Structural and biochemical insights into the cardiac and skeletal muscle excitation-contraction coupling machinery

Excitation-contraction (EC) coupling describes the process whereby the depolarizing action potential is transduced into a rapid increase of cytosolic calcium (Ca²⁺) that initiates muscle contraction. Proper execution of EC coupling relies on the coordinated communication between two calcium channels: plasma membrane-bound, L-type voltage-gated calcium channels (CaVs) and the intracellular Ryanodine Receptors (RyRs). CaVs respond to membrane depolarization by conveying an intracellular signal to the RyR. In skeletal muscle, CaV1.1 mechanically couples to the RyR; in cardiac tissue, extracellular Ca²⁺ entry via CaVs trigger RyR opening. The net effect of RyR activation is elevation of intracellular Ca²⁺ levels, activating the contractile machinery. In skeletal muscle, the nature of the physical CaV-RyR coupling has been an area of intense interest: do the channels directly interact or are auxiliary proteins required? Recently, a novel adaptor protein, STAC3, has been identified as playing a role in trafficking and maintaining components of the EC coupling machinery in a functional state. Indeed, STAC3-null mice and fish exhibit failure of skeletal muscle EC coupling. Chapter 2 presents x-ray crystallographic and isothermal titration calorimetry (ITC) data showing a direct interaction between STAC3 and CaV1.1. EC coupling assays reveal the importance of this interaction in EC coupling. The CaV1.1-STAC3 interaction is perturbed by the Native American Myopathy STAC3 mutation. L-type voltage-gated calcium channels fulfill dual roles as voltage-sensors for EC coupling and calcium ion conduits. In non-muscle cells, STAC3 facilitates CaV1.1’s functional membrane expression and alters the current properties of CaV1.2, suggesting a role of STAC proteins as a CaV regulator. Chapter 3 presents electrophysiology data illustrating the significant effect of STAC3 on modulating CaV1.2 currents. Detection of an interaction to Calmodulin (CaM), a well-known CaV regulator, suggests that STAC proteins may exert its effect on ion conduction via CaM. Genetic defects in the EC coupling machinery underlie numerous congenital myopathies and life-threatening cardiac arrhythmias. Chapter 4 explores the implications of disease-associated mutations within the cardiac Ryanodine Receptor (RyR2) using structural, spectroscopic, and thermal stability assays. An anion binding site within the N-terminal RyR2 region and maintenance of proper domain interfaces are key to RyR2 stability and normal functioning.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat