Interactions between the dihydropyridine receptor beta1a subunit and the ryanodine receptor from skeletal muscle

Abstract

Excitation-contraction (EC) coupling describes the process that links the excitatory action potential to muscle fibre contraction. Essential to this process is the release of Ca2+ from the sarcoplasmic reticulum (SR) via the ligand gated ryanodine receptor (RyR) in the SR membrane. In cardiac muscle fibres, RyR2 (cardiac isoform) activation is initiated by Ca2+ entry through the cardiac L-type voltage-gated dihydropyridine receptor (DHPR). In contrast, EC coupling in skeletal muscle fibres requires a physical interaction between skeletal DHPR and RyR1 (skeletal isoform), although the physical components of this interaction are unclear. It had previously been shown that the C-terminus of the DHPR beta1a subunit strongly influences EC coupling in skeletal myotubes, since mutation of a heptad repeat motif (L478, V485 and V492) and C-terminal truncations > 29 residues reduce EC coupling. It has also been shown that the role of the C-terminal residues in EC coupling is likely via direct interaction with RyR1, because a polybasic K3495-R3502 motif in a RyR1 fragment (M3201-W3661) is important for beta1a association in pull-down assays and this region influences EC coupling in mouse myotubes. My previous work showed that a peptide (beta1a V490-M524) corresponding to the extreme 35 C-terminal residues directly increases RyR1 activity in planar lipid bilayers to the same level as beta1a subunit. One third of this peptide adopts an alpha-helix with a hydrophobic surface (residues L496, L500 and W503) on one side, which provides a putative RyR1 binding site. For this thesis I investigated the relative importance of the beta1a C-terminal heptad repeat and hydrophobic surface residues, and the RyR1 K3495-R3502 polybasic motif in the action of beta1a on RyR1 in lipid bilayers. I also compared the action of beta1a between RyR1 and RyR2, which was of particular interest given their systematic differences in receiving the EC coupling signal from DHPR. Cytosolic exposure of beta1a A474-A508 peptide (containing both the heptad repeat and hydrophobic surface residues) to native RyR1 channels increased channel activity by 2-fold, which was similar to the action of beta1a V490-M524 peptide. Alanine substitution of heptad repeat residues did not alter the action of beta1a A474-A508 peptide on RyR1. In contrast, alanine substitution of hydrophobic surface residues abolished the action of beta1a V490-M524 on RyR1 and reduced pull-down of RyR1 by 85%. Curiously, individual substitution of the hydrophobic surface residues abolished the effect of the beta1a V490-M524 peptide at +40 mV, but not at -40 mV. Overall, the results show that the modulatory action of beta1a on RyR1 depends on all three beta1a hydrophobic surface residues, but not the heptad repeat. The action of beta1a on RyR1 was abolished when the six basic residues in the RyR1 K3495-R3502 region were neutralised by mutation to glutamine residues. In addition and intriguingly, the beta1a subunit increased RyR2 activity in a similar manner as RyR1. This suggests that beta1a may bind to a hydrophobic pocket conserved in RyR1 and RyR2 and that is influenced by the presence of the polybasic K3495-R3502 motif.