Localization of a Disease-associated Mutation Site in the Three-dimensional Structure of the Cardiac Muscle Ryanodine Receptor

Zheng Liu,Ruiwu Wang,Jing Zhang,S.R Wayne Chen,Terence Wagenknecht

doi:10.1074/jbc.m505714200

Abstract

The cardiac muscle ryanodine receptor (RyR2) functions as a calcium release channel in the heart. Up to 40 mutations in RyR2 have been linked to genetic forms of sudden cardiac death. These mutations are largely clustered in three regions of the sequence of the polypeptide: one near the N terminus, one in the central region, and the third in the C-terminal region. The central region includes 11 mutations, and an isoleucine-proline motif (positions 2427 and 2428) in the same region is predicted to contribute to the binding of FKBP12.6 protein. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing. The central mutation site was mapped to a "bridge" of density that connects cytoplasmic domains 5 and 6, which have been suggested to undergo conformational changes during channel gating. Moreover, the location of this central mutation site is not close to that of the FKBP12.6-binding site mapped previously by cryoelectron microscopy.

Highlights

Heart disease is the world’s leading cause of death, accounting for 38% of all deaths in the United States in 2002
The mechanism by which a single-point mutation among the ϳ5000 amino acids of RyR2 affects normal heart function and causes sudden cardiac death remains elusive. These mutations are largely clustered in three regions of the RyR2 primary sequence: one near the N terminus, one in the central region, and the third in the C-terminal region. These three regions are homologous to the three malignant hyperthermia regions in the skeletal muscle RyR: the N-terminal region comprises the first 614 amino acids of RyR1; the central region is located between amino acids 2163 and 2458; and the C-terminal region is between amino acids 4800 and 4900 [13]
Tiso et al [15] have suggested that central region mutations in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2) influence the binding of FKBP12.6 to RyR2, which causes the closed state of mutant RyR2 to become destabilized so that unusually large amounts of Ca2ϩ are released from the zinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase

Summary

Introduction

Heart disease is the world’s leading cause of death, accounting for 38% of all deaths in the United States in 2002. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing.

Results

Conclusion