nerve function in health and in diseases.

[unreadable] DESCRIPTION (provided by applicant): The ATP-binding cassette (ABC) superfamily proteins are important functional transporters in both prokaryotes and eukaryotes, playing the primary roles in mediating the entry and exit of a variety of molecules, which is essential for growth and regulation. Increasing evidence has shown that the native forms of these membrane-bound proteins are highly organized macromolecular complexes where the molecular composition and functional stoichiometry confer the native biology of these proteins and are dynamically regulated.The long-term objective of the proposed research is aimed at investigating the molecular organization and function of ABC transporting by focusing on cystic fibrosis transmembrane conductance regulator (CFTR). In particular, we will focus on biochemical and functional interactions between the CFTR and its associated proteins. The proposed experiments in this application focus on a group of four previously unknown CFTR-Associated proteins (CAPs) that have been purified on the basis of high affinity association with the CFTR protein. The genes encoding these four CAPs have been isolated in our laboratory. The proposed experiments are designed to address their potential molecular and functional roles in the CFTR activities. Through a combination of proposed biochemical, cellular, molecular, and electrophysiological experiments, we wish to obtain important information about the mechanism by which CAPs interact with CFTR as well as the potential functional roles of CAPs in modulation of the CFTR activity. Cystic Fibrosis (CF) is an autosomal recessive disorder caused primarily by mutations of a membrane channel protein known as cystic fibrosis transmembrane conductance regulator (CFTR). The mutations found in CF patients cause changes of both channel activity and subcellular location of the CFTR protein. Interactions between CFTR and other structural and regulatory proteins are essential for its proper function in human. Thus, the knowledge about these various interactions is essential for the full understanding of human diseases caused by the mutated CFTR

DESCRIPTION (provided by applicant): Potassium channels play a central role in cardiac and neuronal functions. Mutations of the human ether-a-go-go (hERG) potassium channel are causal to long QT syndrome. Many drugs that have been withdrawn from the market are due to the inhibitory effects on potassium channels such as hERG, thereby causing prolongation of the QT interval, which ultimately could lead to cardiac sudden death. The failure to express a membrane receptor on a cell surface represents one of the most common and well known mechanisms of action that are caused by genetic mutations in human diseases, including the Long QT syndrome. Although the sequence motifs that dominantly localize protein to intracellular compartments (e.g., ER retention or localization) have been studied extensively, little is known about either motifs or machinery that zipcode the plasma membrane expression of surface receptors. The proposed research is focused on the biogenesis of potassium channel using a combination of molecular, biochemical, and electrophysiological approaches. This proposal is aimed at addressing fundamental questions concerning potassium channel biogenesis. By achieving the proposed goals, much will be learned about the forward trafficking process. This information may provide a general principle concerning functional expression of membrane proteins, and important insights into the molecular basis of human diseases.

[unreadable] DESCRIPTION (provided by applicant): In this proposal we optimize an influenza neutralization assay for easy identification of a broad spectrum of antiviral compounds. We have modified the 'gold-standard' neutralization assay so that it can be used in high-throughput screening of chemical libraries. Wells that contain a monolayer of cells are exposed to a small amount of influenza in the absence or presence of compounds that are being screened. Viral strains A/PR/8/34 (H1N1) and B/HK/73 will be used following BSL2 practices to develop and optimize this assay. We have modified the neutralization assay and reduced the time needed to complete analysis by measuring viral neuraminidase (NA) activity to quantitate the amount of virus present in the well. NA activity is measured within 16 hr of assay start after the addition of a small NA substrate that has a fluorescent product. This assay is therefore fast and sensitive. It has additional advantages over assays that have a single biochemical target: (1) it is based on a well-established neutralization assay that is routinely used as a gold standard for titration of antisera; (2) it can identify compounds that inhibit virus replication at any point of the life cycle. Both NA and M2 inhibitors are used as controls. It is likely that a broad range of antivirals with different mechanisms of action will be identified using this assay; (3) it is easily adapted to all types and subtypes of influenza viruses as the read out (NA enzyme activity) is not strain specific; (4) it is sensitive, with accurate measurement of the inhibitory dose of a compound; (5) it is reproducible - obtaining consistent results within assay replicates as well as assay repeats; (6) it is adaptable to automated liquid handling and screening systems. We will optimize the assay in 96 and 384-well plate formats, validate the assay using automated liquid handling and screening systems and establish the parameters required to identify compounds that specifically inhibit influenza virus replication. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Electrical excitability is primarily mediated by ion channel proteins which selectively conduct different ions. Regulation of ion channels by enzymes is central to confer their biological diversity and respond to physiological changes. The ability to sense both electrical signals and chemical signals endows an ion channel critical roles in diverse biological systems. The large conductance calcium-activated (BK) potassium channels are gated both by membrane potential voltage and by cytoplasmic calcium concentration. Because action potential is central to membrane excitability and the intracellular calcium concentration is coupled with a wide variety of biological processes, the functional roles of BK channels are accordingly diverse and of great significance. One important mechanism of coding functional diversity is through alternative splicing of pore-forming subunit, known as alpha subunit. The current understanding of BK structure and function is, almost exclusively, based on studies of one type of C-terminal splice variants known as BK_ERL. The direct evidence linking native BK channel properties and the corresponding molecular isoforms of BK subunits is not yet adequate to fully establish that the ERL isoform is the most important form for the in vivo function. Another C-terminal splice variant, BK_DEC, has additional 61 aa distinct from commonly studied ERL form. We now show this region is densely packed with functional motifs and represents an enzymatic assembly domain recruiting a number of enzymes and regulatory factors. This research proposal is aimed at investigating newly identified enzymatic complexes organized by BKJDEC. The specific aims include biochemical, cell biological and functional characterization of these newly identified protein complexes. BK channels are critical for a variety of biological processes ranging from hormone secretion to control of neuronal firing properties. Drugs have been developed to regulate these channels'activity to treat human diseases such as stroke, epilepsy and other neurological disorders. Thus, understanding of the function and physiology of different BK subtypes is of therapeutics importance.

[unreadable] DESCRIPTION (provided by applicant): Many biological processes, especially those in the nervous systems that occur on the order of milliseconds, take place with rapid temporal scales. Identification of bioactive compounds or nucleic acids to probe mechanistic processes and development of leads for treating diseases are vital aspects to advancing and expanding research at Johns Hopkins. These projects as well as other biological signaling processes often require specialized instrumentation, expensive but necessary reagents and unique technical expertise. Such prerequisites prohibit individual labs from tackling important problems in these areas. To surmount this barrier, we propose to acquire a fast and high throughput fluorescence kinetics reader system. The shared instrument will be incorporated into already operating high throughput compound screen facility, ChemCORE. The combination of the to-be-acquired instrumentation with existing expertise provides both resources and services that are difficult or impractical for individual laboratories to accomplish on their own. The use of this instrument will leverage the existing robotics capability and chemical libraries at Hopkins and will significantly enhance on-going research programs and stimulate new directions of research as outlined in the proposed core projects by investigators from different departments and campuses. The new experimental opportunities offered by the to-be-acquired instrument complement, but do not duplicate, existing University Core facilities. The core project leaders have unusually strong histories leading research programs and productive collaborations. Their projects as detailed in the proposal address conceptually or clinically important questions in different areas of biomedical sciences that interface basic research and translational applications. Therefore, the capital investment of this instrumentation capability within ChemCORE both enables and expands collaborative research of NIH-funded projects. The combined capability of high throughput compound screens against disease targets and the broadening accessibility of high-end instrument to individual laboratories will facilitate clinical intervention of human diseases. PHS 398/2590 (Rev. 05/01 [unreadable] [unreadable] [unreadable]

Core Functions: 1. Develop and implement management plan to achieve center's production goals 2. Establish coherent and productive personnel teams with results-oriented project management guidelines 3. Maintain instrumentation with productivity and flexibility to accommodate different varieties of channel targets 4. Perform effective outreach to attract high value targets from productive groups 5. Participate in MLPCN management teams to facilitate target selection, establish best practices and support MLPCN goals 6. Identify and rectify potential limitation and execute contingency plans.

DESCRIPTION (provided by applicant): Ion channels and transporters are membrane proteins selectively permeable to ions, small olecular nutrients and metabolites. These proteins are critical to a variety of biological processes and represent a large class of therapeutic targets. Bioactive small molecules are a key source for identification of life-altering Pharmaceuticals and life science discovery probes. Different from many protein classes, ion channels and transporters often require special instruments and unique experimental expertise in order to perform analyses. Thus, carrying out large-scale compound library screens for ion channels has posed a considerable challenge. Johns Hopkins ChemCORE is a fully operational, high throughput compound screening facility with a strong combination of scientific excellence, operational productivity, and ion channel expertise. Hence, the existing operation at Hopkins is ideally suited to contribute the production phase of the Molecular Libraries Probe Production Centers Network (MLPCN). We therefore propose to form the Johns Hopkins Ion Channel Center (JHICC) - "Specialized Screening Center" JHICC will meet the following specific aims: (1) to screen large diverse molecular libraries of 300,000 or more compounds against each of thefive important ion channel/transporter targets assigned by NIH, (2) to actively collaborate with chemists in other network centers to maximize probe development and production, (3) to disseminate the resulting information to the public domain (PubChem), and (4) to perform Center-driven research projects with significant impact on molecular probe discovery and development. By maximizing its quality and productivity and by adhering to MLPCN governance mechanisms, JHICC will be a responsible and highly productive member of the MLPCN and NIH Roadmap Initiative.

D3.2. MAINTAIN A ROBUST INFORMATICS ENVIRONMENT SUPPORTING JHICC ACTIVITIES The major responsible task of the Informatics Core is to handle the life cycle of an entire screening project from the beginning of assay transfer to the endpoint of PubChem update (and final data warehousing). Specific tasks include data acquisition and storage, data analyses and management, PubChem publishing and various distribution functions with NIH, collaborating centers and assay providers. Core Function 1. Register and update the NIH compound collection at JHICC site 2. Maintain a robust informatics environment supporting JHICC activities 3. Perform timely tasks of PubChem deposition

Core Functions: 1. Prepare compounds provided by the NIH 2. Maintain and reconfigure HTS facility with an optimal throughput 3. Perform HTS for different channel targets with either ICR12000 or FDSS-enabled formats 4. Produce data with high quality HTS criteria while containing costs 5. Perform initial validation and counter-screen 6. Perform secondary validation (via a subcontract with Aviva Biosciences) 7. Perform QA and QC.

Assay Development, Adaptation and Implementation (DAI)Core Core Functions 1. Evaluate the suitability of JHICC capability in performing assays assigned by NIH and MLPCN 2. Manage the assay transfer 3. Determine milestones, schedule and deliverables to formulate compound probe development plan (CPDP) 4. Adapt and optimize assays transferred from assay providers 5. Determine and develop secondary assays 6. Prepare and finalize assessment of HTS-readiness 7. Maintain a steady supply of HTS-feasible or HTS-ready assays in pipeline.

Cardiac Toxicity; Cardiotoxicity; Cells; Detection; Development; Evaluation; Ion Channel; Ionic Channels; Label; Membrane Channels; Molecular Bank; NIH; National Institutes of Health; National Institutes of Health (U.S.); Programs (PT); Programs [Publication Type]; Receptor Protein; Research; Research Activity; Services; Sight; United States National Institutes of Health; Vision; base; programs; receptor; technology development

Project Abstract:
The purpose of this project is to build and empirically test a comprehensive approach for developing instructionally sensitive assessments that can provide a better picture of the extent that science instruction impacts student achievement. The project will develop and test an assessment approach that can improve the way that assessments are conceptualized and used for evaluation studies and/or accountability purposes. The large-scale, standardized, assessments students take may not be directly tied to the curriculum they are studying. This situation sets up a tension between the knowledge and competencies students are able to demonstrate on a particular assessment and those they may have which the assessment does not in fact probe. The goal of the project is to address this tension. The project will advance knowledge about validity that is critical to the evaluation of the effectiveness of instruction or instructional programs; it will expand the strategies available for developing and evaluating instructionally sensitive assessments; and it will provide evidence about the importance of implementing assessments varying in instructional sensitivity for accountability or program effectiveness purposes. The proposed research activities will guide future evaluation research and assessment development. The project aims to improve the science assessment practices in the participating school districts, mostly serving disadvantaged student populations. The project will provide training to participating teachers on how to develop and evaluate instructionally sensitive assessments, introduce strategies that can be used in their daily practice to judge the quality of the assessments and link assessments to instruction, and disseminate the approach and relevant materials to teachers in non-participating schools or school districts.

DESCRIPTION (provided by applicant): Voltage-gated potassium channels are critical for neuronal function. There are estimated 160 genes encoding different but highly homologous potassium channels in humans. Because of the considerable sequence homology, gene-specific channel modulators are very rare but they are sought-after reagents both for investigating channel function and developing therapeutics. The KCNQ (or Kv7) channel family includes five members: KCNQ1 to KCNQ5. Evidence from patient studies and animal models has shown that a small change in KCNQ expression by as little as 25% can cause disease conditions such as epilepsy. Therefore, chemical probes would be powerful analytical tools to investigate the structure and function of voltage-gated potassium channels, and in the case of KCNQ, these compounds may be very valuable for therapeutic development. To perform a large-scale compound screen, KCNQ channel cell lines have been generated and an HTS-ready protocol has been developed and optimized. The present application seeks to conduct a large scale compound screen using the developed assay. The specific aims of the proposal are: 1. To effectively work with the NIH-assigned MLPCN center to perform a large compound library (>100,000 compounds) and validate lead compounds that specifically ACTIVATE the heteromultimeric KCNQ2/3 potassium channel 2. To conduct the secondary assays and counter screen against KCNQ1, 4 and 5. 3. To perform initial characterization to allow for selection of potent lead compounds for detail functional analyses. The success of this project will lead to identification of novel compounds that are useful for developing therapeutics and investigating M-current and KCNQ ion channel function. PUBLIC HEALTH RELEVANCE: Small molecules that regulate ion flux are important tools to develop drugs to treat brain and heart diseases. They are also useful research probes to understand structure and function of ion channel proteins which mediate membrane ionic flux. This proposal is aimed at carrying out a screen of large compound library in an effort of finding novel chemical modulators for ion channels.

DESCRIPTION (provided by applicant): Ion channels are membrane proteins, found in both excitable cells and non-excitable cells. They selectively conduct ions across cellular membranes and play a critical role in cellular physiology, including electrical and cellular signaling, ion homeostasis, and hormone secretion. The objective of this proposal is to find potent and specific small molecule chemical probes for TMEM16A that encodes the calcium-activated chloride channel (CaCC). Abnormality of CaCC is thought to be causal to diseases, such as cystic fibrosis, asthma, chronic bronchitis, and hypertension. Its role is implicated in numerous physiological processes including cardiac and neuronal excitation, sensory transduction, trans-epithelial secretion, smooth muscle contraction, and fertilization. In collaboration with Dr. Lily Jan's laboratory, which first cloned the TMEM16A gene, we have developed several cell lines and a fluorescence-based iodide surrogate flux assay. The protocol for high-throughput screening is being tested for the CaCC target TMEM16A. This proposal outlines a plan to conduct a >300,000-compound screen using the TMEM16A expressing cell line with a genetically encoded Cl-/I- biosensor to search potent inhibitor/activator probes for CaCC. The active compounds will then be evaluated by automated patch-clamp recording. In collaboration with Dr. Lily Jan at University of California at San Francisco, our future plan includes testing of the isolated compounds in native preparations, such as neurons and epithelium, respectively. These active compounds, with further efforts in pharmacology and medicinal chemistry, may be exploited either as tools or as lead compounds for developing therapeutic remedies to the CaCC-associated diseases. PUBLIC HEALTH RELEVANCE: The recent discovery of the calcium-activated chloride channel (CaCC) has paved a new path to identify small molecule probes. Abnormality of CaCC activity is thought to be causal to several major diseases, such as cystic fibrosis, asthma, chronic bronchitis, and hypertension. Its role is implicated in numerous physiological processes including cardiac and neuronal excitation, sensory transduction, trans-epithelial secretion, smooth muscle contraction, and fertilization. Therefore, specific modulation compounds are useful to aid therapeutic development.

Abstract

This collaborative project involving the University of Colorado at Denver and the University of Washington at Seattle in conjunction with Facet Innovations, will build a framework for addressing the use of contextualized items in the assessment of STEM learning. The primary goal is to systematically investigate the effects of characteristics of contextualized items on student performance to strengthen practices in science assessments, ensure fairness in science testing, and increase support for both assessment and instructional purposes. Test items with contexts are called contextualized items which include supplemental information that precedes or follows a test item question. Such information may include a description of a lab setup, a natural phenomenon, or a practical problem often depicted as a scenario, background, vignette, or cover story. The project findings will help to understand how students make sense of contextualized items focusing on complex scientific concepts that they usually encounter in science assessments. Currently, contextualized items are constructed from either conventional wisdom or non-contextualized item writing rules. Such items could mislead students to attend to irrelevant information or interfere with the targeted construct, and, therefore lead to inaccurate inferences about student learning. This project will develop a framework for developing items to help address this problem. Approximately, 70 classroom teachers and 4800 students, in secondary grades, will participate in the study.

The project will offer a theoretical articulation of the characteristics of contextualized items and empirically test the effects on student performance. It seeks to address four gaps in the literature on contextualized items: (1) insufficient knowledge about how to conceptualize construct-relevant contextualized items; (2) lack of research on contextualized items in science; (3) lack of research that systematically studies the characteristics of contexts and evaluates their effects on student performance; and (4) the need for studies that examine differential effects related to subgroups of students to gain a greater clarity of what types of contexts affect whom. The research design and data analyses will be guided by three research questions: (1) what are critical context characteristics that may affect student performance and should therefore be considered when developing science test items? (2) what are context characteristics associated with construct-relevant variance? (3) what context characteristics are associated with differential student performance patterns due to gender, ELL status, and socioeconomic-status variables?

The project will take place over three years through a two phase process. During Phase 1 the project will refine a proposed theoretical framework that will identify the item context characteristics and articulate the item development guidelines. During Phase 2, the goal is to apply the framework by selecting, revising, and developing science items with varying profiles of contexts; conduct field tests of the items; and perform a range of psychometric and statistical procedures with test scores, and qualitative analyses of students' cognitive interview responses and teacher interviews. Items resulting from this process will aim to evoke students' stored knowledge relevant to the content and/or process skills targeted. The project will involve a team of researchers specialized in assessment development and validation, science education, content knowledge, linguists, and expert classroom teachers. The item development approach and items generated from this project will have immediate implications for researchers and practitioners in science education nationally and internationally.