Gail A. Robertson - US grants

The long-term objectives of this work are to understand the functional properties and physiological roles of potassium channels derived from the extended gene family known as eag. This proposal focuses on one member of this family, h-erg, which was isolated from the human hippocampus. In contrast to their outwardly-rectifying relatives in the eag family, h-erg ion channels are inward rectifiers, conducting potassium current into the cell when the membrane is hyperpolarized. Like all inward rectifiers, the voltages at which activation occurs depends on the driving force on potassium ions, as though the potassium ions themselves dislodge an internal blocking particle from the conduction pathway. The gating of h- erg channels is complex, however. Activation of the current by a hyperpolarization requires a depolarizing prepulse. The prepulse voltage apparently regulates the availability of channels for activation during the subsequent hyperpolarization. This process is characterized by a voltage dependence that is similar to that of activation of the outwardly- rectifying channels with which h-erg shares significant structural similarity. Thus, the gating of these channels encompasses properties of both inward and outward rectifiers. The first two specific aims are to derive rates for transitions of each gating process from macroscopic current analysis and to develop a model that accounts for the observed complexities. The third specific aim examines the mechanism of inward rectification, to determine whether it arises from (1) an extrinsic blocking particle, such as Mg ions, (2) a polypeptide blocking mechanism that is intrinsic tot he channel itself, or (3) conformational changes in the pore. The fourth specific aim involves mutagenesis studies which take advantage of the considerable sequence homology between h-erg and other channels in the eag family to map functional differences to discrete domains of the channel polypeptide. There is great potential for clinical advances in basic studies of new types of channels such as the inward rectifiers, which are highly expressed in the heart and nervous system. Ion channels are important targets for therapeutic drugs, and effective drug design requires a detailed understanding of channel structure and function. In addition, since a growing number of diseases have been ascribed to defects in ion channel function, inward rectifier channels represent potential disease loci and targets from gene therapy. With these studies we seek to advance our understanding of the basic properties of h-erg and related channels as the groundwork for progress in health-related applications.

9722378 Robertson Ion channels are proteins that allow the flow of ions across cell membranes. The movement of the charged ions produces electrical activity that is the basis of communication in the brain. The long-range goal of this research is to understand how the Eag family of potassium channels contribute to electrical signaling in the nervous system. Although these channels can be detected biochemically, their functional properties and physiological roles are unknown. The specific aims of the project include a combined electrophysiological and pharmacological approach to characterize currents produced by these channels in brain tissue slice. A simpler "expression system" containing pure populations of these channels will be used to further characterize factors that control the opening and closing, or "gating," of the channels. These research activities involve undergraduates in an integrated, supervised program in which students cooperate in the design, execution and analysis of experiments. They will report their findings at scientific meetings and in manuscripts for publication, thus developing writing and communication skills in a scientific setting. The educational goal of the project is to provide these students the opportunity for scientific discovery in an active learning paradigm not typically available to undergraduates, and to develop the skills and self- confidence necessary to pursue careers in science.

Cardiac excitability is determined by the activity of several membrane currents including IKr, which is responsible for the terminal repolarization of the ventricular action potential. When IKr is disrupted by mutations in the HERG gene or blocked by drugs, life-threatening arrhythmias can result. Expression of HERG in frog oocytes produces currents with the biophysical and pharmacological properties of IKr, suggesting that HERG subunits are key components of the native channel. HERG channels exhibit a rapid inactivation that suppresses the current during depolarization; during repolarization the channels recover from inactivation and pass through the open state prior to closing. Because deactivation is slow, HERG current reaches its maximum during repolarization of the cardiac action potential and helps to ensure that repolarization is complete. The long-range goals of this work are to understand the gating mechanisms that specialize HERG for its physiological role in the heart. The N terminus has recently been found to regulate deactivation rate, and naturally-occuring alternative N termini lead to functional diversity among channels encoded by HERG and Merg1, the HERG ortholog in mouse. The modulation of deactivation by the N terminus arises from a domain that is spatially separable from a second domain that promotes C-type inactivation. Both of these functions are disrupted by modifications of the S4-S5 loop, which phenocopies a deletion of the N terminus. The aims of this proposal are to characterize the mechanism by which the N terminus regulates deactivation, to identify modifiers of gating using the yeast two-hybrid system and to characterize the electrophysiological properties and regional distribution within the heart of channels encoded by HERG2 and HERG3, two newly-identified genes within the HERG subfamily. If the aims of this proposal are achieved a more detailed picture of HERG gating will emerge along with a greater understanding of the physiology of HERG-related channels in the heart.

[unreadable] DESCRIPTION (provided by applicant): The human ether-a-go-go-related gene (HERG) encodes an ion channel subunit underlying IKr, a potassium current required for the normal repolarization of ventricular cells in the human heart. More than 90 inherited mutations in HERG cause Long QT Syndrome (LQTS), a leading cause of sudden cardiac death. Some mutations alter gating, but more disrupt trafficking. Because the subunit composition of HERG is uncertain, and the mechanisms underlying HERG biogenesis, processing and targeting to the membrane are unknown, we carried out a yeast two-hybrid screen to identify proteins that interact with HERG. Using the carboxy terminus as bait to screen a human heart library, we isolated five genes encoding HERG-interacting proteins ("HIPs"). Two of these proteins have been previously identified: Tara, an actin-binding protein, and GM 130, a peripheral membrane protein of the Golgi apparatus. Little is known about the function of either. Tara co-localizes with HERG to a region in rat cardiac myocytes corresponding to the T-tubules, as determined by confocal immunocytochemistry. Consistent with a stabilizing role at the membrane, Tara enhances expression in HERG when co-expressed in Xenopus oocytes. GM 130 specifically localizes to the Golgi, where a prominent HERG signal is also observed. In contrast to Tara, GM130 suppresses HERG signal in oocytes. Deletion mapping in binary yeast two-hybrid assays reveals that the C terminus contains distinct domains with which the HIPs selectively interact. Certain LQT2 (HERG) mutations selectively disrupt interactions with only two of the proteins. Three of the proteins, Tara, H17 and H3, interact with each other, implying that they function as an interactive complex. Of the HIPs, Tara alone interacts with another cardiac ion channel protein, KvLQT1, in binary yeast two-hybrid assays, but none interacts with Shaker. Each HIP represents a potential target for LQTS to the extent that its expression is required for the normal expression or targeting of HERG channels. The long-range goal of this research is to elucidate the basic biological processes that are disrupted by the disease process. The specific aims of this proposal are: (1) to demonstrate that HERG and the HIPs interact in vivo: (2) to extend our immunocytochemical and electrophysiological analyses, tests for specificity and domain mapping; (3) to determine the necessity of HIP interactions for HERG channels by reciprocal analysis of HERG C terminal truncations and selective disruption of HIPs in native tissues and heterologous systems; and (4) to screen unmapped LQTS families for disease mutations in the genes encoding the HIPs.

DESCRIPTION (provided by applicant): hERG ion channels are the target for acquired and inherited long QT syndrome (LQTS), diseases leading to polymorphic ventricular fibrillation and sudden death. Previous work in this lab demonstrated that hERGIa subunits are a major component of channels producing the ventricular repolarizing current lKr, thus explaining the underlying cause of LQTS-2 as a loss or reduction of lKr. Most recently, we have identified another subunit of !Kr channels, hERG1b. Encoded by an alternate transcript of the hERG1 gene, hERG1b is identical to hERG1a except for its amino terminus, which is highly divergent. This proposal explores mechanisms by which the differences in the amino termini specify heteromeric assembly, export from the endoplasmic reticulum and stability within the plasma membrane. In addition, we propose to characterize a mutation found in an unmapped LQTS patient potentially representing the first disease mutation specific to hERG1b. These studies are expected to provide novel insights into mechanisms of potassium channel assembly as well as the molecular mechanisms of cardiac arrhythmias.

DESCRIPTION (provided by applicant): Eag-related channels (EAG, hERG, ELK) all share a highly conserved region bearing homology with cyclic- nucleotide binding domains of a variety of other proteins including cyclic nucleotide-gated channels. However, most evidence indicates this domain has evolved to serve a different, but unknown, function in Eag-related channels. This application introduces a new X-ray crystal structure for the EAG1 cyclic nucleotide homology domain (CNBhD) and proposes experiments directed by structural considerations to probe its function as an allosteric modulator of channel gating. Molecules interacting with this domain and modifying channel behavior will be identified through a layered screening approach. A range of biochemical and functional approaches reflecting complementary strengths of the participating laboratories will be employed. This project is expected to address a long-standing question in the field regarding the structure and function of CNBhD's, and produce reagents that will ultimately help define the functional role of EAG1 channels in the brain and facilitate chemotherapeutic drug development.

DESCRIPTION (provided by applicant): A significant bottleneck in ion channel research today is the need to use manual patch clamp recording to obtain high quality data. Research at the University of Wisconsin on safety assays for drug development, mechanisms of therapeutic drug action and the molecular basis for inherited and acquired arrhythmias would be significantly enhanced with the acquisition of new technology that dramatically increases experimental throughput. Whereas just a few data points can be reliably collected per day at the hands of a skilled manual patch technician, the Nanion Patchliner NPC-16 is an automated patch clamp device that allows the collection of up to 500 data points per day, largely unattended. Capabilities include gigaseal recordings with stable access resistance, temperature control to achieve physiologically-relevant conditions, perfusion of both intra- and extracellular compartments, compatibility with cultured cells, primary cells and bilayers containing purified proteins, and the option of using perforated patch to avoid dialysis of intracellular components. Many of these capabilities cannot be replicated using the manual patch (e.g., simultaneous application to extra- and intracellular compartments) or are extraordinarily difficult (patching at physiological temperatures). The users all have extensive experience with a wide range of electrophysiology approaches including patch clamp, as does the operating technician who will oversee daily operations. A management plan is developed allowing for online registration for equipment use and communication among primary users. It is expected the device will not only enhance the productivity toward grant aims of the five NIH-funded users, but will open opportunities for collaborations and for experiments not previously conceivable with traditional technologies.

Project Summary/Abstract The expression of ion channels underlying the rhythmic beating of the heart must be precisely coordinated to fulfill their physiological roles and protect the heart from arrhythmia. Many aspects of how this task is accomplished remain poorly understood. Our preliminary findings suggest a novel way in which ion channel expression is coordinated. The central hypothesis of this proposal is that a ?micro- translatome? of interacting mRNA species encodes functionally related proteins, such as those encoding the ventricular action potential. These ion channels assemble co-translationally into macromolecular complexes that govern higher-order cardiac excitability. We will test this hypothesis using a range of experimental preparations including human ventricular myocardium, cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CM's), animal models and HEK293 cells. We will probe the co- regulation and interaction of transcripts encoding ventricular ion channels, and determine whether such assemblies predict stable macromolecular protein complexes within cardiomyocytes using RNA immunoprecipitation experiments, protein co-immunoprecipitation, patch- clamp electrophysiology and super-resolution microscopy. Using RNA-seq, we will identify other transcripts in the micro-translatome, including those encoding RNA binding proteins that tether the transcripts together, and test their roles using RNAi. We will test the hypothesis that mechanisms of mRNA processing, such as nonsense- mediated decay and miRNA regulation, coordinately control the components of the action potential micro-translatome to modify the disease state and fulfill normal, physiological roles. These experiments will uncover mechanisms that quantitatively regulate the critical balance of cardiac excitability, the perturbation of which triggers catastrophic ventricular arrhythmias.

SUMMARY There is an urgent need to understand the diverse mechanisms of cardiac arrhythmias that lead to sudden death and stroke, which remain major health concerns and place more than 10 million Americans at risk. The 2017 Gordon Research Conference (GRC) on Cardiac Arrhythmia Mechanisms, ?The New Basics: Model Systems, Emerging Technologies and Precision Medicine? addresses this need by emphasizing the importance of basic science discovery, the power of model systems and the new technologies that ultimately enable patient-specific, mechanistic approaches to disease diagnosis and treatment. The long-term objective of this meeting is to provide a forum for investigators ? junior, mid-level and senior ? to engage in focused discussion on the latest advances in the field of cardiac arrhythmia mechanisms. The meeting fosters the development of trainees by supporting an associated Gordon Research Seminar on Cardiac Arrhythmia Mechanisms at which trainees present their research findings and engage in panel discussions with mentors on matters of career pathways and professional development. The specific aims of the conference are: ? To bring together investigators who study the mechanisms of cardiac arrhythmias from different disciplinary perspectives using state-of-the-art methodologies. In the intense and informal atmosphere of the GRC, such interactions can be transformative to participants? research programs. ? To bridge the gap between basic science and clinical applications by highlighting new discoveries and innovations that are translational while preserving the rigor of basic science and exploiting its potential for creating paradigm shifts. ? To provide the opportunity for lively networking interactions that foster the development of junior investigators as they develop lifelong relationships with each other and with more senior investigators. The GRC provides an unparalleled environment for the development and emergence of the next generation of leaders in our field. ? To highlight neglected areas in our understanding of cardiac arrhythmia mechanisms and to identify and prioritize novel research directions. These goals are closely aligned with the mission of NHLBI. They will be achieved during a five-day intensive meeting in which investigators of different professional levels, disciplines and occupations will present their work, engage in group and individual discussions, and establish relationships and collaborations that will transform current practices and advance our understanding of cardiac arrhythmia mechanisms.

KCNH channels such as EAG and hERG serve important physiological roles in the nervous system and are targets for disease such as epilepsy and cardiac arrhythmia. They are emerging biomarkers for malignancy and proliferation in a wide range of blood cancers and tumors. Unique to this family of channels are highly conserved intracellular domains that have evolved over the millennia to serve unique physiological roles. In the previous project period, the PI and Co-I resolved details about the role of the C-terminal cyclic nucleotide- binding homology domain (CNBhD) in gating, and provided first insights into dynamic behavior of the Per-Arnt- Sim (PAS) domain. We generated new reagents in the form of single-chain fragment (scFv) antibodies, which we showed exerted therapeutic potential with a beneficial ceiling effect that could confer protection against arrhythmia. Here we have developed a new model for dynamic modulation of gating via the interactions of the PAS domain, CNBhD and the C-linker based on recent cryo-EM structures of closed and open channels. We will test hypotheses emerging from the EAG1 cryo-EM structure with calmodulin (CaM) to elucidate the mechanism by which CaM inhibits EAG1 channel function. We will test a new hypothesis for how the PAS-cap modulates channel gating scFv antibodies as tools to monitor state-dependent changes in accessibility and by immobilizing the domain by crosslinking substituted unnatural amino acids. We will count the number of PAS domains in heteromeric hERG channels comprising PAS-containing (1a) and PAS-less (1b) subunits in both heterologous systems and native tissues. To answer this long-standing question of hERG stoichiometry, we will use isoform-specific scFvs in combination with a novel single-molecule technology that can detect individual binding events of antibodies with modest affinities to untagged channel subunits and is equally applicable to native tissues. The broad range of biochemical, biophysical and functional approaches reflect highly complementary strengths of the participating laboratories. Given the importance of the KCNH family of channels to so many physiological and disease processes, the advances expected from the work here, made possible by recent cutting-edge conceptual and technical developments, will have broad implications.

? DESCRIPTION (provided by applicant): This application requests continuing support for Years 16-20 for the Training Program in Translational Cardiovascular Science (TPTCS) within the School of Medicine and Public Health Cardiovascular Research Center (CVRC) at the University of Wisconsin-Madison. The CVRC is a campus-wide interdisciplinary research center with faculty and research members from 31 departments in seven schools and colleges. All are dedicated to developing programs in basic research, clinical investigation, diagnosis and therapy, and/or public education concerning the basis for cardiovascular diseases. The TPTCS training program provides basic research training for postdoctoral MDs and PhDs (four positions) as well as predoctoral trainees (five positions) with the goal of training scientists wh will be capable of thinking and working from molecule to bedside. The practical immediate aims are to attract and train clinicians in basic research, and to attract and train graduate and postdoctoral (PhD) students in clinically motivated basic science. This application requests support for research training of physicians as part of their resident and fellow training. The training program also funds predoctoral and postdoctoral trainees in translational cardiovascular sciences. The 25 trainers in this program have been selected from members of the CVRC for the clinical relevance of their science in the focus areas (contractility/heart failure, ion channels/arrhythmias and vascular biology/atherosclerosis), their training records, and the overall robustness and activity of their peer-reviewed research programs. Training takes place at the University of Wisconsin-Madison and exploits the strong institutional support and environment for such training, as well as the established working relationships between clinical and basic science departments and faculty both within the School of Medicine and Public Health and the University at large. Trainers and trainees all participate in core activities that define te program as a cohesive entity. The scientists and physician scientists trained in this translational cardiovascular science program are in unique positions to apply basic scientific knowledge to benefit patients with cardiovascular disease, the foremost cause of mortality among Americans. (End of Abstract)