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.
Lee
Eun Hui
EH
Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea.
Song
Dong-Woo
DW
Lee
Jae Man
JM
Meissner
Gerhard
G
Allen
Paul D
PD
Kim
Do Han
DH
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D047468:Immunoprecipitation; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000032:analysis / Q000378:metabolism*; D009154:Mutation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D002352:Carrier Proteins; D002413:Cations, Divalent; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin; D002110:Caffeine; D002118:Calcium