Identification of triadin 1 as the predominant triadin isoform expressed in mammalian myocardium.
Triadin is an integral membrane protein of sarcoplasmic reticulum shown to interact with the ryanodine receptor/Ca(2+) release channel, junctin, and calsequestrin. Several triadin isoforms have been postulated to exist in cardiac muscle, but to date none has been conclusively identified. Here, we show that only triadin 1 is significantly expressed. We cloned and sequenced cDNAs encoding canine cardiac triadin 1 and 3 but found no evidence for triadin 2. From deduced primary structures, antibodies against domains common to all triadins and an antibody against the unique C terminus of triadin 1 were raised. All antibodies detected two prominent proteins of molecular masses 35 and 40 kDa on immunoblots from cardiac microsomes, including the antibody that recognizes only triadin 1. The 40-kDa mobility form was shown to correspond to the glycosylated form of triadin 1, not a distinct triadin 2 isoform as previously hypothesized. Confirming this, overexpression of triadin 1 in transgenic mouse hearts produced both the 35-kDa deglycosylated and the 40-kDa glycosylated mobility forms. The glycosylation site of triadin 1 was localized to asparagine residue 75, and its bitopic arrangement in the membrane was confirmed. Although a 92-kDa immunoreactive protein could be tentatively identified in myocardium as triadin 3, its expression level was insignificant (</=5%) compared with that of triadin 1. We conclude that triadin 1 is the triadin isoform most likely to play a role in Ca(2+) release in heart.
Kobayashi
Y M
YM
Department of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Jones
L R
LR
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004285:Dogs; D006168:Guinea Pigs; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C498269:TRDN protein, human; C498270:Trdn protein, mouse; C498271:Trdn protein, rat; C070048:triadin