DUAL REGULATION OF THE SKELETAL-MUSCLE RYANODINE RECEPTOR BY TRIADIN AND CALSEQUESTRIN

Triadin, a calsequestrin-anchoring transmembrane protein of the sarcop lasmic reticulum (SR), was successfully purified from the heavy fracti on of SR (HSR) of rabbit skeletal muscle with an antitriadin immunoaff inity column. Since depletion of triadin from solubilized HSR with the column increased the [H-3]ryanodine binding activity, we tested a pos sibility of triadin for a negative regulator of the ryanodine receptor /Ca2+ release channel (RyR). Purified triadin not only inhibited [H-3] ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kas ai (1994) Biochem. Biophys. Res. Commun. 199. 1120-1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181-1185] potentiated [H-3]ry anodine binding to the solubilized HSR. Ca2+ dependency of [H-3]ryanod ine binding to the solubilized HSR was reduced by triadin, whereas tha t was enhanced by calsequestrin. Interestingly, [H-3]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by tr iadin. Immunostaining with anti-triadin antibody proved that calseques trin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR i s regulated by both triadin and calsequestrin, probably through an int eraction between them, In this paper, triadin has been first demonstra ted to have an inhibitory role in the regulatory mechanism of the RyR.