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

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

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance

Skeletal muscle excitation-contraction coupling: Who are the dancing partners?

There is an overwhelming body of work supporting the idea that excitation-contraction coupling in skeletal muscle depends on a physical interaction between the skeletal muscle isoform of the dihydropyridine receptor L-type Ca2+ channel and the skeletal isoform of the ryanodine receptor Ca2+ release channel. A general assumption is that this physical interaction is between "critical" residues that have been identified in the II-III loop of the dihydropyridine receptor alpha subunit and the ryanodine receptor. However, despite extensive searches, the complementary "critical" residues in the ryanodine receptor have not been identified. This raises the possibility that the coupling proceeds either through other subunits of the dihydropyridine receptor and/or other co-proteins within the large RyR1 protein complex. There have been some remarkable advances in recent years in identifying proteins in the RyR complex that impact on the coupling process, and these are considered in this review. A major candidate for a role in the coupling mechanism is the beta subunit of the dihydropyridine receptor, because specific residues in both the beta subunit and ryanodine receptor have been identified that facilitate an interaction between the two proteins and these also impact on excitation-contraction coupling. This role of beta subunit remains to be fully investigated as well as the degree to which it may complement any other direct or indirect voltage-dependent coupling interactions between the DHPR alpha II-III loop and the ryanodine receptor

An alpha-helical C-terminal tail segment of the skeletal L-type Ca2+ channel beta1a subunit activates ryanodine receptor type 1 via a hydrophobic surface

Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the β1a subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β1aV490-M524, corresponding to the C-terminal 35 residues of β1a, reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in β1a binding to RyR1 does not contribute to RyR1 activation. Wild-type β1aA474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β1a C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling

CaMKIIδ post-translational modifications increase affinity for calmodulin inside cardiac ventricular myocytes

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)-including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation-have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ-the primary cardiac isoform-has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca]free and deactivates upon Ca-CaM dissociation when [Ca]free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Förster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca]free. This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca]free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca]free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics-measured here in myocytes-are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart