Negatively Charged Amino Acids within the Intraluminal Loop of Ryanodine Receptor Are Involved in the Interaction with Triadin

Abstract

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 Ca 2؉ , but also to the active Ca 2؉ 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 intralu- 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 Ca 2؉ release process of excitation-contraction coupling in muscle cells.Ultrastructural and biochemical evidence suggests that a protein complex exists at the junctional SR 1 membrane in cardiac and skeletal muscle to facilitate the Ca 2ϩ release that occurs during muscle contraction (1-4). These include the ryanodine receptor (RyR), triadin, junctin, and calsequestrin, which may associate into a stable complex at the junctional membrane (5). The RyR contains a large cytoplasmic foot region plus a transmembrane domain at the carboxyl-terminal end. The transmembrane domain of RyR has been shown to contain the conduction pore of the Ca 2ϩ release channel (6). According to the hydropathy plot of Takeshima et al. (7), four putative transmembrane segments (TM1 to TM4) are predicted to span the SR membrane, with residues connecting TM1 and TM2 forming the first intraluminal loop (aa 4581-4640) and residues connecting TM3 and TM4 contributing to the second intraluminal loop (aa 4860 -4917). Elegant studies from other investigators have shown that the second intraluminal loop of RyR participates in the overall ion conduction and selectivity process of the Ca 2ϩ release channel (8 -10). Calsequestrin is the high capacity Ca 2ϩ storage protein located in the lumen of the SR (11). In striated muscles, calsequestrins are largely tethered to the terminal cisternae of SR, leading to the suggestion that they could sequester Ca 2ϩ to sites of Ca 2ϩ release (12). Triadin and junctin are structurally similar integral membrane proteins having a short aminoterminal cytoplasmic domain and a long stretch exposed to the luminal side of the SR (13,14). The luminal region is especially enriched in multiple clusters of alternating lysine and glutamic acid residues, named as the KEKE motif (14 -16).Previous studies show that triadin could directly modulate the activity of the RyR1 channel in a reconstitutional system (17, 18). Calsequestrin has been proposed to indirectly regulate the RyR function presumably through its interaction with triadin and junctin (5,19). Recently, the binding regions of calsequestrin and the histidine-rich Ca 2ϩ -binding protein to triadin were identified in our group (20,21). However, the nature of interaction between triadin ...

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

“…1 B). Identical results were obtained in the absence of calcium (as in Lee et al, 2004) and in the presence of 1 mM Ca 2+ . In addition, identical results were also observed in reverse coimmunoprecipitation experiments in which purifi ed WT RyR1 or ∆M 1 , 2,3 were coupled to protein A/G sepharose via anti-RyR and their ability to pull down purifi ed triadin and junctin was detected (unpublished data).…”

“…Purifi ed skeletal triadin or junctin were coupled to protein A/G sepharose via antitriadin or anti-junctin antibodies (respectively), and association with WT and mutated RyR1 constructs was determined by incubation with purifi ed recombinant RyR1 constructs. In contrast to Lee et al (2004), triadin binding to RyR1 was not altered by any of the single mutants (D4878A, ∆M 1 ; D4907A, ∆M 2 ; E4908A, ∆M 3 ) or for D4878A/E4908A (∆M 1,3 ) ( Fig. 1 B), with similar amounts of each mutant binding to triadin as that observed for WT RyR1.…”

“…Lee et al (2004) identifi ed three negatively charged amino acids (D4878, D4907, and E4908) in an RyR1 luminal loop that were each required for the isolated loop to bind a specifi c positively charged triadin KEKE motif (residues Fig. 1 A).…”

“…A similar protocol was used to immunostain and obtain images from WT RyR1-and ∆M 1,2,3 -expressing HEK293 cells using antibody 34C (1:20) and a rhodamine-conjugated goat anti-mouse IgG (1:2,000). To determine the relative importance of the three negatively charged RyR1 residues identifi ed by Lee et al (2004) (Fig. 1 A) for triadin and junctin binding to fulllength RyR1, we assessed the effects of a comprehensive series of mutations to these residues in triadin and junctin coimmunoprecipitation.…”

“…A putative triadin binding site was recently identifi ed in the terminal intraluminal loop of RyR1 between residues 4860 and 4917 (Lee et al, 2004). More recently, alanine substitution of three specifi c negatively charged residues within this region (D4878, D4907, and E4908) was found to disrupt triadin binding to fulllength RyR1 and also alter the magnitude and kinetics of caffeine-induced Ca 2+ release (Lee et al, 2006).…”