Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin.
In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin. Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin. Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1. Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells.
Lee
Jae Man
JM
Department of Life Science, Kwangju Institute of Science & Technology, Gwangju, 500-712, Korea.
Rho
Seong-Hwan
SH
Shin
Dong Wook
DW
Cho
Chunghee
C
Park
Woo Jin
WJ
Eom
Soo Hyun
SH
Ma
Jianjie
J
Kim
Do Han
DH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D007477:Ions; D008297:Male; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D007477:Ions; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin; D001224:Aspartic Acid; D005982:Glutathione Transferase; D002118:Calcium