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

Triadin isoforms, splice variants of one gene, maintain healthy Ca²⁺ homeostasis in skeletal muscle by subserving several functions including an influence on Ca²⁺ 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 K²¹⁸, K²²⁰, and K²²⁴ together facilitate normal Trisk 95 binding to RyR1 and channel activation. Neither RyR1 binding nor activation are altered by alanine substitution of K²²⁰ alone or of K²¹⁸ and K²²⁴. Therefore K²¹⁸, K²²⁰, and K²²⁴ 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 D⁴⁸⁷⁸, D⁴⁹⁰⁷, and E⁴⁹⁰⁸ and Trisk 95 residues K²¹⁸, K²²⁰, and K²²⁴ facilitate Trisk 95 binding to RyR1 and channel activation. Since K²¹⁸, K²²⁰, and K²²⁴ 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.