Activation of skeletal muscle fibers, which is a prerequisite for all bodily movement as well as for respiration, is initiated by electrical depolarization of the transverse tubules (TTs), causing membrane voltage (V) sensors in the TT dihydropyridine receptor (DHPR) to trigger Ca2+ release via the abutting skeletal muscle ryanodine receptor (RyR1)/Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. However, the molecular mechanisms coupling the TT V sensor to SR RyR1 release activation are poorly understood, and the roles of various modulatory molecules, including S100A1 and calmodulin (CaM) are not clear. These issues are important since any pathologic interference with the Ca2+ release activation process may modify or disrupt muscle function. Here in Aim 1 we identify a previously totally unsuspected marked suppression of muscle Ca2+ release in a transgenic mouse model expressing a hypokalemic periodic paralysis (hypoPP) CaV1.1 V sensor charge mutation, and characterize the mechanism(s) underlying this defect in muscle activation. Muscle Ca2+ release is also modulated by a variety of accessory proteins. During the current grant cycle we have made the novel finding that the Ca2+ binding protein S100A1 binds to the previously identified calmodulin (CaM) binding domain (CaMBD) in RyR1, which should now be referred to as a CaM/S100A1 binding domain since these molecules interact for binding at this site.
In Aims 2 and 3 we utilize shRNA techniques to suppress the protein expression of S100A1, CaM or both S100A1 and CaM to investigate the effects of each of these ligands as well as their competitive interaction at sites other than the CaMBD of RyR1. We will use high speed (<50 us/line) line-scan confocal imaging of fibers containing the Ca2+ indicator fluo-4 to monitor Ca2+ signals and calculate the underlying Ca2+ release flux from the SR during single or trains of action potentials in intact fibers, or during voltage clamp depolarization of whole cell voltage clamped fibers with high levels of EGTA in the patch pipette solution. We will use adult muscle fibers with molecular biologically manipulated expression of endogenous or exogenous proteins. Parallel NMR and binding studies will examine the structures and binding affinities of S100A1 and/or CaM binding to peptides corresponding to the identified binding sites. This project will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of voltage sensor charges that are mutated in hypoPP in muscle Ca2+ release. It will characterize the modulation of SR Ca2+ release by S100A1 and CaM, which might be compromised in generalized or specific muscle disease states, or in aging muscle. Thus, this project has high impact for multiple disciplines, and for problems of both locomotion and breathing common to a variety of advanced diseased states and aging.

Public Health Relevance

Release of calcium ions from their intracellular storage location in skeletal muscle due to muscle fiber depolarization is a prerequisite for all skeletal muscle activity, including that involved in locomotion and breathing. Here we identify and study a previously unsuspected drastic suppression of muscle calcium release during depolarization of muscle fibers from a mouse genetically engineered to have one of the mutations responsible for hypokealemic periodic paralysis in humans. We also identify a previously unrecognized interaction of 2 proteins, calmodulin and S100A1, in modulation of both calcium release and L-type calcium channels that could influence muscle activation in both generalized or muscle-specific disease states, or in aging muscle.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR055099-36
Application #
9875266
Study Section
Skeletal Muscle Biology and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
1985-07-01
Project End
2022-01-31
Budget Start
2020-02-01
Budget End
2021-01-31
Support Year
36
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Liu, Yewei; Russell, Sarah J; Schneider, Martin F (2018) Foxo1 nucleo-cytoplasmic distribution and unidirectional nuclear influx are the same in nuclei in a single skeletal muscle fiber but vary between fibers. Am J Physiol Cell Physiol 314:C334-C348
Hernández-Ochoa, Erick O; Melville, Zephan; Vanegas, Camilo et al. (2018) Loss of S100A1 expression leads to Ca2+ release potentiation in mutant mice with disrupted CaM and S100A1 binding to CaMBD2 of RyR1. Physiol Rep 6:e13822
Au, Dianaly T; Ying, Zhekang; Hernández-Ochoa, Erick O et al. (2018) LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins. Arterioscler Thromb Vasc Biol 38:2651-2664
Hernández-Ochoa, Erick O; Schneider, Martin F (2018) Voltage sensing mechanism in skeletal muscle excitation-contraction coupling: coming of age or midlife crisis? Skelet Muscle 8:22
Banks, Quinton; Pratt, Stephen Joseph Paul; Iyer, Shama Rajan et al. (2018) Optical Recording of Action Potential Initiation and Propagation in Mouse Skeletal Muscle Fibers. Biophys J 115:2127-2140
Hernández-Ochoa, Erick O; Banks, Quinton; Schneider, Martin F (2017) Acute Elevated Glucose Promotes Abnormal Action Potential-Induced Ca2+ Transients in Cultured Skeletal Muscle Fibers. J Diabetes Res 2017:1509048
Robison, Patrick; Sussan, Thomas E; Chen, Hegang et al. (2017) Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke-induced mouse model of COPD. Muscle Nerve 56:282-291
Iyer, Shama R; Shah, Sameer B; Valencia, Ana P et al. (2017) Altered nuclear dynamics in MDX myofibers. J Appl Physiol (1985) 122:470-481
Melville, Zephan; Hernández-Ochoa, Erick O; Pratt, Stephen J P et al. (2017) The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP. Biochemistry 56:2328-2337
Hernández-Ochoa, Erick O; Vanegas, Camilo; Iyer, Shama R et al. (2016) Alternating bipolar field stimulation identifies muscle fibers with defective excitability but maintained local Ca(2+) signals and contraction. Skelet Muscle 6:6

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