TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle

Abstract

Rationale: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca 2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. Objective: In cardiomyocytes, spontaneous Ca 2+ waves, triggered by store overload–induced Ca 2+ release mediated by the RyR 2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR 2 -mediated Ca 2+ release machinery that directly modulates store overload–induced Ca 2+ release activity via CTT. Methods and Results: We show that cardiomyocytes derived from the TRIC-A −/− (TRIC-A knockout) mice display dysregulated Ca 2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR 2 . Modeling and docking studies reveal potential sites on RyR 2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR 2 , transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca 2+ oscillations. Ca 2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca 2+ leak across the endoplasmic reticulum by directly targeting RyR 2 to modulate store overload–induced Ca 2+ release. Moreover, synthetic CTT-A peptide facilitates RyR 2 activity in lipid bilayer reconstitution system, enhances Ca 2+ sparks in permeabilized TRIC-A −/− cardiomyocytes, and induces intracellular Ca 2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A −/− mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. Conclusions: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR 2 to modulate sarcoplasmic reticulum Ca 2+ handling in cardiac muscle.