Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

Abstract The NH2-terminal region (residues 1-543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca2+ release. The three dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca2+ release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca2+-induced Ca2+ release and store overload induced Ca2+ release (SOICR) in HEK293 cells. We found that all deletion mutants are capable of forming caffeine and ryanodine sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca2+ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination, but had no effect on caffeine or Ca2+ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca2+ activation and lowered the threshold for SOICR activation and termination. On the other hand, deleting domain C suppressed caffeine activation, abolished Ca2+ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.