Independent Actions Of Junctin And Triadin On Skeletal Muscle RyR1 Channels

Abstract

Two similar proteins, junctin and triadin, are associated with the membrane of the sarcoplasmic reticulum (SR) Ca2+ store in skeletal muscle and have been thought to serve a common function in anchoring the Ca2+ binding protein calsequestrin (CSQ1) to the type 1 ryanodine receptor (RyR1). The resulting CSQ1/junctin/triadin/RyR1 complex is thought to mediate a functional response in the ion channel which conserves the Ca2+ stored within the SR. The individual actions of triadin and junctin on RyR1 and their contribution to the functional interaction between CSQ1 and RyR1 have not previously been examined and are reported here. Highly purified triadin or junctin added to the luminal side of purified RyR1 channels in lipid bilayers caused an increase in channel open time and stabilized channel openings to the maximum conductance. Competition studies indicated that triadin and junctin exert independent actions on RyR1. In addition, purified CSQ1 inhibited junctin/triadin-associated or junctin-associated, but not triadin-associated, RyR1 channels in the presence of 1mM luminal Ca2+. As with native RyR1, purified RyR1 channels associated with either CSQ1/triadin/junctin, or CSQ1/junctin were further inhibited when luminal [Ca2+] was reduced from 1mM to ≤100μM, in the same way as the native RyR1 channel complex. In marked contrast, the channel activity of the CSQ1/triadin/RyR1 complex increased when luminal Ca2+ was lowered, in a similar manner to the un-associated purified RyR1. These results indicate that junctin alone is responsible for mediating signals between luminal Ca2+, CSQ1 and RyR1 and that triadin does not contribute to this function. Other evidence indicates that triadin plays an independent role in supporting Ca2+ release during excitation-contraction coupling (Goonasekara et al. 2007 J. gen. Physiol. 130, 365; Wang et al. 2008 Cell Calcium in press).