Role of C-Term Tail of DHPR β1A in the DHPR/RyR1 Interaction

Although the mechanism by which the DHPR β1a subunit supports EC-coupling is still debatable it is apparent that C-terminal domain of β1a (β-Ct) is an intricate component of the interaction between the DHPR complex and RyR1. To characterize the molecular components of β-Ct involved in DHPR/RyR1 signaling we tested the effect of progressive truncation of β1a subunit on EC-coupling and RyR1 activity. To do this cDNA constructs carrying truncations of either the 52 (β-52), 38 (β-38) or 14 (β-14) most C-terminal amino acid residues of β1a were expressed in β-null myotubes and then tested for their ability to restore depolarization-induced Ca2+ release in Fluo-4 loaded cells. Whereas β-null myotubes expressing constructs β-52 and β-38 were unresponsive to K+ depolarization the cells expressing β-14 displayed EC-coupling that was indistinguishable from that of cells expressing wt-β1a (K+EC50: β1a = 23mM, β-14 =21mM). Thus, these results identify a segment of β-Ct of up to 24 amino acids that appears to be critical for the functional interaction between DHPR and RyR1 during EC-coupling. To test for specific interactions between the β-Ct and RyR1 we then studied the effect of purified β subunits on RyR1 activity. Using 3H-ryanodine binding to SR vesicles from skeletal muscle we found that purified wt-β1a subunit had an inhibitory effect in RyR1 activity in a dose-dependent manner (IC50=38nM). However, purified β-38, a construct that failed to restore EC-coupling in myotubes, failed to inhibit 3H-ryanodine binding to RyR1. Overall, these results are consistent with the hypothesis that DHPR β1a subunit functionally interacts with RyR1 through a domain in the final 38 amino acids in its C-terminal tail.Supported by NIH Grants 5K01AR054818-02 (to CFP) and 1P01AR044750 (to PDA)