Mitsugumin-29 Regulates RyR1 Activity In Mouse Skeletal Myotubes

Abstract

Canonical-type transient receptor potential cation channel type 3 (TRPC3) in plasma membrane allows the entry of Ca2+ ions into various cells. In skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor1, a Ca2+ channel in sarcoplasmic reticulum (SR) membrane) regulates the gain of skeletal excitation-contraction coupling (J. Biol. Chem., 2006). Mitsugumin-29 (MG29) is a four membrane-spanning protein and is found in both plasma and SR membrane. MG29 has been known as a TRPC3-interacting protein in skeletal myotubes (Biochem. J., 2008).To identify critical region(s) of MG29 that participate in binding to TRPC3 or the role of MG29 in skeletal muscle, N-terminus, three intervenient loops among four transmembrane regions, and C-terminus of MG29 were expressed in E. coil as N-terminal GST-fused forms, and subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. Cytoplasmic N-terminus and a loop between first and second transmembrane domains of MG29 effectively bound to TRPC3. Two deletion mutants of MG29 (missing the TRPC3-binding sites: deleting the N-terminus only or longer N-terminus covering the loop region) was expressed in mouse skeletal myotubes, and the myotubes was subjected to the measurement of Ca2+ transients with Fura-2 or Fluo-4. The later mutant showed significantly decreased responsiveness of RyR1 to caffeine, suggesting that MG29 may be a mediator between the functional interaction between TRPC3 and RyR1.