Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β(1a) subunit

BACKGROUND: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β(1a) subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β(1a) activation of RyRs and the β(1a) binding site on RyR1. METHODS: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca(2+) currents and Ca(2+) transients in myotubes. RESULTS: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10–100 nM of the β(1a) subunit. Activation of RyR2 by 10 nM β(1a) was less than that of RyR1, suggesting a reduced affinity of RyR2 for β(1a). A reduction in activation was also observed when 10 nM β(1a) was added to the alternatively spliced (ASI(−)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β(1a) activation observed for both RyR2 and ASI(−)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca(2+) transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β(1a) subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β(1a) subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca(2+) release generated during skeletal EC coupling. CONCLUSIONS: We conclude that the β(1a) subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13395-015-0049-3) contains supplementary material, which is available to authorized users.