The Cardiac Ryanodine Receptor N-Terminal Region Contains an Anion Binding Site that Is Targeted by Disease Mutations

Abstract

Ryanodine receptors (RyRs) are calcium release channels located in the membrane of the endoplasmic and sarcoplasmic reticulum and play a major role in muscle excitation-contraction coupling. The cardiac isoform (RyR2) is the target for >150 mutations that cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and other conditions. Here, we present the crystal structure of the N-terminal region of RyR2 (1-547), an area encompassing 29 distinct disease mutations. The protein folds up in three individual domains, which are held together via a central chloride anion that shields repulsive positive charges. Several disease mutant versions of the construct drastically destabilize the protein. The R420Q disease mutant causes CPVT and ablates chloride binding. The mutation results in reorientations of the first two domains relative to the third domain. These conformational changes likely activate the channel by destabilizing intersubunit interactions that are disrupted upon channel opening.

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

“…However, how the NH 2 -terminal region regulates the activation and termination of Ca 2ϩ release and how mutations in this region impair these processes are unclear. The NH 2 -terminal region of RyR2 encompasses three well defined domains: domain A (residues 1-217), domain B (residues 218 -409), and domain C (residues 410 -543) (9,13). In the present study, we assessed the role of these individual domains in Ca 2ϩ release activation and termination.…”

“…The intra-and intersubunit interactions between domains A and B are believed to be important for stabilizing the closed state of the channel. Disease mutations located in interfaces between domains A and B would weaken these interactions, thus facilitating channel opening (7)(8)(9)(10)(11)(12)(13)(14). Del-A would be expected to remove both intra-and intersubunit interactions between domains A and B, leading to destabilization of the closed state and channel activation.…”

“…The recently solved crystal structures of the NH 2 -terminal region of RyR have provided novel insights into the structural basis of disease mechanisms associated with the NH 2 -terminal mutations (7)(8)(9)(10)(11)(12)(13)(14). The three-dimensional structure of the NH 2 -terminal region of RyR contains three domains: domain A (residues 1-217), domain B (residues 218 -409), and domain C (residues 410 -543) (9).…”

RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation induces bradycardia by disturbing the coupled clock pacemaker mechanism

“…Thus, the 420 site seems to be important for the RyR 2 function. Structural analyses have shown that this residue highly conserved among species plays an important role in the channel conformation (45,46). The RyR 2 R420Q loses the capacity to bind Cl -, which maintains the three regions of the N terminal domain in place (45), although the functional consequences remained to be elucidated.…”

Effective cascade screening through identification of a mutation in RYR2 in a large family with a history of sudden death

“…The p.(Arg420Gln) mutation lies within the N terminal region. Kimlicka et al have shown that this mutation results in the loss of chloride binding and removal of links between two domains of RyR2, domain A and domain B [8]. This in turn results in alterations in the domain orientations.…”