Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit.

Robyn T Rebbeck,Philip G Board,Linda Groom,Nicole A Beard,Hermia Willemse,Marco G Casarotto,Angela F Dulhunty,Robert T Dirksen

doi:10.1186/s13395-015-0049-3

Abstract

BackgroundAlthough excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.MethodsWe used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes.ResultsWe demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10–100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a 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 β1a 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 Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling.ConclusionsWe conclude that the β1a 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 materialThe online version of this article (doi:10.1186/s13395-015-0049-3) contains supplementary material, which is available to authorized users.

Highlights

Excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown
Ability of the β1a subunit to activate different RyR isoforms The β1a subunit activates RyR1 and cardiac ryanodine receptor (RyR2) channels As we reported previously [15], when added to the cytoplasmic cis chamber, the full-length β1a subunit increases the activity of native RyR1 channels incorporated into planar lipid bilayers (Fig. 1a)
Our results demonstrate that the functional effect of 100 nM β1a subunit is conserved between RyR1 and RyR2, the activation by 10 nM β1a was lower in RyR2 than in RyR1

Summary

Introduction

Excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. Overexpression of a β subunit interacting protein, Rem, in adult mouse skeletal muscle fibers was recently shown to reduce voltage-induced Ca2+ transients by ~65 % without substantially altering α1S subunit membrane targeting or intramembrane gating charge movement or SR Ca2+ store content [20]. This suggests that the DHPR-RyR1 interaction may be uncoupled by virtue of direct interference of β1a subunit. It is unlikely that basic residue binding to the hydrophobic residues could contribute to EC coupling, both basic residues and hydrophobic residues in the β1a C-terminus influence EC coupling [16, 19]

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Results

Discussion

Conclusion