Occurrence of atypical Ca2+ transients in triadin-binding deficient-RYR1 mutants

Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994-7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca(2+) release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50-60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin-wtRYR1 complex. Ca(2+) imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca(2+) transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca(2+) release.