1985 — 1989 |
Beam, Kurt G |
K04Activity Code Description: Undocumented code - click on the grant title for more information. |
Physiological Control of Cellular Excitability
The long-term objective of this research is to elucidate physiological regulation of cellular excitability. In the studies proposed, the three-microelectrode and macro-patch voltage clamp techniques will be used to probe electrical excitability in mammalian skeletal muscle. In initial studies of this tissue, a calcium current and slow outward current have been identified. Proposed studies will continue the characterization of these slow ionic currents by establishing the absence or presence of a calcium-activated potassium current and by determining which of three alternative mechanisms account for the inactivation of the calcium current. Also to be tested is the hypothesis that activation of the current is fast enough at physiological temperature to promote significant calcium entry during normal fiber electrical activity. Circumstantial evidence suggests that calcium currents and calcium-activated potassium currents are much more prominent in embryonic and neonatal muscle than in adult muscle and that these currents become more prominent again following denervation of adult muscle. This hypothesis will be tested directly by measuring these slow ionic currents in immature muscle and in denervated adult muscle. The action of beta-adrenergic amines on slow ionic currents will be established, both in normal and denervated muscle. Experiments are proposed to probe the hypothesized role of voltage-dependent charge movement in the regulation of calcium release from the sarcoplasmic reticulum (SR). As part of this effort, it will be determined whether a pharmacologically-labile component of charge which has been identified in frog muscle is also present in rat muscle. Charge movement will be measured before and after treatment with catecholamine to test the hypothesis that the hormone's potentiation of the twitch occurs at the level of charge movement. Charge movement will be measured in developing muscle in order to determine whether its presence in a muscle correlates with the developmental appearance of specific morphological structures and proteins. Monoclonal antibodies will be raised against specific proteins residing in the junctional region between transverse tubules and SR and tested for effects on contractile activation and charge movement. This approach should permit the identification of the proteins involved in charge movement and calcium release from the SR as well as clarifying the relationship between these two processes.
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0.976 |
1985 — 2020 |
Beam, Kurt G |
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. |
Regulation of Membrane Excitability @ Colorado State University-Fort Collins
The long-term objective of this project is to identify the molecular interactions underlying skeletal-type excitation- contraction (EC) coupling, the process which links electrical excitation to muscular contraction. EC coupling hinges upon a functional interaction between the ryanodine receptor (RyR), a Ca2+-release channel located in the sarcoplasmic reticulum (SR), and the dihydropyridine receptor (DHPR), a voltage-gated Ca2+ channel which is located in the plasma membrane and contains alpha1S as its principal subunit. A number of experimental approaches will be used to probe the interaction between the DHPR and RyR, including the use of patch clamping, Ca2+ indicator dyes, electron microscopy and molecular biology. The proposal's first specific aim is to establish the determinants that cause DHPRs to target to junctions between the plasma membrane and sarcoplasmic reticulum. This will be accomplished by expression in dysgenic (alpha1S-null) myotubes of green fluorescent protein (GFP) tagged chimeras of alpha1S and alpha1H (a distantly related Ca2+ channel), and by a yeast two-hybrid screen of a muscle library using a potential targeting domains of alpha1S as baits. The second aim is to use alpha1S/alpha1H chimeras expressed in dysgenic myotubes as a means of testing whether the beta subunit of the DHPR is required for skeletal-type coupling, to determine whether the primary sequence of cytoplasmic domains outside the II-III loop are critical for coupling, and to identify the sequence(s) of alpha1S that cause DHPRs to be organized into "tetrads". The third aim is to test whether EC coupling depends upon conformational changes of the alpha1S II-III loop, which will be examined by means of introducing structural perturbations into the regions of the loop which surround the "critical domain" of the loop. One perturbation to be tested is the introduction of the biotin acceptor domain, which specifies the metabolic addition of biotin to a small number of native enzymes containing this essential cofactor. The fourth aim is to attempt to reconstitute skeletal-type coupling by expressing a minimal set of muscle proteins in a non-muscle cell.
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1 |
1988 — 1994 |
Beam, Kurt G |
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. |
Ontogeny of Neuronal Ion Channels @ Colorado State University-Fort Collins
The long-term goal of this research is to understand the developmental expression of the neuronal genome. The proposal focuses on the development of ion channels in motoneurons. motoneurons were selected for examination because (1) They represent a relatively homogeneous population which, with recent technical advances, can be experimentally identified after enzymatic dissociation, (2) The timing of important events in their in vivo developmental history has been established, and (3) Mutations have been identified that profoundly alter neuromuscular development. One project aims to characterize ionic currents in motoneurons from animals at important in vivo developmental stages: prior to the period of motoneuron death, immediately after this period during which half to two-thirds of these cells die, before and after the period of synapse elimination when about half the neuromuscular contacts made by a motoneuron are withdrawn, and after the postnatal maturation of the neuromuscular system.. after retrograde labeling by injection of dye into hindlimbs, identified motoneurons will be isolated by ezymatic dissociation of the spinal cords of both chicks and mice. ionic currents will be measured with the whole-cell patch clamp technique. The developmental expression of motoneuronal ion channels will be further dissected using mutants: crooked neck dwarf chicks and muscular dysgenic mice both suffer a fatal mutation that abolishes skeletal muscle contraction and largely eliminates motoneuronal death. The experiments will determine whether motoneurons in these animals retain the electrophysiological features displayed by motoneurons before cell death in normal animals. A pharmacological approach, that complements this genetic approach, will be to characterize ion channel development in chicks paralyzed by in ovo injections of curare, which others have shown largely abolishes motoneuron death. Another mutation that will be experimentally exploited is motor endplate disease (med) in mice, a fatal mutation that causes the failure of evoked transmitter release at the motor endplate. The experiments will test the hypothesis that a defect in a motoneuronal calcium current is responsible for the pathophysiology of this disease. The in vitro development of motoneuron ion channels will be determined by measuring ionic currents at varying times after plating motoneurons in culture. The effects of motoneuron growth factors on ionic currents will be characterized since this will help provide a more mechanistic understanding for the action of these growth factors.
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0.951 |
1993 — 1994 |
Beam, Kurt G |
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. |
Regulation of Mebrane Excitability @ Colorado State University-Fort Collins
The long-term goal of this research is to understand the molecular basis of calcium channel function and of excitation-contraction (E-C) coupling in skeletal muscle. Many of the experiments make use of cultured skeletal myotubes from mice with the muscular dysgenesis mutation. In mutant mice, the gene encoding the skeletal muscle dihydropyridine (DHP) receptor is altered, and skeletal muscle lacks slow L-type calcium current and E-C coupling. Complementary DNAs encoding different kinds of DHP receptor constructs will by expressed in cultured dysgenic myotubes, and three different physiological parameters of this expression will be measured. Intramembrane charge movement and L-type calcium conductance will be quantified with the patch clamp technique and calcium release from the sarcoplasmic reticulum will be assessed by microspectrofluorometry with the indicator dye Indo-1. The cDNA constructs to be examined will be chimaeras in which one or more of the internal repeats of the skeletal muscle DHP receptor are replaced by the equivalent portion of the cardiac DHP receptor. By establishing correlations between changes in the identity of repeats and changes in one or more of the measured physiological parameters, it will be possible to begin assigning these physiological important signals to specific structural components of the DHP receptor. To enable biochemical analysis of the expressed DHP receptor cDNAs, new methods appropriate for efficient transfection of primary myotube cultures will be established. As a test of whether junctional tetrads that are seen in freeze fracture of normal muscle represent DHP receptors, it will be determined whether expression of DHP receptor cDNAs in dysgenic myotubes leads to the appearance of these structures. To establish the relationship between junctional tetrads, E-C coupling and L-type calcium channels, focal stimulation and current recording techniques will be applied to developing muscle fibers. To gain insight into the reason for the generalized over-expression of receptors for calcium channel antagonists in mutant, cardiomyopathic hamsters, whole cell calcium currents will be measured in neurons, cardiac cells and skeletal muscle. Finally, it will be determined whether cDNAs encoding two new representatives of calcium channel proteins, the carp skeletal muscle DHP receptor and a rabbit brain calcium channel, produce calcium currents and E-C coupling in dysgenic myotubes.
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0.951 |
2013 — 2016 |
Beam, Kurt G |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Effects of Mh Mutations On Function of Dihydropyridine Receptor @ University of California At Davis
PROJECT SUMIV1ARY (See instructions): In skeletal muscle, the interaction between two proteins, the dihydropyridine receptor (DHPR) in the plasma membrane/transverse-tubules and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR), is essential for linking electrical excitation to contraction (excitation-contraction coupling, EC coupling). In particular, it is thought that conformational changes of the DHPR (containing Cav1.1 as its principal subunit) in response to depolarization cause RyR1 to open and release calcium from the SR, and that this signaling depends on physical links between the two proteins. Significantly, mutations of the DHPR or RyR1 in humans can result in the inherited disorder of malignant hyperthermia susceptibility (MHS), whereby volatile anesthetics cause dysregulation of calcium release that can lead to a fatal rise in body temperature unless there is rapid intervention. With the long-term objective of understanding the interactions between the DHPR and RyR1, and the mechanisms of MH, the specific aims of Project 3 are: Aim 1.1: To use whole cell patch clamping to measure L-type Ca2+ current and charge movement (which arise directly from the DHPR), together with Ca release from the SR, to determine how MHS mutations (RyR1-R2435H and -T4826I; Cav1.1-R174W) in mouse and human myotubes affect bi-directional signaling. Aim 1.2: To determine the effect of dantrolene on bi-directional signaling in MHS myotubes. Aim 1.3: In collaboration with Core D, to determine if MHS mutations alter the frequency or disposition of DHPR tetrads in myotubes. Aim 2: To measure membrane currents and myoplasmic Ca2+ transients in dissociated FDB fibers from adult (3-6 mo) and aged (12-18 mo) male and female MHS mice (Cav1.1-R174W and RyR1-R163C,-R2435H, and -T4826I) to determine the heterogeneity that results from differences in gender, age and locus. Aim 3: To measure membrane currents and Ca2+ transients in FDB fibers from 3-6 month old male Het RyR1-T4826I MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling) or dnTPRC6 (reduced SOCE), or which were administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (?KA scavenger) to determine if modification of one of the 4 key elements associated with MHS can mitigate or abrogate alterations in RyR-DHPR bi-directional signaling. Aim 4.1: To determine whether Ca2+ currents, charge movements or voltage-gated Ca2+ transients are differentially affected by volatile anesthetics in WT or MHS mutant (RyR1-R1630, -R2435H, -T4826I; Cav1.1-R174W) FDBs. Aim 4.2: To determine whether effects of volatile anesthetics on Ca2+ channel function are prevented by treatment with dantrolene. Aim 5: To use expression in myotubes of proteins harboring MHS mutations newly discovered by Core C in order to determine their effects on bi-directional signaling and hypersensitivity to volatile anesthetics.
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0.945 |
2014 — 2017 |
Beam, Kurt G Kraft, Mary L (co-PI) [⬀] Lidke, Keith Allan (co-PI) [⬀] Tamkun, Michael M. [⬀] |
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. |
Structural and Functional Interactions Within the Neuronal Er/Pm Junction @ Colorado State University
DESCRIPTION (provided by applicant): Endoplasmic reticulum/plasma membrane (ER/PM) junctions are best understood in muscle and immune cells where they mediate contraction and lymphocyte activation. Despite the early electron microscopic detection of sub-surface cisterns in neurons the function and molecular interactions responsible for the maintenance of this membrane junction are poorly understood. Our preliminary data demonstrate that the Kv2.1 delayed rectifier K+ channel plays a central role in the formation of neuronal ER/PM junctions. This channel forms highly stable cell surface clusters on the neuronal soma that reside in close apposition the ER membrane. Most of these localized channels are non-conducting and play a direct structural role by enhancing the ER/PM junctions and dramatically increasing the junction surface area, likely by binding unknown ER membrane proteins. Our published data indicate that the Kv2.1 enhanced ER/PM junctions are trafficking hubs, providing platforms for delivery and retrieval for multiple types of membrane proteins. In addition, our preliminary data indicate that calcium signaling proteins localize to the neuronal Kv2.1/ER/PM junction. We propose the Kv2.1-stabilized ER/PM junctions represent a macromolecular plasma membrane complex that functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. Given that this complex is regulated by stroke-related neuronal insults, an improved understanding of the components, function and dynamics within this cell surface microdomain is needed. This proposal assembles an interdisciplinary team from four institutions with combined expertise in intracellular Ca2+ dynamics, ion channel electrophysiology, molecular and cell biology, nanoSIMS cellular imaging technology, high-resolution real time imaging, optics, and quantitative analysis of single molecule diffusion. Aim 1 seeks to identify the proteins and lipids involved in the Kv2.1/ER/PM complex, Aim 2 examines L-type calcium channel function at the Kv2.1/ER/PM junction and Aim 3 studies calcium channel/beta2 adrenergic receptor dynamics within this domain.
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0.979 |