The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis

Abstract

Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree.

Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients

“…A C-terminal cysteine was added to enable conjugation via maleimide activation to keyhole limpet hemocyanin (Pierce Chemical, Rockford, Illinois). New Zealand White rabbits were immunized with the conjugated peptide as described 45 , and specificity was confirmed by ELISA and immunoblot analysis with whole brain, cerebellum and skeletal and cardiac muscle microsomes 46 . For immunoblot analysis, sarcoplasmic and endoplasmic reticulum-enriched microsomes prepared from fast skeletal muscle, cardiac tissue, whole brain and cerebellum 47,48 were separated on a 5% SDS polyacrylamide gel (30 µg protein per lane), the proteins were electrophoretically transferred to PVDF membrane and then probed with either unpurified or affinity-purified antibody at a dilution of 1:1000 (ref.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…The conformation of the open RyR has so far only been proposed to resemble the conformation of the open K þ channel (Samsó et al 2009). The pore region has been predicted to consist of between 4 and 12 transmembrane segments (Takeshima et al 1989;Zorzato et al 1990;Tunwell et al 1996;Du et al 2002). Most of these models place both amino-and carboxy-termini in the cytoplasm.…”

Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia

“…The P2328S mutation is located in the large, footlike cytoplasmic domain of RyR2, and the latter 2 mutations occur in the carboxy-terminal part of the receptor, which contains several membrane-spanning, presumably critical regions of RyR2. 15 All 3 amino acid alterations cosegregated with the clinical phenotype in the 3 pedigrees and were absent in at least 100 healthy control individuals (200 alleles). Only individuals without drug treatment that could possibly affect heart rate and QT interval were included in this table.…”