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

Abstract

Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca²⁺ 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. In cardiomyocytes, spontaneous Ca²⁺ waves, triggered by store overload-induced Ca²⁺ release mediated by the RyR₂ (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 RyR₂-mediated Ca²⁺ release machinery that directly modulates store overload-induced Ca²⁺ release activity via CTT. We show that cardiomyocytes derived from the TRIC-A^-/- (TRIC-A knockout) mice display dysregulated Ca²⁺ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR₂. Modeling and docking studies reveal potential sites on RyR₂ that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR₂, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca²⁺ oscillations. Ca²⁺ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca²⁺ leak across the endoplasmic reticulum by directly targeting RyR₂ to modulate store overload-induced Ca²⁺ release. Moreover, synthetic CTT-A peptide facilitates RyR₂ activity in lipid bilayer reconstitution system, enhances Ca²⁺ sparks in permeabilized TRIC-A^-/- cardiomyocytes, and induces intracellular Ca²⁺ 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. In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR₂ to modulate sarcoplasmic reticulum Ca²⁺ handling in cardiac muscle.