Susan L. Hamilton, Ph.D. - US grants

Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of excitable cell types. In some cell this permeability increase is either blocked or enhanced by dihydropyridines, leading to the hypothesis that the dihydropyridine binding site is part of or associated with the voltage-dependent calcium channel. We propose to purify the DHP binding protein from bovine ventricular tissue and to characterize its subunit composition. This will be accomplished using the binding radioactive DHPS to follow the purification. Purification procedures will utilize conventional column chromatography, HPLC and FPLC techniques, affinity chromatography, and ultracentrifugation. In a collaborative effort, we will use reconstitution techniques to establish whether the DHP binding protein is also the voltage- dependent calcium channel. Important to this purification and characterization of the cardiac DHP binding protein will be the production of antibody and toxin probes specific for this protein. One approach to the preparation of specific antibodies will be to use the subunits of the skeletal muscle DHP binding protein as antigens. We will also use the partially-purified cardiac preparation. Probes of the DHP binding protein will be used to purify it further, to determine the subunit composition, and to analyze the arrangement of the subunits in the membrane.

FKBP12 is a small immunophilin that binds with high affinity to sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors, RyRs) and transforming growth factor ¿1 receptors (T¿I RI). In this application we are going to test the hypothesis that the gain of E-C coupling is regulated in a biphasic manner by FKBP concentration. Specifically we will: 1) Demonstrate that FKBP12 binding to RyR1 controls the gain of E-C coupling in a biphasic manner, thereby, modulating Ca2+ stores, force production, fatigue, and recovery from injury; 2) Evaluate the ability T¿RI activation to increase the gain of E-C coupling via FKBP12; 3) Evaluate the ability of drugs that decrease FKBP12 binding to RyR1 to slow the development of fatigue and enhance recovery from injury and 4) Identify the amino acids on RyR1 that contribute to FKBP12 binding. The research described in this application will clarify the role of FKBP12 in skeletal muscle function and lay the groundwork for the development of new therapeutic interventions to slow diaphragm fatigue and enhance recovery from injury.

DESCRIPTION: The overall goal of the proposal is to define the molecular events involved in the regulation of Ca release from ryanodine sensitive intracellular stores and to determine the mechanisms by which defects in these pathways give rise to Malignant Hypothermia and Central Core Disease. The general hypothesis on which the proposal is based is that changes in the activity of the Ca-release channel are accompanied by global conformation changes that can be characterized biochemically and with cryoelectron microscopy and image reconstruction. In support of this hypothesis preliminary data is included showing the images of closed and open Ca-release channels and Ca-release channels treated with rapamycin to remove FKBP12. Extensive biochemical evidence is also presented for peptide mapping and immunochemical identification and characterization of the ryanodine receptor (RyR) in intact rightside out SR and in 28S and 14S proteolytic complexes. These studies will be extended in the five specific aims of the current proposal. The first aim is to generate different functional states of the Ca release channel (opened by caffeine and AMP-PCP, inactivated by mM Ca and inhibited by ruthenium red or neomycin) and to characterize these states by 3-H ryanodine binding and single channel analysis. The structures of these functional states will then be determined by electron cryomicroscopy and angular reconstruction. The second is to use different proteolytic mapping approaches combined with surface and membrane interior labeling, immunological and peptide binding (high affinity peptides identified using a phage display library) to map the topology of Ca release channel domains and structural changes that relate to different functional states. The 3-dimensional structure of a 14S proteolytic fragment of the protein which contains the channel and the ryanodine binding site will also be determined. The third aim is to map the binding site for FKBP12 and determine its effects on the structure of the Ca release channel. The 3-dimensional structure of the channel will be determined before and after the removal of FKBP12 by rapamycin treatment and upon readdition of purified FKBP12 to the depleted channel. The FKBP12 binding site will be mapped on the Ca release channel by examining label transfer from 125-I-APDP or 125-I-SASD and peptide mapping and sequencing of the 28S complex. The fourth and fifth aims both use electron cryomicroscopy to compare Ca release channel structures. In aim four the 3-dimensional structures of normal pig and malignant hypothermic pig Ca2+ release channels will be compared under conditions where (1) affinity differences for ryanodine are greatest, (2) in the absence of Ca, where both channels should be closed, and (3) in the presence of Ca and ryanodine where both channels should be open. In the final aim, the three dimensional structures of the skeletal and cardiac Ca release channels will be compared in the open and closed conformations. This may help provide a structural basis for the differences between cardiac and skeletal muscle EC coupling. The studies outlined in this proposal may lead to important information on Ca channel structure, topology and mechanism as well as identifying structural or mechanistic alterations found in disease states such as Malignant Hyperthermia and Central Core Disease.

In skeletal muscle the voltage sensor in the transverse-tubule membranes is thought to control the opening of the Ca2+ release channel in the sarcoplasmic reticulum. Human diseases such as malignant hyperthermia and central core disease may arise from alterations in the activity of the Ca2+ release channel, the voltage sensor, or proteins that modulate the activity of these two proteins. The Ca2+ release channel, also known as the ryanodine receptor, controls the release of Ca2+ from the sarcoplasmic reticulum; an event that triggers the sequence of events that leads to muscle contraction. The voltage sensor, which is also a voltage dependent Ca2+ channel that binds dihydropyridines, appears to directly control the gating of coupled Ca2+ release channels. Other modulatory proteins may regulate the interactions between the voltage sensor and the Ca2+ release channel. Calmodulin, for example, may regulate the conformation of the Ca2+ release channels in a Ca2+ dependent manner. Determining how these proteins regulate each other's activity is the overall goal of the research described in this application. We propose to 1) identify candidate sites on RYR1 for direct binding to the voltage sensor and test the role of these domains in mechanical E-C coupling, 2) evaluate the role of calmodulin in skeletal muscle E-C coupling, and 3) map both the voltage sensor interaction sites in the 3 dimensional reconstruction of RYR1 and the RYR1 interaction sites in a 3D reconstruction of the voltage sensor. Techniques to be used in this application include: protein biochemistry, proteolysis, radioligand binding, IASys biosensor technology, analysis of interactions of GST labeled DHPR fragments with polyHis tagged/Flag labeled fragments of RYR1, the yeast interaction trap system, phage display, site directed mutagenesis, generation of RYR1-RYR2 chimeras, measurement of whole cell Ca2+ currents and Ca2+ transients and cryoelectron microscopy and angular reconstitution.

Abstract Not Provided.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Ca2+ release channel (also known as the ryanodine receptor, RyR) belongs to a family of integral membrane Ca2+ channels and is the largest known ion channel. In mammals, there are three homologous RyR isoforms. Their 70% sequence homology accounts for the many functional similarities between them. RyR1 is found primarily in skeletal muscle and mediates the release of Ca2+ from sarcoplasmic reticulum (SR) stores into the cytoplasm during contraction. RyR1 is the most studied due to its high concentration in the SR membrane and its relatively simple purification. The channel is composed of four subunits, each ~565 kDa, constituting a single homotetrameric cation-selective channel pore in the SR membrane. Mutations in this channel are associated with several human muscular genetic diseases such as malignant hyperthermia (MH) and the myopathy called central core disease (CCD). We are using single particle cryoEM to determine its highest possible resolution structure at different physiological states and cryoEM restrained modeling to determine folds of different domains.

? DESCRIPTION (provided by applicant): Cardiovascular disease remains the leading cause of morbidity and mortality in t h e industrialized world. As such, there is a pronounced need to educate and train the next generation of scientists and physician-scientists focused on increasing our basic understanding of cardiovascular disease and translating these discoveries from the bench to the clinic. The Training Program in Molecular Physiology of the Cardiovascular System (MPCS) at Baylor College of Medicine is a multidisciplinary program composed of 26 faculty from 5 basic science and 5 clinical departments. The MPCS program has trained 41 pre-doctoral and 82 postdoctoral trainees since its inception in 1989. Although the majority of MPCS faculty members have primary appointments at BCM, several are at Rice University and UTHSC. MPCS's mission is to train biomedical scientists (5 pre-doctoral and 5 postdoctoral trainees) to work at the interface of basic and clinical research in one of three specialized areas related to cardiovascular research. These themes include: 1) Electrophysiology and cardiac arrhythmias, 2) Cardiovascular development and congenital disease, and 3) Tissue engineering, regenerative cardiology, and development of innovative methods for analysis and treatment of cardiovascular disease. The incredible collaborative research infrastructure at Baylor College of Medicine and partner institutions in the Texas Medical Center, combined with the diverse background of our mentors (basic scientists and practicing clinicians), provide an unrivalled environment for the scientific development, both in the classroom and at the bench, for MPCS trainees. The MPCS program consists of formal didactic courses, experimental planning and grant writing workshops, journal clubs, seminar series, ethics training, and regular interactions with training faculty for career guidance and development. Moreover, the pre- and postdoctoral fellows will be trained as leaders of teams of scientists and physicians that collaborate in translational research.

? DESCRIPTION (provided by applicant): Mutations in the type I ryanodine receptor (RyR1) are associated with a variety of human muscle diseases including malignant hyperthermia (MH), MH with cores, central core disease (CCD), multi-minicore disease, and others. RyR1-related myopathies are among the most common group of non-dystrophic muscle diseases and are associated with significant clinical disabilities, often including wheelchair dependence, severe scoliosis, respiratory failure, and can result in premature death in childhood. Currently there are no therapies for these devastating myopathies. MH mutations in RyR1 are frequently associated with heat stress, and/or exercise-induced heat stroke and/or rhabdomyolysis. CCD is a congenital myopathy associated with metabolically inactive central cores in skeletal muscle fibers, but the presence of cores is highly variable (despite the name) and the severity of the disease does not correlate with cores. We have shown that some CCD mutations increase Ca2+ leak from the sarcoplasmic reticulum, while others decrease Ca2+ permeation through RyR1. These finding raise the question of how opposing functional effects on RyR1 can result in related diseases. To answer this central, unresolved question and aid in the development of new interventions, we created mouse models of MH with cores (Y524S, YS) and CCD (I4895T, IT). Heterozygous YS mice are susceptible to anesthetic and heat-induced sudden death and also exhibit an age-dependent myopathy characterized by mitochondrial damage and the formation of amorphous cores. Heterozygous IT mice are not heat sensitive, but display decreased exercise capacity, deceased muscle fiber cross-sectional area, and a myopathy that increases with age. In this renewal application, we propose to define the cellular and molecular mechanisms by which functionally opposing RyR1 mutations produce these distinct phenotypes and then use these findings to develop new, mechanism-based therapeutic interventions for MH and CCD. Our working hypothesis is that the YS mutation drives the disease via increased RyR1 Ca2+ leak, activation of the mitochondrial permeability transition pore (mPTP), and oxidative stress, while the IT mutation enhances SR Ca2+ content, which leads to increased ER stress, p53 expression, mitochondrial damage and apoptosis. To test this hypothesis, we propose to: 1) Define the role of altered cytosolic, mitochondrial, and SR lumenal Ca2+ signaling and mPTP activation in the YS and IT myopathies. 2) Define the roles of RyR1 post- translational modifications. 3) Define the role of p53 in the IT myopathy and the enhanced heat sensitivity of YS mice. 4) Assess the potential of S107, NAC, 4PBA, and pifithrin-µ as interventions for MH and CCD. There are currently no therapies to treat RyR1-related myopathies. This multi-PI application is designed to address this need by carefully delineating specific cellular pathways that underlie disease processes and then to use this information to develop and test several novel therapeutic interventions with distinct mechanisms of action.

Abstract Mice with a skeletal muscle (SkM)-specific decrease in the small 12 kDa FK506 binding protein, FKBP12, (FKD mice) display improved endurance, insulin-mediated glucose clearance, and bone mineral density, as well as decreased body fat and resistance to weight gain on a high fat diet. Low doses of rapamycin and SLF (synthetic ligand for FKBP12) that displace FKBP12 from its binding partners mimic the effects of FKBP12 deficiency in SkM, suggesting these drugs have potential as interventions to improve muscle function and metabolism. The primary target of FKBP12 in SkM is the sarcoplasmic reticulum (SR) Ca2+ release channel, RyR1, which controls the release of Ca2+ from intracellular stores during excitation-contraction coupling (ECC). Partial removal of FKBP12 from RyR1 (genetically or by treatment with low doses of rapamycin or SLF) increases both the amplitude of the myoplasmic Ca2+ transient and Ca2+ influx into the muscle fiber during repetitive stimulation. Both enhanced SR Ca2+ release and increased Ca2+ influx associated with partial removal of FKBP12 from RyR1 are blocked by inhibitors of calmodulin-dependent protein kinase II (CaMKII) and store-operated Ca2+ entry (SOCE). However, the mechanisms by which increases in Ca2+ release and influx result in improved muscle function and metabolism remain unknown. We hypothesize the existence of a tunable feedback loop that functionally couples ECC, SOCE, and mitochondrial Ca2+ uptake to modulate muscle function and metabolism. The specific aims of this application are to: A1. Define the roles of FKBP12 and RyR1 phosphorylation in regulating the amplitude of the Ca2+ transient during repetitive stimulation and improving SkM performance and metabolism. 2. Define the feedback loop that enhances Ca2+ store refilling and ATP production to sustain the improved muscle performance and metabolism in FKBP12 deficient mice. 3. Evaluate the therapeutic potential of SLF to improve muscle function and metabolism. Our long-term goal is to develop interventions to improve muscle function in people who cannot perform strenuous exercise due to age, muscle disease, obesity and/or have type II diabetes.