Abstract

This grant examines the participation of the dihydropyridine receptor (DHPR) beta1a subunit in skeletal muscle excitation-contraction (EC) coupling. In response to depolarization, the DHPR produces a signal that briefly opens ryanodine receptor (RyR) channels leading to the release of stored Ca2+. Considerable progress in the understanding of DHPR-RyR interactions has been made by the availability of mouse models for the expression of DHPRs and RyRs. These are the dysgenic mouse line lacking alpha1S and knockout mice lacking the skeletal muscle RyR1 isoform or lacking the beta1a subunit of the skeletal muscle DHPR. The proposed experiments will use these mutants to identify domains of the beta1a subunit that participate in EC coupling. A C-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C-terminus domain could bring about a stronger colocalization of DHPRs and RyRs or could be essential for the generation of the signal that opens the RyR. Both possibilities will be tested. To address these questions, we make extensive use of double-null myotubes, (alpha1S/beta1)-null and (beta1/RyR1)-null, generated by mouse breeding. Double-null skeletal muscle cells should permit studies of alpha1S/beta and beta/RyR interactions in expression systems in which the two missing subunits can be expressed and modified.
The specific aims of the application are:
Aim 1. Establish the role of beta1a in the expression of DHPRs specifically required for EC coupling;
Aim 2. Identify molecular domains of alpha1a required for EC coupling;
Aim 3. Test functional interactions between beta1a and RyR1 controlling Ca2+ sparks;
and Aim 4. Determine whether beta1a triggers a component of the Ca2+ transient. The latter is investigated by expression of alpha1S constructs lacking the II-III loop. The main methods include a) expression of cDNA constructs of alpha1, beta and RyR subunits in single subunit-deficient or in double-null myotubes in culture; b) transgenic overexpression of beta1 constructs; c) macroscopic measurements of Ca2+ currents and charge movements in voltage-clamped myotubes; and d) confocal imaging of Ca2+ transients and Ca2+ sparks. DHPR-RyR interactions are crucial for understanding the molecular basis of EC coupling in normal and diseased states of skeletal muscle.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR046448-02
Application #
6349972
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Lymn, Richard W
Project Start
2000-02-16
Project End
2005-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
2
Fiscal Year
2001
Total Cost
$365,678
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715

  Publications

Bhattacharya, Dipankar; Mehle, Andrew; Kamp, Timothy J et al. (2015) Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) ?1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes. PLoS One 10:e0131399
Balijepalli, Ravi C; Delisle, Brian P; Balijepalli, Sadguna Y et al. (2007) Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol. Channels (Austin) 1:263-72
Carbonneau, Leah; Bhattacharya, Dipankar; Sheridan, David C et al. (2005) Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle. Biophys J 89:243-55
Cheng, Weijun; Altafaj, Xavier; Ronjat, Michel et al. (2005) Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling. Proc Natl Acad Sci U S A 102:19225-30
Coronado, Roberto; Ahern, Chris A; Sheridan, David C et al. (2004) Functional equivalence of dihydropyridine receptor alpha1S and beta1a subunits in triggering excitation-contraction coupling in skeletal muscle. Biol Res 37:565-75
Pouvreau, Sandrine; Berthier, Christine; Blaineau, Sylvie et al. (2004) Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells. J Physiol 555:365-81
Sheridan, David C; Cheng, Weijun; Carbonneau, Leah et al. (2004) Involvement of a heptad repeat in the carboxyl terminus of the dihydropyridine receptor beta1a subunit in the mechanism of excitation-contraction coupling in skeletal muscle. Biophys J 87:929-42
Sheridan, David C; Carbonneau, Leah; Ahern, Chris A et al. (2003) Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes. Biophys J 85:3739-57
Sheridan, David C; Cheng, Weijun; Ahern, Chris A et al. (2003) Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes. Biophys J 84:220-37
Ahern, Chris A; Sheridan, David C; Cheng, Weijun et al. (2003) Ca2+ current and charge movements in skeletal myotubes promoted by the beta-subunit of the dihydropyridine receptor in the absence of ryanodine receptor type 1. Biophys J 84:942-59

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