Article

Toward a molecular understanding of the structure-function of ryanodine receptor Ca2+ release channels: perspectives from recombinant expression systems.

Wales Heart Research Institute, Department of Cardiology, College of Medicine, Cardiff University, UK CF14 4XN.
Cell Biochemistry and Biophysics (Impact Factor: 1.91). 02/2005; 42(2):197-222. DOI: 10.1385/CBB:42:2:197
Source: PubMed

ABSTRACT Identification of the genetic basis of human diseases linked to dysfunctional free calcium (Ca2+) signaling has triggered an explosion of interest in the functional characterization of the molecular components regulating intracellular Ca2+ homeostasis. There is a growing appreciation of the central role of intracellular ryanodine-sensitive Ca2+ release channel (RyR) regulation in skeletal and cardiac muscle pathologies, including malignant hyperthermia, heart failure, and sudden cardiac death. The use of cloned RyR isoforms and recombinant expression techniques has greatly facilitated the elucidation of the molecular basis of RyR Ca2+ release functionality. This review will focus on the recombinant techniques used in the functional characterization of recombinant RyR isoforms and the insights that these approaches have yielded in unraveling the mechanistic basis of RyR channel functionality.

0 Bookmarks
 · 
59 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca(2+). Ca(2+) is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca(2+) transient by activation of a class of Ca(2+) release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca(2+)-ATPases (SERCAs) to terminate the Ca(2+) transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR(NTD)) at 2.5 A. The RyR(NTD) structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)Rsup), but lacks most of the long helix-turn-helix segment of the "arm" domain in IP(3)Rsup. The N-terminal beta-trefoil fold, found in both RyR and IP(3)R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation "hot spot" loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyR(NTD), supporting previously described mechanisms whereby mutations perturb protein-protein interactions.
    Proceedings of the National Academy of Sciences 01/2009; 106(27):11040-11044. · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ion channels underlie the electrical activity of cells. Calcium channels have a unique functional role, because not only do they participate in this activity, they form the means bywhich electrical signals are converted to responses within the cell. Calcium concentrations in the cytoplasm of cells are maintained at a low level, and calcium channels activate quickly such that the opening of ion channels can rapidly change the cytoplasmic environment. Once inside the cell, calcium acts as a “second messenger” prompting responses by binding to a variety of calcium sensitive proteins. Calcium channels are known to play an important role in stimulating muscle contraction, in neurotransmitter secretion, gene regulation, activating other ion channels, controlling the shape and duration of action potentials and many other processes. Since calcium plays an integral role in cell function, and since excessive quantities can be toxic, its movement is tightly regulated and controlled through a large variety of mechanisms.
    12/2006: pages 241-299;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation-contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR21-606 N-terminal fragment. Soluble hRyR21 606 was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.
    Protein and Peptide Letters 06/2013; · 1.99 Impact Factor

Full-text

Download
13 Downloads
Available from
May 28, 2014