Distinct regions of triadin are required for targeting and retention at the junctional domain of the sarcoplasmic reticulum

Abstract

Ca2+ release, which is necessary for muscle contraction, occurs at the j-SR (junctional domain of the sarcoplasmic reticulum). It requires the assembly of a large multiprotein complex containing the RyR (ryanodine receptor) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multiprotein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1 (targeting region 1), TR2 and TR3, that contribute to the localization of triadin at the j-SR. FRAP experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Taken together, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multiprotein complex associated with RyR.

Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

“…After 2 d, cells were transfected with the Lipofectamine-Plus method (Invitrogen) following the manufacturer's instructions. Myoblasts were induced to differentiate with α-MEM containing 2 mM L-glutamine (Lonza), 100 μg/mL streptomycin (Lonza), 100 U/mL penicillin (Lonza), 1 mM sodium pyruvate (Lonza), 1 mM dexamethasone (Sigma-Aldrich), 0.05 mM hydrocortisone, supplemented with 10% heat-inactivated FBS (Sigma Aldrich), and 5% heat-inactivated horse serum (Sigma-Aldrich) (49). HeLa cells and HEK293T were cultured in DMEM supplemented with 10% heatinactivated FBS (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 100 μg/mL streptomycin (Sigma-Aldrich), 100 U/mL penicillin (Lonza), and 1 mM sodium pyruvate (Lonza).…”

A Mutation in theCASQ1Gene Causes a Vacuolar Myopathy with Accumulation of Sarcoplasmic Reticulum Protein Aggregates

“…Serial 8‐μm‐thick transversal cryosections or single fibers from human skeletal muscle were fixed with 3% (v/v) paraformaldehyde (Sigma‐Aldrich, St. Louis, MO) in phosphate‐buffered saline (PBS) and permeabilized in HEPES–Triton Buffer (20 mM HEPES pH 7.4, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl 2 , 0.5% Triton X‐100). Cryosections or single fibers were incubated with the following primary antibodies: anti‐CASQ1 mouse monoclonal antibody diluted 1:300 (clone MA3‐913; Thermo Scientific), anti‐RyR mouse monoclonal antibody at a final concentration of 1 μg/ml (clone 34C; Thermo Scientific, Waltham, MA), anti‐RyR1 rabbit polyclonal antibody [Rossi et al., ], and anti‐SERCA monoclonal antibody diluted 1:1 (clone Y/IF4 kindly provided by Prof. M. East). Cy3‐ or Cy2‐conjugated antimouse or antirabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME) were used for immunofluorescence detection.…”

“…Myoblasts were obtained from hind limb muscles of 2‐day‐old rats (Sprague–Dawley; Harlan Laboratories, Indianapolis, IN) as described in Rossi et al (). Cell suspension was plated on 0.025% laminin (Sigma–Aldrich) coated glass coverslips.…”

Physiology and pathophysiology of excitation–contraction coupling: the functional role of ryanodine receptor

“…RyRs are also regulated by oxidation and nitrosylation (Shan et al 2010; Andersson et al 2011; Santulli 2017; Fauconnier et al 2010). Other modulatory proteins complex directly and indirectly with RyR, including sorcin (Farrell et al 2004), calmodulin (Meissner and Henderson 1987), homer (Feng et al 2002), histidine-rich Ca 2+ binding protein (Lee et al 2001), triadin (Rossi et al 2014), junctin (Zhang et al 1997), and calsequestrin (Ohkura et al 1998). …”