In Vitro Interactions Between the Beta1a Subunit of the Skeletal Muscle Dhpr and RyR1

Abstract

The beta1a subunit of the skeletal muscle DHPR plays two important roles in excitation-contraction (EC) coupling in skeletal muscle. Beta1a was originally found to target the alpha1S subunit of the DHPR to sarcolemmal tetrads which oppose skeletal ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. It was later found to also aid in transmission of EC coupling between the alpha1S subunit and RyR1 (1,2,3), through its C-terminal tail residues (1,2). This presumably depends on an interaction between the C-terminal tail of beta1a and RyR1. The full beta1a subunit binds to RyR1 in affinity chromatography experiments (4), but direct binding of the C-tail to RyR1 has not been reported, nor have direct functional interactions between the proteins. We show here that a peptide corresponding to the native sequence of residues in the C-terminus of beta1a, but not a scrambled sequence, bind to RyR1. In addition the peptide and the full β1a subunit significantly increase both [3H]ryanodine binding and single RyR1 channel activity at concentrations of 0.1 to 1nM, with maximum 3- to 5-fold activation at a concentration of ∼10nM. The increase in RyR1 channel activity with both constructs is essentially irreversible within the lifetime of the bilayer, indicating tight binding. These results show that the C-terminal tail of the beta1a subunit is capable of directly binding to and activating RyR1 and hence suggest that beta1a may enhance EC coupling by virtue of its ability to activate RyR1.1. Beurg M et al. Biophys J1999 77, 2953-2967.2. Zhou W et al. Cell Calcium 2006 39, 227-236.3. Schredelseker J et al. J Biol Chem 2009 284, 1242-51.4. Cheng W et al. Proc Natl Acad Sci 2005 102, 19225-19230.