1994 — 2002 |
Cannon, Stephen C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Physiology of Neuromuscular Diseases @ Massachusetts General Hospital
Many inherited disorders of muscle are caused by an abnormality in the electrical excitability of the sarcolemmal hyperkalemic periodic paralysis (HPP) episodes of weakness occur in association with an elevation in extracellular potassium. During an attack, muscles are depolarized and electrically inexcitable. A related disorder, paramyotonia congenita (PMC), is characterized by localized cold-induced stiffness (myotonia) and mild weakness. Myotonia arises from repetitive after-discharges that originate in affected muscle independent from neuronal input. A combination of physiologic and genetic evidence has established that HPP, PMC and an equine form of periodic paralysis are all caused by mutations in the alpha subunit of the adult skeletal muscle isoform of the sodium channel. We have shown previously that the primary functional defect in HPP is a disruption of Na current inactivation. The loss of inactivation in human HPP myotubes was enhanced by raised extracellular [K]. This provided an explanation for the episodic nature of the attacks, but was unexpected biophysically. A major aim of this proposal is to determine whether extracellular K directly alters gating in mutant channels, to explore how K exerts its influence, and to elucidate the kinetic basis for the persistent Na current. Gating behavior of Na channels has never been measured in the temperature-sensitive phenotype, PMC. The functional defects produced by PMC mutations will be defined by heterologous expression of mutant cDNAs in mammalian cells. Many PMC mutations occur in the III-IV cytoplasmic loop, and additional site- directed mutagenesis will be performed to define how this domain participates in the process of inactivation. The aberrant Na channel behaviors in HPP and PMC will be incorporated into both a computer simulation and an animal model to explore the pathophysiologic basis for the dominant expression of these phenotypes. In equine periodic paralysis all affected animals have the same point mutation in the alpha subunit, and the frequency of attacks is reduced by phenytoin. Unitary Na currents will be recorded from equine myotubes to define the functional defect and to measure the effects of phenytoin on aberrant channel gating. The proposed studies are designed to provide a complete understanding of the molecular physiologic basis of two human neuromuscular diseases. In addition, these results will further our understanding of Na channel function at the molecular level, will provide insights from which to design rational therapy for these diseases, and will serve as a model system for understanding other disorders of altered electrical excitability (epilepsy, cardiac dysrhythmias).
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0.913 |
2003 — 2007 |
Cannon, Stephen C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Physiology of Neuromusclar Diseases @ University of Texas SW Med Ctr/Dallas
DESCRIPTION (provided by applicant): The myotonias and periodic paralyses are heritable diseases of skeletal muscle in which mutations of voltage-gated ion channels alter the electrical excitability of the sarcolemma. The long-term goals of this project are to characterize the functional defects of mutant channels and to determine how abnormal channel behavior produces symptoms. For these disorders, muscle dysfunction is caused by intermittent derangements in electrical excitability, which may be pathologically enhanced or depressed. Myotonia is a disorder of enhanced excitability wherein a single stimulus elicits a burst of action potentials that produces involuntary persistent muscle contraction lasting seconds. Conversely, periodic paralysis results from a depolarization-induced loss of muscle excitability. Chloride channel mutations cause myotonia, whereas mutations in potassium or calcium channels give rise to periodic paralysis. Missense mutations in a skeletal muscle sodium channel (NaV1.4 encoded by SCN4A) may cause myotonia, periodic paralysis, or of both in the same individual. The pathophysiological basis for this variation in clinical phenotype, all arising from mutations in a common sodium channel gene, is a major focus of the studies in this proposal. Our experimental approach is to identify alterations in the behavior of mutant channels by measuring ionic current, and then use computer or animal-based models to explore how specific alterations in channel function cause myotonia or periodic paralysis. Aim 1 is to identify the functional defects for additional, as-yet uncharacterized, NaV1.4 mutants and thereby define further the biophysical profile of gating defects that give rise to specific forms of altered membrane excitability. Prior work, by us, and others, has identified slow-inactivation gating as a critical determinant in the predisposition to attacks of weakness. In comparison to other gating transitions, relatively little is known about slow inactivation. Aim 2 seeks to improve our understanding of slow inactivation through a combination of refined voltage-dependent gating protocols and structural studies based on cysteine-scanning mutagenesis. Aim 3 explores how functional defects in Na channel behavior cause myotonia or periodic paralysis by characterizing a mouse model with a targeted mutation in NAY1.4 and using computer-based models of muscle excitability. More than 30 human disorders are known to be caused by mutations in voltage-gated ion channels. The proposed studies are designed to provide a more complete understanding of the pathophysiological basis for a group of human channelopathies: from gene defect to clinical symptoms. These studies will also further our knowledge of Na channel function at the molecular level, will lead to an improved understanding of the determinants of muscle excitability, will provide mechanistic insights for a more rational design of therapeutic strategies, and will serve as a model system for understanding more common disorders of excitability such as epilepsy or cardiac arrhythmia.
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0.957 |
2008 — 2018 |
Cannon, Stephen C. |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Molecular Physiology of Myotonia and Periodic Paralysis @ University of California Los Angeles
DESCRIPTION (provided by applicant): The myotonias and periodic paralyses are heritable diseases of skeletal muscle in which mutations of voltage-gated ion channels alter the electrical excitability of the fiber. The long-term goals of this project are to characterize the functional defects of mutant channels in these disorders and to determine how abnormal channel behavior produces symptoms in affected individuals. In these disorders, muscle dysfunction is caused by intermittent derangements in the electrical excitability of the fiber, which may be pathologically enhanced or depressed. Myotonia is a disorder of enhanced excitability wherein a single stimulus elicits a high- frequency burst of action potentials that produces involuntary persistent muscle contraction lasting seconds. Conversely, periodic paralysis results from a depolarization -induced loss of muscle excitability. Missense mutations in the adult skeletal muscle sodium channel (NaV1.4) may cause myotonia, periodic paralysis, or a combination of both in the same individual. The pathophysiological basis for this variation in clinical phenotype, all arising from mutations in a common sodium channel gene is a major focus of the studies in this proposal. Our experimental approach is to identify alterations in the behavior of mutant channels by measuring ionic current, and then use computer or animal-based models to explore how specific alterations in channel function give rise to myotonia or periodic paralysis. Aim 1 is to characterize the gating behavior of NaV1.4 channels, with a new focus on characterizing these properties for channels expressed in their native skeletal muscle environment. The availability of two mouse lines generated in our lab with knock-in point mutations in NaV1.4 (M1592V and R669H) offers a unique opportunity to characterize mutant channel behavior as occurs in muscle. Our studies on gating of disease- associated mutations of NaV1.4 will also explore the exciting new finding that missense mutations of arginines within S4 voltage-sensor domains may give rise to gating pore currents through an alternative permeation pathway different from the central pore. The propagation of action potentials into the transverse tubular system (TTS) and the activity-dependent accumulation of K+ therein are critical determinants of susceptibility to myotonia. Aim 2 will provide greater understanding for this important feature of muscle excitability by using state-of-the-art optical methods to measure TTS voltage transients and analytical models to estimate K+ accumulation both in normal mammalian muscle and for mouse models of myotonia and periodic paralysis. Aim 3 is a comparative analysis of the clinical phenotypes and electrophysiological properties of muscle from mice harboring either the M1592V or R669H mutations, as a model for gaining further insight on the mechanistic basis for the divergent phenotypes observed in humans for these allelic disorders of NaV1.4 (hyperkalemic periodic paralysis with myotonia contrasted by hypokalemic periodic paralysis without myotonia).
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0.993 |
2012 — 2021 |
Cannon, Stephen C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Disease Pathogenesis and Modification For Cav1.1-Associated Hypokalemic Periodic @ University of California Los Angeles
DESCRIPTION (provided by applicant): Hypokalemic periodic paralysis (HypoPP) is a dominantly inherited disorder of skeletal muscle in which recurrent attacks of weakness are caused by intermittent failure of fiber electrical excitability. Episodes occur in association with hypokalemia (K+ < 3 mM) and may be triggered by carbohydrate ingestion, exercise, or stress. The molecular defect in HypoPP is heterogeneous, with 60% of families having missense mutations in CACNA1S encoding the L-type Ca channel CaV1.1, another 20% have missense mutations in SCN4A encoding the voltage-gated Na channel NaV1.4, and the remainder undetermined. Despite this scientific advance, the pathogenic basis for the transient attacks of fiber depolarization with loss of excitability is not fully established. Curiously, all 8 mutationsin NaV1.4 and 6 of 7 in CaV1.1 occur at arginine residues in S4 voltage-sensor domains. Thus far, all 6 NaV1.4-HypoPP mutations studied in the cut-open oocyte have revealed a small anomalous cation current activated at hyperpolarized potentials, via conduction through a gating pore between the mutated S4 segment and the channel protein. We recently reported a gating pore current in muscle fibers from NaV1.4-R669H mice. This gating pore conductance is hypothesized to be the source of the inward current that triggers the paradoxical depolarization of HypoPP fibers in low K+. A major unanswered question is whether the homologous R/X mutations in CaV1.1 associated with HypoPP also produce a gating pore current, thereby providing supportive evidence for a common pathomechanism for HypoPP arising from mutations in NaV1.1 or CaV1.1. The overall goal of this project is to gain a greater understanding for the pathologic basis of HypoPP resulting from CaV1.1 mutations. We have used a gene-targeting approach to generate an R528H knockin mutation of CaV1.1 as a model for HypoPP. The Aims of this project are: (1) to extend the phenotypic characterization of the CaV1.1-R528H mouse for features of hypokalemic periodic paralysis, (2) to test the hypothesis that the CaV1.1-R528H channel conducts an anomalous gating pore current (3) to characterize the integrity of Ca2+- release in CaV1.1-R528H muscle fibers, (4) to explore potential disease-modifying agents in the mouse model of CaV1.1-HypoPP. This work will extend our understanding of the pathogenesis for attacks of weakness in HypoPP and will provide a model system to test the efficacy of therapeutic strategies, both as a means to reduce or ameliorate the burden of disease and to provide confirmatory experimental support for the proposed mechanism of disease.
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0.993 |
2021 |
Cannon, Stephen C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology of Myotonia and Periodic Paralysis @ University of California Los Angeles
Project Summary / Abstract Periodic paralysis and myotonia are ion channelopathies of skeletal muscle with debilitating episodes of severe weakness lasting hours to days and activity-dependent muscle stiffness. The long-term goal of this project is to advance our understanding of disease mechanism in these disorders of muscle excitability and to apply this knowledge in the design and pre-clinical testing of therapeutic interventions. Much progress has been made in establishing a causal relationship between the biophysical defect of a mutant channel and the clinical phenotype. For example, over 80 missense mutations have been identified in the NaV1.4 sodium channel, and we have shown by functional expression studies, coupled with simulations of fiber excitability, that mutations with gain of function changes (e.g. impaired inactivation) cause hyperkalemic periodic paralysis (HyperPP) with myotonia. Alternatively, the NaV1.4 mutations in hypokalemic periodic paralysis (HypoPP) are all R/X substitutions in S4 segments of voltage sensor domains that share a common functional defect - the anomalous gating pore leakage current. In all forms of periodic paralysis, the transient attacks of weakness result from sustained depolarization of ????? and loss of excitability, which are often triggered by stress, diet (carbohydrate, salt content, fasting), cold temperature, or exercise. The mechanisms by which these triggers destabilize ?????, in the setting of a static defect for a mutant channel, are fundamental open questions in the field and also represent opportunities for therapeutic intervention. A major impediment to progress has been the scarce availability of affected muscle. We created three knock-in mutant mouse models of PP that have robust phenotypes for HyperPP (NaV1.4-M1592V) or HypoPP (NaV1.4-R669H; CaV1.1-R528H). These mouse models have led to new insights on disease mechanism (e.g. recovery from acidosis is a potent trigger of HypoPP) and have led to novel therapeutic interventions that are now in clinical trials (bumetanide inhibition of the NKCC1 cotransporter prevents HypoPP). We will extend our investigations of periodic paralysis by focusing on the impact of ion gradients. Changes in extracellular [K+]o are established triggers for HypoPP (low) or HyperPP (high), but relatively little is known about Na+ and Cl- shifts in PP. Limited human data suggest an acute rise of [Na+]in during an episode of HyperPP or chronically high [Na+]in for HypoPP. In addition, we showed that reducing Cl- influx completely prevents HypoPP attacks. We have developed improved ion-selective microelectrodes, that in combination with the unique resource of our knock-in mutant mice, will enable us to (1) characterize muscle fiber Na+ and Cl- content at rest and during an attack of PP, (2) define the contribution of specific ion transport systems (mutant NaV1.4, NKCC1, Na/K-ATPase, Cl- exchangers) in setting ion concentrations in muscle channelopathies, (3) define the functional consequences of ion gradient perturbations in PP, based on computational modeling and simulation, and (4) use these insights in the design and pre-clinical testing of disease-modifying interventions. .
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0.91 |