Three residues in the luminal domain of triadin impact on Trisk 95 activation of skeletal muscle ryanodine receptors

Abstract

Triadin isoforms, splice variants of one gene, maintain healthy Ca homeostasis in skeletal muscle by subserving several functions including an influence on Ca release through the ligand-gated ryanodine receptor (RyR1) ion channels. The predominant triadin isoform in skeletal muscle, Trisk 95, activates RyR1 in vitro via binding to previously unidentified amino acids between residues 200 and 232. Here, we identify three amino acids that influence Trisk 95 binding to RyR1 and ion channel activation, using peptides encompassing residues 200-232. Selective alanine substitutions show that K, K, and K together facilitate normal Trisk 95 binding to RyR1 and channel activation. Neither RyR1 binding nor activation are altered by alanine substitution of K alone or of K and K. Therefore K, K, and K contribute to a robust binding and activation site that is disrupted only when the charge on all three residues is neutralized. We suggest that charged pair interactions between acidic RyR1 residues D, D, and E and Trisk 95 residues K, K, and K facilitate Trisk 95 binding to RyR1 and channel activation. Since K, K, and K are also required for CSQ binding to RyRs (Kobayashi et al. 17, J Biol Chem 275, 17639-17646), the results suggest that Trisk 95 may not simultaneously bind to RyR1 and CSQ, contrary to the widely held belief that triadin monomers form a quaternary complex with junctin, CSQ and RyR1. Therefore, the in vivo role of triadin monomers in modulating RyR1 activity is likely unrelated to CSQ.

Core skeletal muscle ryanodine receptor calcium release complex

“…Both insert into the SR membrane via a single alpha‐helix, with short cytoplasmic domains that interact with the cytoplasmic “foot” of RyR1 . In the SR lumen, basic residues in the longer C‐terminal domains bind to CSQ and to the minute luminal domain of RyR1 which is formed by the short luminal loops linking the transmembrane helices and contains <1.5% of the total protein mass. In bilayers, luminal addition of either protein increases purified RyR1 activity .…”

“…3C). The three Trisk95 residues that are implicated in binding to, and activation of, RyR1 are K218 (lightest blue) is shown interacting with RyR1 E4908; K220 (mid‐shade blue) is shown interacting with RyR1 D4907, while K224 (darkest blue) interacts with RyR1 D4878…”

“…In contrast, the junctin/RyR1 complex is inhibited by adding luminal CSQ1 and the channel responds rapidly to changes in luminal [Ca 2+ ] . The inability of CSQ1 to influence the Trisk 95/RyR1 complex may be explained by overlapping Trisk95 binding sites for RyR1 and CSQ1, preventing CSQ1 binding if the site is occupied by RyR1. Junctin can bind to CSQ1 and RyR1 due to multiple binding sites for both proteins.…”

“…Purified RyR1 is similarly activated by luminal addition of Trisk95 and a peptide (aa218-232) containing a KEKE motif from its C-tail, indicating that activation by Trisk95 depends solely on this luminal motif. 70,71 The interactions with junctin are more complex as RyR1 is activated by luminal junctin, but inhibited by luminal addition of C-jun. 67 Additionally, the massive 5-fold activation by cytoplasmic N-jun is opposite to RyR1-inhibition by Trisk95 N-tail.…”

“…76 Single channel and binding data suggest that the accepted model of a quarternary complex between Trisk95 and junctin monomers with RyR1 and CSQ1 is unlikely to exist in isolated systems as CSQ and RyR1 bind to the same site on Trisk95. 68,70,71 However Trisk95 in the muscle likely exists as a polymer, held together by disulphide interactions involving C270 and C649, that are well removed from the common CSQ/RyR binding site (around K220). 83,84 If only one molecule in the Trisk95 polymer bound to RyR1, the common RyR1/CSQ binding site in the other molecules would be available for CSQ binding.…”