Understanding the Structure-Function Relationships in Cardiac Ryanodine Receptor (RyR2)

Abstract

The cardiac ryanodine receptor (RyR2) is an intracellular Ca2+ release channel and plays a critical role in the process of Ca2+ induced Ca2+ release (CICR) that underlies excitation-contraction coupling in the heart. Activation of RyR2 by cytosolic Ca2+ is an essential step in CICR, but the exact mechanism of RyR2 Ca2+ activation is unclear. Recent breakthroughs in cryo-electron microscopy and single-particle analysis have led to the determination of three-dimensional structures of RyRs at near-atomic resolutions. Although some detailed structural information of RyRs has recently been revealed, the structure-function relationships in RyRs remain largely undefined. The overall objective of this work is to understand the molecular basis of Ca2+ activation of RyR2 and the impact of RyR2 disease-causing mutations. RyR2 is a large protein that is comprised of a number of domains. Structural analyses suggested that the EF-hand Ca2+ binding domain may function as the Ca2+ sensor of RyRs, but its functional significance is unclear. Our site-directed mutagenesis studies revealed that the EF-hand domain is not required for cytosolic Ca2+ activation, but it is critical for luminal Ca2+ activation of RyR2. Structural analyses also identified potential Ca2+-, ATP-, and caffeine-binding sites in the central domain of RyRs, but the functional importance of the central domain in RyR2 activation and regulation remains unclear. Our systematic structure-function relationship analysis revealed that the central domain controls not only Ca2+ activation and basal activity of RyR2, but also modulation of the channel by ATP, caffeine and Mg2+. The central domain is a disease mutation hotspot, but the mechanisms of action of disease mutations are not well understood. We showed that central domain disease-associated mutations enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. The highly conserved C-terminal domain (CTD) is another disease mutation hotspot. We showed that mutations located in the CTD inter-subunit interface destabilized the closed-state of RyR2 by probably altering electrostatic interactions between adjacent CTDs, resulting in spontaneous channel openings and enhanced channel activation. Collectively, the present work sheds novel insights into the structure-function relationship in the activation, regulation, and stability of the RyR2 and the pathogenic mechanisms of disease-associated RyR2 mutations.