Trisk 32 regulates IP3 receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation-contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6. G8 myoblasts. Colocalization of Trisk 32 and IP3 receptors (IP3R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP3-mediated Ca2+ release were assessed by measuring changes in [Ca2+](i) following the stimulation by bradykinin or vasopressin. The amplitude of the Ca2+ transients evoked by 20 mu M bradykinin was significantly higher in Trisk 32-overexpressing (p< 0.01; 426 +/- 84 nM, n= 27) as compared to control cells (76 +/- 12 nM, n= 23). The difference remained significant (p< 0.02; 217 +/- 41 nM, n= 21, and 97 +/- 29 nM, n= 31, respectively) in the absence of extracellular Ca2+. Similar observations were made when 0.1 mu M vasopressin was used to initiate Ca2+ release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca2+ entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin-or vasopressin-evoked Ca2+ transients; rather, they were due to the enhanced activity of IP3R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca2+ release via IP3R.