Dual Role of Functionally Intact Dyadic Junctions in Cardiac Excitation-Contraction Coupling

Abstract

Cardiac excitation-contraction (EC) coupling relies on Ca2+-induced Ca2+ release (CICR) enabled by an intimate relationship between L-type (CaV1.2) channels and ryanodine receptors (RyRs) at dyadic junctions. How CaV1.2 channels target in proximity to RyRs at dyads is unknown, but likely involves protein interactions mediated by one or more intracellular loops of CaV1.2 pore-forming α1C subunit. We hypothesized that over-expressing α1C intracellular loops that play a critical role in CaV1.2/RyR functional co-localization would disrupt CICR in cardiomyocytes. We over-expressed fluorescent-protein-tagged α1C intracellular loops and termini (NT, I-II, II-III, III-IV, CT) in adult rat cardiomyocytes and assessed their impact on field-stimulation-evoked rhod-2 reported Ca2+ transients and other determinants of CICR (ICaL, t-tubule structure, SR Ca2+ content, spontaneous Ca2+ sparks). Over-expressed I-II and CT uniquely disrupted EC coupling as characterized by two distinct signatures: a sharp augmentation in CICR failure, and an increased propensity for arrhythmic Ca2+ transients. Surprisingly, both I-II and CT paradoxically induced a substantial rise in frequency of spontaneous Ca2+ sparks. The effects of I-II on CICR and Ca2+ sparks were phenocopied by over-expressing the 18-residue AID peptide responsible for auxiliary CaVβ binding to the I-II loop in α1C. Over-expressing a mutated AID (YWI/AAA) that no longer binds CaVβ produced normal CICR and spontaneous Ca2+ sparks. The effects of CT on CICR and spontaneous Ca2+ sparks were phenocopied by the distal-CT that is unique to α1C, and contains binding sites for several signaling and scaffold proteins. Altogether, the results shed new light on molecular determinants important for CaV1.2 functional targeting in cardiomyocytes, and suggest a new unconventional role for functionally intact dyads– as being necessary to quell spontaneous openings of RyRs.