Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca²⁺

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca ²⁺([Ca²⁺ _rest), suggesting a role for triadins in modulating global intracellular Ca²⁺ homeostasis. To understand this mechanism, we study here how triadin alters [Ca²⁺ _rest, Ca²⁺ release, and Ca²⁺ entry pathways using a combination of Ca²⁺ microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca²⁺, and Mn²⁺ imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca²⁺] _rest that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn²⁺ quench rates under resting conditions indicate that triadin-null cells also have higher Ca²⁺ entry rates and lower sarcoplasmic reticulum Ca²⁺ load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca²⁺ entry, recovering sarcoplasmic reticulum Ca²⁺ content levels, and restoring near normal [Ca²⁺]_rest. Exogenous FKBP12.6 also reduced the RyR1 channel P_o but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P_o nor recovered multiple subconductance gating. These data suggest that elevated [Ca²⁺] _rest in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca²⁺ entry at rest.