Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer.

Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.

N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.

MDC1A, the second most prevalent form of congenital muscular dystrophy, results from laminin-α2 chain deficiency. This disease is characterized by extensive muscle wasting that results in extremely weak skeletal muscles. A large percentage of children with MDC1A are faced with respiratory as well as ambulatory difficulties. We investigated the effects of overexpressing insulin-like growth factor-1 (IGF-1) as a potential therapeutic target for the disease in the Lama2(Dy-w) mouse, a model that closely resembles human MDC1A. IGF-1 transgenic Lama2(Dy-w) mice showed increased survivability, body weight and muscle weight. In addition, these mice showed better ability to stand up on their hind limbs: a typical exploratory behavior seen in healthy mice. Histology and immunohistochemistry analyses revealed increased regenerative capacity and proliferation in IGF-1 transgenic Lama2(Dy-w) muscles. Western blot analysis showed increased phosphorylation of Akt and ERK1/2, both known to enhance myogenesis. Additionally, we saw increases in the expression of the regeneration markers MyoD, myogenin and embryonic myosin (myosin heavy chain 3, MYH3). We conclude that overexpression of IGF-1 in Lama2(Dy-w) mice increases lifespan and improves their overall wellbeing mainly through the restoration of impaired muscle regeneration, as fibrosis or inflammation was not impacted by IGF-1 in this disease model. Our results demonstrate that IGF-1 has a promising therapeutic potential in the treatment of MDC1A.