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.