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 application 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 will 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 11S-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1).
This application 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.
| Polster, Alexander; Perni, Stefano; Filipova, Dilyana et al. (2018) Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain. J Gen Physiol 150:293-306 |
| Polster, Alexander; Nelson, Benjamin R; Papadopoulos, Symeon et al. (2018) Stac proteins associate with the critical domain for excitation-contraction coupling in the II-III loop of CaV1.1. J Gen Physiol 150:613-624 |
| Perni, Stefano; Lavorato, Manuela; Beam, Kurt G (2017) De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release. Proc Natl Acad Sci U S A 114:13822-13827 |
| Polster, Alexander; Nelson, Benjamin R; Olson, Eric N et al. (2016) Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation. Proc Natl Acad Sci U S A 113:10986-91 |
| Perni, Stefano; Close, Matthew; Franzini-Armstrong, Clara (2016) Design Principles of Reptilian Muscles: Calcium Cycling Strategies. Anat Rec (Hoboken) 299:352-60 |
| Bannister, Roger A; Sheridan, David C; Beam, Kurt G (2016) Distinct Components of Retrograde Ca(V)1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1. Biophys J 110:912-21 |
| Polster, Alexander; Perni, Stefano; Bichraoui, Hicham et al. (2015) Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels. Proc Natl Acad Sci U S A 112:602-6 |
| Ohrtman, Joshua D; Romberg, Christin F; Moua, Ong et al. (2015) Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus. J Gen Physiol 145:303-14 |
| Monk, Kelly R; Voas, Matthew G; Franzini-Armstrong, Clara et al. (2013) Mutation of sec63 in zebrafish causes defects in myelinated axons and liver pathology. Dis Model Mech 6:135-45 |
| Bannister, Roger A; Beam, Kurt G (2013) Ca(V)1.1: The atypical prototypical voltage-gated Ca²? channel. Biochim Biophys Acta 1828:1587-97 |
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