Identification of triadin 1 as the predominant triadin isoform expressed in mammalian myocardium

Abstract

Triadin is an integral membrane protein of sarcoplasmic reticulum shown to interact with the ryanodine receptor/Ca2+ 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 Ca2+ release in heart.