Skeletal muscle excitation-contraction (EC) coupling depends upon interactions at triad junctions between L- type Ca2+ channels (dihydropyridine receptors, DHPRs) in the plasma membrane and type 1 ryanodine receptors (type 1 RyRs, RyR1) in the sarcoplasmic reticulum (SR). The DHPR, in response to plasma membrane depolarization, activates RyR1 to release Ca2+ from the SR, which is thought to occur via conformational coupling between the two proteins. Such coupling is strongly supported by freeze-fracture electron microscopy, which reveals that DHPRs are arranged into groups of four apposed to the four subunits of every other RyR1. However, despite a wealth of functional, biochemical and structural evidence, there is little consensus on the identity of the protein-protein interactions linking the DHPR and RyR1. This proposal advances four specific aims with the long-range goal of establishing the identity of these interactions.
Aim 1 is to use site-directed binding of streptavidin and FRET to identify conformational changes of the DHPR that are essential for EC coupling. Constructs encoding DHPR subunits containing a biotin acceptor domain (BAD) and/or fluorescent proteins at sites of interest will be analyzed after expression in myotubes null for the endogenous subunit.
Aim 2 is to identify the localization of specific sites within the three-dimensional structure of the DHPR. Biotin-containing DHPRs will be purified from muscle of transgenic mice and subjected to electron-microscopic analysis as single, frozen-hydrated particles.
Aim 3 is to define the orientation of specific domains of the DHPR in relation to the RyR. Freeze-fracture and thin-section electron microscopy wiil be used to visualize the disposition of gold-streptavidin bound to targeted sites of DHPRs.
Aim 4 is to identify junctional proteins that neighbor functionally important sites within the DHPR by exploiting both proteomics and metabolic biotinylation. Accomplishing these specific aims should reveal essential new information about a basic muscle function, EC coupling. The proposed experiments will also provide knowledge essential for understanding the inherited human muscle diseases hypokalemic periodic paralysis (caused by mutations of ?1S-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1). PROJECT NARRATIVE: This proposal will characterize the interaction between two proteins (the "DHPR" and "RyR1") that are essential for muscle contraction. Mutations in the DHPR and RyR1 cause inherited, human muscle diseases. Thus, the proposed research will be important for understanding both normal muscle function and the pathology of human muscle diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR055104-25
Application #
8292044
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Boyce, Amanda T
Project Start
2008-09-05
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
25
Fiscal Year
2012
Total Cost
$612,546
Indirect Cost
$115,995
Name
University of Colorado Denver
Department
Physiology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Bannister, R A (2013) Dantrolene-induced inhibition of skeletal L-type Ca2+ current requires RyR1 expression. Biomed Res Int 2013:390493
Eltit, Jose Miguel; Bannister, Roger A; Moua, Ong et al. (2012) Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor. Proc Natl Acad Sci U S A 109:7923-8
Beam, Kurt G; Bannister, Roger A (2010) Looking for answers to EC coupling's persistent questions. J Gen Physiol 136:7-12
Bannister, Roger A; Papadopoulos, Symeon; Haarmann, Claudia S et al. (2009) Effects of inserting fluorescent proteins into the alpha1S II-III loop: insights into excitation-contraction coupling. J Gen Physiol 134:35-51