Robert S. Kass - US grants

This project is designed to continue to provide a quantitative description of cardiac ionic currents and contractile activity and their modulation by neurohormones and drugs. Several investigations are planned that will provide further information about two important ionic channels in the heart: the calcium channel, a pathway for calcium ion influx during the action potential, and a calcium sensitive channel which carries a transient outward current. The project will combine the most recent procedures for pharmacological separation of ionic currents and for tissue preparation. Particular emphasis will be placed on studying channel properties that might influence the response of the channels to neurohormones or block of the channels by certain drugs. A second aim of this project is to determine the mechanisms of action of an important group of drugs: dihydropyridine calcium channel blockers. Experiments are planned to determine the manneer in which membrane potential modulates blockade of calcium channels by, and binding to receptors of, these drugs. These results will be crucial to our understanding of the modes of action of these compounds in cardiac and other cells, and will help direct the use of these drugs towards specific tissues and/or towards the treatment of specific conduction disorders. Experiments will be carried out using voltage-clamp techniques in preparations obtained from cardiac Purkinje fibers and ventricular muscle. A conventional two microelectrode voltage clamp technique will be used to measure membrane currents in isolated segments of shortened Purkinje fiber cell bundles in some experiments. In other experiments, membrane currents will be measured in enzymatically-dispersed Purkinje fiber and ventricular muscle cells. In the latter experiments, a suction pipette will be used to measure whole cell and single channel currents. Optical techniques will be used to monitor contractile activity and drug binding.

This project is designed to continue to investigate the properties of ion channels in the heart using electrophysiological techniques in combination with chemical probes of the channels. Some of the experiments will focus on one type (L-Type) of cardiac calcium channel and the modulation of this type of channel by 1,4 dihydropyridine (DHP) compounds. This important group; of drugs includes compounds that enhance (agonists) or inhibit (antagonists) Ca+2 influx by modulating channel gating instead of by plugging or unplugging the channel. This part of the proposal is a continuation of work begun in this laboratory during the previous funding period. The proposed experiments will focus on the influence pH on the actions of these compunds, and will include optical measurement to directly monitor the effects of membrane potential on drug binding. In addition, experiments are also planned to provide more information about the nature of the gating changes these drugs cause. Other experiments are planned to study two important potassium channels: the inward rectifier and the delayed rectifier channels. The properties of the instantaneous current/voltage (I/V) relationships of these two K channels will be characterized and compared over a wide range of voltages and in the presence of different permeant ions to provide information about their relative permeabilities. A series of quaternary ammonium molecules will be used to block the channels and provide information about their structures. The results of this set of experiments will generate new information about the channels that regulate action potential duration in the heart, and will be useful in the design of therapeutic compounds targeted to regulate specific stages of cardiac action potential repolarization. The usefulness of these drugs as potential Class IV antiarrhythmic agents is pointed out. Experiments will be carried out in enzymatically-dispersed guinea pig ventricular cells. Membrane currents will be measured with the patch clamp technique arranged in whole cell and single channel configurations. An optical procedure will be used to monitor the fluorescence of a 1,4-DHP Ca channel antagonist in a set of experiments designed to monitor the influence of membrane potential on drug binding.

The research described in this proposal is designed to provide information about the relationship between the structure and function of cardiovascular L-type calcium channels. Functional characteristics of native and recombinant L-type channels will be studied using patch clamp procedures in combination with specific chemical compounds that interact with the channel protein and regulate channel gating. There are four specific aims of the project. The first is to use electrophysiological techniques to identify the location of the high affinity dihydropyridine (DHP) binding site on native heart L-type channels relative to the membrane surface. The second aim is to use an electrophysiological approach to describe the biophysical properties of recombinant channel activity expressed by Chinese Hamster Ovary cells stably transfected with cDNAs for specific heart and smooth muscle calcium channel subunits. The third aim of the project is to investigate the modulation of recombinant channel activity by a quaternary DHP compound to identify the location of the high affinity DHP binding site in homooligomeric alpha1, and if necessary multimeric alpha1,beta channels. The final aim is to investigate the modulation of recombinant channels by neutral DHPs to identify structural components that underlie DHP voltage-dependent regulation of L-channel gating, and to test the hypothesis that structural differences in cardiac and smooth muscle alpha1 subunits contribute to the selective targeting of smooth muscle L-type calcium channels by this important family of drugs. The results of this work will provide insight into the molecular components that constitute the DHP binding site and regulate L-channel gating and into the molecular basis of tissue selectivity of DHP compounds. Because DHP regulation of calcium channel activity is associated with control of channel gating, this information will provide insight into the molecular mechanisms that underlie the opening and closing of these channels. In turn, this information will thus provide a framework to understand how calcium entry into heart cells is regulated in normal and diseased states, and how this regulation can be specifically controlled by therapeutic drugs.

One of the fundamental properties of heart cells is their ability to alter their electrical characteristics in response to neuro-hormonal stimulation. This property allows the heart to adapt its physiological function to the range of activity it must meet. The work in this proposal focuses upon a delayed potassium channel in the sino atrial node and ventricle of the mammalian heart. This channel provides outward current (IK) that contributes to control of action potential duration, and thus changes in it will be critical to maintaining the proper relationship between systole (contraction) and diastole (filling time) as heart rate changes. In addition, because calcium ions enter heart cells during the period of depolarization of the action potential known as the plateau phase, changes in IK will indirectly affect calcium entry by controlling duration. In previous investigations, we have found that IK of the guinea pig ventricle is regulated by two enzyme systems (protein kinase A and C) and that this regulation has unique properties that distinguishes control of ventricular IK from that of the cardiac (L-type) calcium channel. The first aim of the present research proposal is to extend this work from guinea pig ventricle to the Sino Atrial (SA) node by characterizing the voltage dependence, channel selectivity, and single channel properties of the dominant SA node delayed rectifier in the guinea pig. The second aim is to test the hypothesis that IK channels are regulated differently by neurohormones in the ventricle and SA node. The third aim of the project is to characterize the functional properties of recombinant delayed rectifier K channels expressed in mammalian cells that have been transfected with the gene that encodes a unique, slowly activating potassium channel, minK. These experiments will use functional properties of the recombinant channels to provide further evidence identifying the protein underlying native IK channels. Then experiments will be designed to compare neurohormonal regulation of the recombinant and native channels and then to identify the molecular sites of action that underlie channel regulation. The results of this work will be of interest to the field of cardiac electrophysiology and to the more general area of neuromodulation of ion channel proteins.

DESCRIPTION (adapted from abstract): This proposal focuses on elucidation of the molecular, pharmacological and regulatory properties of an important K+ channel which is believed to be an essential repolarizing current during the cardiac action potential. As such, this particular delayed rectifier K+ channel, IKs, represents an important target site for several new class III antiarrhythmic agents. The applicant proposes to test the hypothesis that alterations in the genetic message encoding this channel may be directly responsible for at least one type of potentially lethal idiosyncratic drug response to class III agents observed in a small percentage of otherwise healthy patients. The specific aims of the proposal include: (1) to measure and compare the biophysical properties of native and recombinant forms of IKs to determine whether or not the minK gene encodes an ion channel protein or instead represents a regulator or cofactor of an unidentified protein responsible for IKs in native cardiac cells and heterologous expression systems, (2) to test the hypothesis that a single amino acid mutation in minK (A112G) recently discovered by the applicant to be a polymorphism of the human minK gene, changes the sensitivity of expressed minK channels to the class III antiarrhythmic drug, NE-10064, by identifiable changes in expressed channel structure, and (3) to investigate the expression of native and recombinant forms of IKs in wild type and genetically altered murine embryonic cells and stem cell derived cardiocytes to study the developmental regulation of functional minK channels in mammalian heart. This last specific aim will test the hypotheses that expression of functional minK channels in the developing heart is regulated by the beta-adrenergic signaling cascade, and that the presence of message for minK alone in early stage murine embryonic cells is insufficient to cause expression of functional minK channels. The results of these studies should provide the most detailed description to date of the molecular and genetic properties of IKs and its relevance to human cardiovascular pharmacology and disease.

[The experiments that are planned in this proposal are designed to provide insight into the molecular processes underlying 1,4 dihydropyridine (DHP) Ca2+ channel antagonist regulation of cardiovascular L-type Ca2 channel function with a particular emphasis on the distinct modulatory properties of ionized 1,4-DHP derivatives. Recent work in other laboratories has identified specific residues on the sixth membrane spanning segment of domain IV (IVS6) of the L-type Ca2+ channel alpha1c subunit that are key to modulatory actions of neutral DHP compounds, and has shown that Ca2+ binding to physically distinct pore region glutamate residues in each alpha1c domain allosterically modifies neutral drug interactions in this laboratory, studies of native cardiac L-type channels have revealed unique functional properties that distinguish charged from neutral DHP derivatives, and preliminary data summarized in this application suggest that these differences in activity are not simply due to restrictions in access to a common DHP receptor site. The purpose of the work proposed in the present application is to determine the structural basis of charged DHP actions and to use a combination of neutral and charged drugs to distinguish between allosteric and direct ionic interactions. The planned experiments will employ custom-synthesized 1,4 DHP derivatives in which a test head group (charged or neutral) is separated from an active DHP moiety by a hydrocarbon spacer chain of variable length in combination with path clamp studies of recombinant wild type (WT) and mutant-type Ca2+ channels to study the interrelationship between channel protein and modulating drug structure and function. There are three specific aims of this proposal. The first is to study the modulation of recombinant WT L- type Ca2+ channels by charged DHPs of variable spacer chain lengths in order to define the role of spacer chain length in changed drug interactions and to establish the background necessary for structural studies of aims 2 and 3. The second aim is to study in detail the calcium-dependence of changed and neutral DHP modulation of recombinant Ca2+ channels and to distinguish between high and low affinity Ca2+- dependent interactions. The third aim of the project is to use site- directed mutagenesis of pore region glutamates and specific residues in IVS6 of the alpha 1c L-channel subunit to determine the structural basis of charged DHP interactions. This work will provide new insight into the molecular architecture of functionally key residues of the calcium channel alpha1c subunit, and mechanistic data that will account, for the first time, for differences between the modes of action of changed and neutral DHP derivatives which most likely underlie unique therapeutic advantages of long-lasting tertiary DHP calcium channel antagonists.]

The overall goal of the research proposed in this application is to understand the molecular basis of cardiac arrhythmias caused, at least in part, by inherited mutations of the SCN5A gene, and to determine novel gene-targeted therapeutic strategies to treat them. The central hypothesis is that one step in the genesis of these arrhythmias is the perturbation of membrane electrical activity caused by alteration in the biophysical properties of the SCN5A gene product, the principal cardiac Na+ channel alpha subunit, by diseased-linked mutations, but that similar functional perturbations may be linked to distinct clinical disorders. Altered ion channel properties may also confer unique pharmacological properties upon the encoded ion channels making them unique targets for therapeutic intervention but, perhaps less effective in unmasking distinct inherited syndromes. We will focus on identified SCN5A mutations linked to the long QT syndrome (LQT-3) and Brudaga's syndrome (BrS) as paradigms to test this hypothesis. Structural analysis of the alpha subunit and site directed mutagenesis will complement the analysis of inherited mutations to provide a structural framework to interpret alteration in channel function. There are two aims of this project. Aim 1 is to test the hypothesis that there can be functional overlap caused by inherited mutations of the SCN5A gene linked either to BrS or LQT-3. Aim 2 is to test the hypothesis that there can be overlap in inherited BrS and LQT-3 SCN5A mutation-specific pharmacology due to overlap in mutation induced gating changes of expressed channels. Experiments that are proposed will combine patch clamp measurement of recombinant channel activity transiently expressed in mammalian cells. Theoretical testing of our predictions will be carried out using computer-based simulations of ion channel gating and cardiac action potentials that incorporate our patch clamp data. We hypothesize that information gained from these cellular and molecular experiments can be translated directly to improved therapeutic intervention in humans based on specific properties of mutant gene products, and also shed light on the possible interrelationship of these two inherited disorders.

Description (Adapted from Applicant's Abstract) The overall goal of the research proposed in this project is to identify molecular properties of cardiac ion channel proteins and organic drug molecules that will allow targeted control of calcium entry in cardiac myocytes in general and in myocytes surviving in the border zone of infarcted hearts in particular. Motivation for this goal comes from data in other projects of this program where it was shown that increasing L-type calcium channel current may prevent reentrant tachycardia in the infarcted canine heart, and that functional and molecular properties of key ion channels (Na+, Ca2+, and K+) are altered in epicardial cells that survive in the epicardial border zone (EBZ) of infarcted hearts. The overall goal of this project is thus to provide molecular insight into mechanisms that would permit more precise targeting of drugs to control calcium entry in these cells. There are thus three specific aims of this project. (1) to identify molecular determinants that target potentiation of calcium entry to cardiac vs. smooth muscle L-type calcium channels: (2) to test the hypothesis that drug-induced changes in L-type Ca2+ channel deactivation kinetics is a powerful mechanism of modulating calcium entry into targeted cells; and (3) to test the hypothesis that subunit assembly of 1Ks channel, which may differ between normal and EBZ cells, confers unique pharmacological and regulatory properties upon expressed channels. Together this information will provide a molecular basis for targeting control of calcium entry into cells of the EBZ which, in combination with the data obtained from other Projects of this program will provide the framework for the development of novel anti-arrhythmic therapy to control reentrant arrhythmias in ischemia.

DESCRIPTION (provided by applicant): The work proposed in this application is designed to provide insight into the molecular mechanisms that underlie a fundamental regulatory pathway in the heart: control of cardiac electrical activity by the sympathetic nervous system (SNS). To ensure adequate diastolic filling time between heartbeats during exercise and stress when SNS activity is increased, the duration of depolarization of the ventricular chambers, the QT interval of the electrocardiogram must be shortened. This occurs in large part through an increase in repolarization reserve of the heart by a protein kinase A (PKA) mediated regulation of the slowly activating IKS potassium (K+) channel, a process that requires assembly of a multi protein signaling complex coordinated by the A-Kinase anchoring protein Yotiao. That this K+ channel and its regulation are critical to human cardiac electrophysiology is evident from the range of heritable arrhythmias linked to mutations in genes coding for its principle subunits or for channel-associated proteins that coordinate its regulation. The long-term objective of this project is to identify additional signaling molecules that comprise the IKS multi protein complex, to unravel the fundamental processes by which these molecules control the IKS channel, and to relate them to our understanding of the basis and treatment of human disease. There are four aims of the proposed work. Aim 1 is to identify additional signaling molecules and their binding motifs in the IKS signaling complex which we postulate will serve as templates for discovery of, as yet unidentified, inherited Yotiao mutations that underlie multiple heritable arrhythmia syndromes. Aim 2 is to test the hypothesis that the SNS-mediated increase in repolarization reserve in the heart is due in part to an increase in the number of functional IKS channels regulated by a phosphorylation-sensitive trafficking pathway. Aim 3 is to test the hypothesis that SNS regulation of the IKS channel, physiologically essential in healthy individuals, can be a critical contributor to arrhythmia susceptibility in inherited atrial arrhythmia syndromes. Aim 4 is to test the hypothesis that IKS channels are expressed and regulated in cardiac myocytes (CMs) differentiated from human embryonic stem cells (hESCs) and that these cells can serve as a novel model system for investigating the expression and regulation of this and other critical channels in a human cardiac cellular environment. We propose that characterization of these channels in hESC-derived CMs will serve as an important baseline for future studies of these channels in inducible pluripotent stem cells (iPSCs) derived from patients suffering from heritable arrhythmia syndromes. The proposed project will combine biochemistry, imaging, and single cell electrophysiology in human cardiac cells to provide insight into the mechanisms causing congenital arrhythmias and, consequently, more specific and effective strategies to treat them. PUBLIC HEALTH RELEVANCE: The experiments that are proposed in this application are planned to build upon an emerging picture of an important signaling complex in human heart that is required for control of heart function during exercise and stress. Identifying the molecular components of this complex, that includes a potassium ion channel and several signaling molecules that couple sympathetic nerve stimulation to altered cardiac function, is essential to understanding the mechanisms of, and treatment strategies for, at least three different heritable human cardiac rhythm diseases. Thus the goal of this work is to define new molecular motifs that are responsible for, and can be targeted to treat, specific congenital cardiac arrhythmias.

Action Potentials; adrenergic; Adrenergic Agents; Arrhythmia; base; Calcium; Cardiac Myocytes; Cell membrane; Chronic; Collaborations; Complement; Complex; computerized data processing; Data; Death, Sudden, Cardiac; Diastole; Event; Exhibits; Goals; Heart; Heart failure; Homeostasis; Human; improved; Ion Channel; Lead; Link; Measures; Mediating; Molecular; Molecular Target; Mus; mutant; Mutation; novel; novel therapeutics; Patients; Physiological; Plasma; Play; Potassium Channel; Predisposition; programs; Regulation; research study; Role; Ryanodine Receptors; RyR2; Secondary to; Sodium; Sodium Channel; Sympathetic Nervous System; Testing; Therapeutic; Therapeutic Intervention; Translating; Variant; Work

DESCRIPTION (provided by applicant): This interdisciplinary training program in the pharmacological sciences is designed to teach novel approaches to molecular pharmacology to meet the challenges of conducting biomedical research in the era of post genomic science that we are rapidly approaching. It is our view that this is best approached by building on the explosion in genetic, molecular, and structural information that is evolving in modern biology to provide students with training that will enable them to identify molecular biological targets and to learn principles that guide in the design of novel compounds that modulate them and to analyze the consequences of drug-receptor interactions at the systems level of whole animal physiology and behavior. The program aims to produce scientists broadly trained in pharmacology with specific interest and expertise in one of the subspecialties emphasized in the program as a "research track". The four "tracks" are signal transduction; molecular cardiology; neuropharmacology; and structural pharmacology. The trainees are exposed to areas of study that interrelate broadly with neurobiology, cardiovascular biology, immunology, cancer, genetics, chemistry, biochemistry, and molecular biophysics. This interdisciplinary approach is not only desirable but necessary for modern pharmacologists in the post genomic era. The program is interdisciplinary in terms of its curriculum, participating faculty and students in training. The core curriculum includes courses in biochemistry and molecular biophysics, physiology, molecular biology and molecular pharmacology. In addition, students elect courses that focus on identified specialized areas (tracks) of interest. The 28 participating faculty are affiliated with 7 basic science departments, 4 centers and numerous clinical departments. These faculty work in diverse areas, including but not limited to: signal transduction in cancer, immunology, neurobiology and cardiovascular biology; learning, memory, and behavior; chemistry; molecular biophysics; functional genomics (bioinformatics); molecular genetics of inherited cardiac arrhythmias; and the structure and function of ion channels and G-protein-coupled receptors. Many areas of research have direct translational opportunities for interactions with clinically relevant problems, e.g. the molecular genetics of sudden cardiac death and drug discovery in the treatment of cystic fibrosis. Students are admitted only as candidates for the Ph.D. degree. An average of 3-4 students is admitted each year, and completion of degree requirements takes 5-6 years. The primary training facilities are the research laboratories and other facilities of the participating laboratories, as well as those of all other faculty and the many Centers, Institutes and special research facilities of the Health Sciences Campus of Columbia University.

DESCRIPTION (provided by applicant): This interdisciplinary training program in the pharmacological sciences is designed to teach novel approaches to molecular pharmacology to meet the challenges of conducting biomedical research in the era of post genomic science that we are rapidly approaching. It is our view that this is best approached by building on the explosion in genetic, molecular,, chemical, and structural information that is evolving in modern biology to provide students with training that will enable them to identify molecular biological targets and to learn principles that guide in the design of novel compounds that modulate them and to analyze the consequences of drug-receptor interactions at the systems level of whole animal physiology and behavior. The program aims to produce scientists broadly trained in pharmacology with specific interest and expertise in one of the subspecialties emphasized in the program as a research track. The four tracks are signal transduction; molecular cardiology; neuro & psycho pharmacology, and chemical & structural pharmacology. The trainees are exposed to areas of study that interrelate broadly with neurobiology, cardiovascular biology, immunology, cancer, genetics, chemistry, biochemistry, computational biology, and molecular biophysics. This interdisciplinary approach is not only desirable but necessary for modern pharmacologists in the post genomic era. The program is interdisciplinary in terms of its curriculum, participating faculty and students in training. The core curriculum includes courses in biochemistry and molecular biophysics, physiology, molecular biology and molecular pharmacology. In addition, students elect courses that focus on identified specialized areas (tracks) of interest. The 37 participating faculty are affiliated with 6 basic science departments, 3 centers and numerous clinical departments. These faculty work in diverse areas, including but not limited to: signal transduction in cancer, immunology, neurobiology and cardiovascular biology; learning, memory, and behavior; chemistry; molecular biophysics; functional genomics (bioinformatics); high throughput screening techniques, molecular genetics of inherited cardiac arrhythmias; and the structure and function of ion channels and G-protein-coupled receptors. Many areas of research have direct translational opportunities for interactions with clinically relevant problems, e.g. the molecular genetics of sudden cardiac death and drug discovery in the development of psychiatric disorders. Students are admitted only as candidates for the Ph.D. degree. An average of 3-4 students are admitted each year, and completion of degree requirements takes 5-6 years. The primary training facilities are the research laboratories and other facilities of the participating laboratories, as well as those of all other faculty and the many Centers, Institutes and special research facilities of the Health Sciences Campus of Columbia University.

DESCRIPTION (provided by applicant): Electrophysiological studies of electrically excitable cells, such as cardiac myocytes, skeletal myoblasts, neurons, osteoblasts, are critically important for the derivation of these cells from various stem cell sources and for their applicatio in regenerative medicine, fundamental biological research, and study of disease. For example, only electrophysiological measurements can determine if functional cardiomyocytes and neurons have been generated, as marker expression is necessary but not sufficient for the establishment of cell phenotype. These measurements need to be done at high speed (short time constants for changes of bath solutions) and in high-throughput fashion to provide realistic information about the cell properties in a large parameter space. This request is to purchase a SyncroPatch 96 (Nanion Technologies), an automated giga- seal patch clamp (APC) system for studies of electrophysiological properties of single cells with the highest throughput presently available. This core instrument would provide advanced technical capabilities to seven diverse NIH-funded laboratories across both campuses of Columbia University, by rapidly generating high quality patch clamp data for accurate pharmacological and physiological characterization of electrically excitable cells. Full dose response curves are rapidly measured in individual cells with a fast exchange of the bath solutions (within 100 ms), in a 96-well plate format. The system would provide unique capabilities not presently available either at the Columbia University Medical Center, the Columbia University Morningside Campus, or at any other University in the New York Metropolitan area. As detailed in the descriptions of the individual research projects in Section B, the SyncroPatch 96 would greatly accelerate our ongoing research in seven different areas: (i) High throughput screening of iPS-cardiomyocytes derived from patients carrying heritable arrhythmias, (ii) Determination of the impact of pacing and stretch on the maturation of cardiac myocytes derived from human iPS and embryonic stem cells (hESCs), (iii) Screening for novel drugs to treat intracellular calcium leak in heart failure, (iv) Modulation of voltage-dependent potassium channels; (v) Determination of the structural inter-subunit interface in a key smooth muscle potassium channel, (vi) Identification of new genetically encoded Ca channel blockers, and (vii) Novel therapies for amyotrophic lateral sclerosis (ALS) through small molecule screens of patient-derived iPS cell motor neurons (iPS-MNs). Preliminary data were collected using the SyncroPatch 96 in all five areas of proposed research. All Major Users of this equipment are seasoned NIH PIs with extensive expertise in analysis of cellular responses. We expect the instrument to be used at its full capacity, by investigators throughout the Columbia University and collaborators in the New York City area, promoting collaboration.

DESCRIPTION (provided by applicant): The KCNQ1 voltage gated K+ channel is expressed in many different tissues and plays widely different roles in these different tissues. Mutations and polymorphisms in KCNQ1 have been implicated in multiple diseases, including cardiac arrhythmias, inherited deafness, and susceptibility to type 2 diabetes. In the heart, KCNQ1 is co-expressed with the beta subunit KCNE1 to form the slowly activating, voltage gated IKs channels that contribute critically to the repolarization of the cardiac action potential at the cellular level and the QT interval of the electrocardiogram. In other cell types, such as epithelia and kidney cells, KCNQ1 is co-expressed with the beta subunits KCNE2 or KCNE3 to form a voltage independent K+ channel that is important for K+ and Cl- secretion. The critical role of KCNQ1 has been revealed by inherited mutations which have been associated with such diverse cardiac rhythm disturbances as the Long QT syndrome, the Short QT Syndrome, and atrial fibrillation. In many cases, co-assembly with the KCNE1 ¿ subunit dictates pathological function. How different beta subunits and mutations alter the function of KCNQ1 channels is not completely understood. We will here simultaneously measure the movement of the voltage sensor and the activation gate in KCNQ1 channels. This will allow us to determine whether a specific beta subunit or mutation mainly affects the voltage sensor or the gate. We will also test the effects of different modulators, both activators and inhibitors, of KCNQ1 on the voltage sensor movement and the activation gate, in order to understand how these molecules affect KCNQ1 activity and whether these molecules can restore the function of mutant KCNQ1 channels. The completion of these aims will generate a better understanding of how KCNE beta subunits modulate KCNQ1 channel functions, how small molecule modulators affect KCNQ1 channels, and how the defects of disease- causing KCNQ1 mutations can be overcome in a mutation- and small molecule dependent manner. This would be a first step in generating mutation specific treatments of diseases, such as cardiac arrhythmias, caused by mutations in KCNQ1.

DESCRIPTION (provided by applicant): Long QT Syndrome Type 3 (LQT3) is an inherited channelopathy associated with a high-risk of life-threating cardiac events across the entire age spectrum from infancy through adolescence to adults and seniors with unclear therapeutics. The central theme of our Multiple-PI LQT3 R01 grant is that joint collaborative investigations into the clinical, phenotype, genotype, and mechanistic aspects of LQT3 will yield novel insights and innovative targets for existing (beta- blocker) and new (sodium/calcium channel blockers) therapeutic approaches for this incompletely studied disorder, with potential extrapolation of the findings to other SCN5A-related medical conditions. This grant application involves three major research activities, each involving genotype-phenotype-therapeutic investigations at different basic and clinical levels involving: 1) Clinical: continue the enrollment and long-term follow-up o patients with LQT3 mutations in our ongoing LQT3 Registry that currently involves over 400 LQT3 patients and carry out population-based LQT3 risk stratification studies involving clinical, phenotype, gender, genotype, and therapy factors, with the information providing lead-ins to mechanistic basic science studies and gene-specific therapies; 2) Basic science: conduct biophysical and pharmacological functional investigations on specific LQT3 mutations utilizing: a) innovative, patient-specific induced pluripotent stem cells (iPSC) derived from patients in the LQT3 Registry harboring high-risk LQT3 mutations refractory to conventional therapy; b) relevant LQT3 mouse models investigating genetic risk and therapy in the intact animal; c) specific cellular expression studies to cross-validate the findings from the iPSC and mouse model studies and to evaluate dose-response therapies on disordered sodium-channel kinetics; and d) cardiomyocyte studies to complement beta-blocker and Na+ channel investigation in Aim 2c; and 3) Data management/analysis: provide centralized data management and coordinated biostatistical analyses to optimize the integration and science of the clinical and basic laborator studies. The successful collaborative efforts of the two PIs (Drs. Moss and Kass) extend over 10 years of professional interactions. This Multiple-PI R01 will be carried out at the University of Rochester Medical Center in Rochester, NY and the Columbia University Medical Center in New York City.