Biochemical Characterization and Molecular Cloning of Cardiac Triadin

Abstract

Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling. Ca2ϩ release from the sarcoplasmic reticulum of skeletal and cardiac muscle is regulated by similar but distinct mechanisms (1-3). In cardiac muscle, depolarization leads to the opening of voltage-gated Ca 2ϩ channels. Ca 2ϩ influx through L-type Ca 2ϩ channels (the dihydropyridine receptor) triggers the opening of the ryanodine receptor/Ca 2ϩ release channel in the sarcoplasmic reticulum. However, in skeletal muscle, entry of external Ca 2ϩ is not needed for this signal transduction process. The skeletal muscle dihydropyridine receptor interacts either directly or indirectly with the ryanodine receptor, thereby activating the Ca 2ϩ release channel without a requirement for extracellular calcium. Despite this difference, many factors that modulate the channel properties of the skeletal muscle ryanodine receptor also affect the cardiac ryanodine receptor in a similar fashion (4, 5). Identification of protein components in the junctional sarcoplasmic reticulum is fundamental to our understanding of the mechanisms of Ca 2ϩ storage and release in muscle cells. So far, the major components of the excitationcontraction coupling, such as the ryanodine receptor, the dihydropyridine receptor, and calsequestrin, have been extensively studied in skeletal muscle, and their counterparts have later been identified and characterized in cardiac muscle.Triadin is an intrinsic membrane protein originally identified in skeletal muscle (6 -8). It is specifically enriched in the junctional sarcoplasmic ...

Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants

“…In striated muscle, protein kinases and phosphatases modulate both contraction and relaxation by phosphorylating and dephosphorylating numerous SR proteins that include the luminal Ca 2ϩ -binding protein calsequestrin (37), the Ca 2ϩ pump (38), its regulatory protein phospholamban (39), the RyR-associated proteins triadin (40,41) and sorcin (42), and the Ca 2ϩ release channel/ryanodine receptor (11). Changes in the phosphorylation levels are of interest due to the possibility that these contribute to an impaired SR and contractile function during heart failure.…”

Cardiac Hypertrophy and Impaired Relaxation in Transgenic Mice Overexpressing Triadin 1

“…Ca 2ϩ release in myocardium is controlled by a complex of proteins localized to the junctional SR. These proteins include (a) the Ca 2ϩ release channel or ryanodine receptor, which forms the foot structure on the cytoplasmic surface of the junctional SR membrane; (b) the 55-kDa high capacity Ca 2ϩ -binding protein calsequestrin, located in the lumen of the junctional SR; and (c) the junctional SR transmembrane proteins junctin and triadin, which have been proposed to anchor calsequestrin to the Ca 2ϩ release channel from the lumenal side of the junctional face membrane (1)(2)(3).…”

“…Triadin 1 (cardiac triadin) is a shorter variant of skeletal muscle triadin, which appears to arise from alternative splicing of mRNA transcribed from a common triadin gene (2,10). On SDS-PAGE, cardiac triadin runs as a doublet of 35-and 40-kDa molecular mass proteins, corresponding to deglycosylated and glycosylated mobility forms of the protein, respectively (10).…”

“…On SDS-PAGE, cardiac triadin runs as a doublet of 35-and 40-kDa molecular mass proteins, corresponding to deglycosylated and glycosylated mobility forms of the protein, respectively (10). Both cardiac and skeletal muscle triadin are identical for the first 257 amino acids, then cardiac triadin 1 ends abruptly, incorporating a short C-terminal tail of 21 amino acids that is unique (2,10). The regions common to cardiac and skeletal muscle triadin are the short cytoplasmic segments (residues 2-47), the transmem-* This work was supported by National Institutes of Health Grant HL-28556 (to L. R.…”

“…Notably, the highly charged lumenal domains, which are common to all triadins, are particularly enriched in repeating lysine and glutamic acid residues, organized into "KEKE motifs," which have been proposed to facilitate protein-protein interactions (11)(12)(13). It is not surprising, then, that, like skeletal muscle triadin, the lumenal domain of cardiac triadin has also been shown to bind to both calsequestrin (2, 3, 14 -16) and the ryanodine receptor (2,3). Based on results of immunoprecipation and fusion-protein pulldown assays, a quarternary complex between triadin, junctin, calsequestrin, and the ryanodine receptor was proposed, which may be important for the orchestrated operation of Ca 2ϩ release in both cardiac and skeletal muscle (3).…”