Colin G. Nichols - US grants

Project Summary ATP-sensitive potassium (KATP) channels are a major link between cell metabolism and and electrical activity, and in the heart, these channels underlie actiojn potential change sin response to ischemia. Recent evidence also supports a role in early repolarization syndrome (ERS). We have developed innovative new approaches to determine the moelcuelar details of KATP channel regulation, and to assess channel localization and function in the intact heart. During the previous period of support, we developed novel FRET approaches to assessing protein structure dynamics during gating, and to assess channel domain and subunit organization. We also developed novel transgenic animals that raise new questions regarding the role of Kir6.1 subunits in the heart. These studies now lead to three experimental series, addressing the questions regarding (1) the molecular basis of nucleotide gating in KATP, (2) the association rules between Kir6 subunits and (3) the analysis of an animal model of Kir6.1-dependent ERS. The results of proposed experiments will bring insight to the regulation and role of KATP channels in cardiac arrhythmias and will provide information that will ultimately underlie the development of rational therapies for the treatment of cardiac ischemia and arrhythmias.

DESCRIPTION (provided by applicant): Inward rectifying potassium (Kir) channels regulate excitability in many tissues, and multiple diseases result from mutations of Kir channel genes. The long-term goal of this project is to understand the molecular details of Kir channel function. Previously, we discovered that soluble cytoplasmic polyamines cause inward rectification and demonstrated their mechanism and sites of action in the channel. We have developed novel systems for large-scale purification of bacterial and human Kir channels, and have succeeded in functional analysis of these recombinant channel proteins in reconstituted membrane systems. Together with ideas generated by recent crystal structures of both pro- and eukaryotic Kir channels, our novel approaches to biochemical and functional analysis of these channels allow us to develop and address exciting new questions and hypotheses regarding the fundamental basis of Kir channel activity. We will determine the molecular mechanisms by which lipids regulate gating in model Kir channels, and the dynamic structural changes that accompany gating, by combinations of biochemical and electrophysiological recordings.

Achieving euglycemia in diabetics ultimately requires understanding of the cellular and molecular mechanisms controlling insulin secretion. We have recently confirmed the central role of ATP-sensitive potassium (KATP) channels in linking blood glucose levels to the secretion of insulin, and demonstrated that inherited hyperinsulinemia is causally associated with lack of pancreatic KATP channel activity. We have cloned and expressed the high affinity sulfonylurea receptor (SUR1) and inward rectifier K channel subunits that are the necessary molecular components of KATP channel activity, and are performing extensive mutagenic analysis to determine the structural basis of channel activity and regulation by intracellular nucleotides. We have also used transgenic approaches to define the role of glucose transporters in diabetes and insulin resistance diseases. Together these efforts provide the basis for the current proposal in which we will generate transgenic mice with altered pancreatic KATP channel activity, in order to understand the consequences of altered pancreatic KATP channel activity for regulation of blood glucose. Transgenic animals expressing mutated KATP channels with altered nucleotide sensitivity, and gene knockout animals will allow a new and powerful approach to understanding the role of KATP channels in pancreatic function within the intact animal. The availability of KATP transgenic animals, and answers to the specific questions posed below, will provide a critical foundation for the development of new treatment approaches to infant-onset diabetes and hyperinsulinemia.

[unreadable] DESCRIPTION (provided by applicant): Metabolism-excitation coupling in the heart is mediated by ATP-sensitive potassium (KATP) channels. This project seeks to understand the cellular and molecular basis for metabolic control of ATP-sensitive potassium channels by sulfonylurea receptors in the intact heart. [unreadable] [unreadable] Preliminary studies provide significant new discoveries regarding the molecular basis of KATP channel function. We have made use of these discoveries to develop novel transgenes that allow us to express overactive mutant KATP channels in cardiac sarcolemmal membranes. We have generated transgenic mice in which pancreatic and cardiac sarcolemmal KATP channel properties are significantly changed through expression of mutant Kir6.2 subunits. These studies demonstrate dramatic differences in the response of pancreatic and cardiac KATP channels to cellular metabolism. In order to explain these differences, and to further understand metabolism-excitation coupling in the intact heart, we now propose experiments to address the following question: How do SUR2A and cellular environment confer the cardiac KATP channel phenotype? [unreadable] [unreadable] Previous work has contributed substantially to current understanding of the role and molecular basis of KATP channels. The results of the proposed experiments will bring insight to the regulation of these channels in the heart in vivo. The work will provide information that will ultimately underlie the development of rational therapies for the treatment of cardiac ischemia and arrhythmias.

DESCRIPTION (provided by applicant): Inward rectifying K (Kir) channels regulate excitability in many tissues, and several diseases result from mutations of Kir channel genes. During previous award periods we demonstrated that soluble cytoplasmic factors cause intrinsic rectification in strong inward rectifiers, identified these factors as polyamines, and confirmed that they act as 'long-pore plugs' to block the channel permeation pathway. Recent crystallization of two different K channel pore structures has illuminated mechanisms of permeation and gating in K channels. However, the structural details of rectification and of gating in Kir channels remain unclear. Preliminary data lead us to specific hypotheses regarding the molecular details of polyamine-induction of rectification and of ligand-gating mechanisms in Kir channels. These hypotheses will be critically examined in three experimental Aims, utilizing a combination of biochemical and biophysical techniques, together with molecular modeling to gain previously unobtainable insight to the mechanisms of inward rectification and gating. The work will provide information that will form the background to the development of rational therapies for cardiac arrhythmias, epilepsy and other disorders of cell excitability through modulation of Kir channel activity.

? DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the mechanisms, role and significance of electrical control of insulin secretion and development of diabetes. Much previous work has demonstrated the central role of the K-ATP channel in electrical activity of the pancreatic ?-cell and has revealed how defective K-ATP channel activity can have profound effects on insulin secretion and cause neonatal diabetes. This has led to transfer of these patients from insulin to sulfonylurea therapy, with marked improvements in lifestyle and disease outcome. Based on new preliminary data, we propose that failure of insulin secretion due to altered electrical activity may be more prominent than previously realized, and that multiple unrecognized molecular components are likely to be relevant to excitation-secretion control and the development of diabetes. To pursue these ideas, we will utilize unique animal models to assess quantitative relationships between islet K-ATP activity and insulin secretory control, and we will utilize innovative forward genetic approaches in model organisms to search for novel regulators of metabolism-excitation coupling. In the process, the proposed experiments will test mechanistic hypotheses regarding metabolism-excitation-secretion coupling in islets and in the etiology of different forms of diabetes and will provide mechanistic information that will impact treatment approaches.

DESCRIPTION (provided by applicant): Sulfonylurea receptors (SURs) provide a unique link between cardiac metabolism and excitability. Previous efforts on this project have demonstrated that both SUR1 and SUR2 genes are expressed in the heart, with spatial and pathological variability in different regions. Because these subunits confer different metabolic and pharmacologic sensitivities to KATP channels, this realization means that cardiac KATP channel activation may vary regionally and with disease state. Additional studies suggest that differentially spliced versions of the proteins may be expressed in the heart and confer differential functional consequences. The development of novel cell biological tools and transgenic animals have allowed us to generate extensive preliminary data that lead us to propose experiments using biochemical, cell biological and physiological approaches to reach a full understanding of the nature and role of SUR subunit variation in the heart. They are motivated by our discovery of differential subunit expression and consequent functional differences within the mouse heart based on analysis of genetically modified animals, and encompass both mechanistic studies of expression control, and studies of the functional consequences in model systems and human hearts.

? DESCRIPTION (provided by applicant): Despite recent advances in blood glucose control, the continually rising incidence of diabetes leads to an ever- increasing burden of disease complications. Major limitations to treatment include inability to bioengineer beta cell replacement for both type 1 and 2 diabetes, as well as to monitor and image the development of the disease, and to engineer repair of diabetic tissues. Such advances depend on innovations and discoveries throughout the physical and biological sciences, and the ability to bring these advances to the relevant clinical problems. The following examples describe current relevant challenges that are intimately dependent on advances in basic and translational sciences. (1) Progress in cell therapy and tissue engineering promises major breakthroughs in repair of diabetic tissues, but model systems as well as noninvasive imaging of potential replacement cells will be needed to ascertain delivery to target sites. (2) Novel and hybrid imaging systems need to be developed, including microscopic and macroscopic approaches such as PET and optical or photoacoustic imaging are needed to gain deeper and finer assessment of tissue pathologies. (3) Nanoparticles with multimodality imaging capabilities and drug payloads need to be optimized and moved into clinical trials. To transform diabetes outcomes, these and other challenges must be met by interdisciplinary teams of engineers, physicists, computer scientists, biologists, and physicians working together at the interfaces between biology, technology, and medicine. This program seeks to enable these processes, in order to bring advances in technology and engineering to bear on the challenges of research and translation to improved therapeutic outcome, by training a new generation of researchers with strength in physical science and engineering, and with intimate exposure to and involvement in relevant areas of biomedical research. Dually mentored pre-Doctoral and post-Doctoral researchers will develop and apply novel imaging, modeling (both computational and experimental) and engineering approaches to key questions in diabetes and associated pathologies.

ABSTRACT My lab studies the mechanistic basis, and functional consequences, of ion channels, particularly background inward rectifier and ATP-sensitive potassium channels, that are found throughout the cardiovascular system. Our work integrates studies at multiple levels, from the fundamental molecular basis of channel activity to animal models of pathologies associated with human disease. We are interested in how channels are constructed and function, how they regulate individual smooth and cardiac muscles, and how altered channel function contributes to the pathological consequences of aberrant function in the cardiovascular system. Previously, we discovered that soluble cytoplasmic polyamines cause inward rectification and demonstrated their mechanism and sites of action in Kir channels. We have developed the capability to purify Kir and ATP-sensitive (KATP) channels and to analyze these proteins structurally, biochemically and functionally. This allows us to develop and address exciting new questions and hypotheses regarding the fundamental basis of Kir and KATP channel activity, including the molecular mechanisms by which lipids regulate gating and the dynamic structural changes that accompany gating. KATP channels link metabolism to electrical activity in cardiac and smooth muscle. Our recent findings regarding a causal role of KATP channel mutations in human Cantu Syndrome (CS) reveal multiple pathological consequences of underexcitability, including persistence of fetal circulation, pericardial effusion, lymphedema, decreased vascular compliance and decreased gut motility. Development of unique and novel genetically modified animals, as well as a unique research CS clinic, has allowed us to generate extensive preliminary data that begin to explain such features, and leads us to novel hypotheses which will be explored using multiple cell biological and physiological approaches in animals and humans to reach a full understanding of the nature and role of KATP dependent excitability in regulation of cardiovascular function. These studies will form the background to the testing of relevant pharmacological approaches to CS therapy in animal models and in humans, with the ultimate goal of developing a specific therapy for CS and related pathologies.

PROJECT SUMMARY Cantu syndrome (CS) is a rare genetic condition for which there is currently no directed therapy. CS patients suffer from multiple pathologies, but cardiovascular complications, and neurological features are major concerns. CS results from gain-of-function mutations in two specific genes that encode cardiovascular ATP-sensitive (KATP) channels. FDA-approved blockers of these channels are potential treatments. In preparation for a clinical trial, this project will validate cardiovascular and neurological features as markers of disease in a unique cohort of CS patients, and will validate KATP channel inhibitors as appropriate therapy in unique animal models of CS and in patient-derived cells. Successful accomplishment of the project will complete clinical trial readiness for the proposed therapeutic approach.