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An α-helical C-terminal tail segment of the skeletal L-type Ca2+ channel β1a subunit activates ryanodine receptor
Yamuna Karunasekara1, Robyn T Rebbeck, Llara M Weaver
1Department of Translational Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia.
Abstract:
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 β(1a) 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 β(1a)V490-M524, corresponding to the C-terminal 35 residues of β(1a), 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 β(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type β(1a)A474-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 β(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.
Insights
Specific hydrophobic residues on the beta-1a subunit C-terminus bind to the ryanodine receptor 1 (RyR1) and enhance its activity, crucial for skeletal muscle excitation-contraction coupling.
Area of Science:
- Muscle physiology
- Molecular biology
- Biochemistry
Background:
- Excitation-contraction coupling in skeletal muscle relies on interactions between the dihydropyridine receptor (DHPR) and the ryanodine receptor 1 (RyR1).
- The precise molecular mechanisms of DHPR-RyR1 interaction and RyR1 activation remain incompletely understood.
Purpose of the Study:
- To investigate the role of the C-terminal 35 residues of the beta-1a subunit in binding to and activating RyR1.
- To identify specific residues within the beta-1a C-terminus critical for RyR1 interaction and function.
Main Methods:
- Synthesized peptides corresponding to the C-terminal region of the beta-1a subunit.
- Performed binding assays with isolated rabbit skeletal muscle RyR1.
- Measured RyR1 channel activity in lipid bilayers using wild-type and mutated peptides.
Main Results:
- A nascent alpha-helix in the beta-1a C-terminus contains a hydrophobic surface formed by residues L496, L500, and W503.
- Mutation of these hydrophobic residues significantly reduced peptide binding to RyR1 and abolished RyR1 activation.
- An upstream heptad repeat in beta-1a, while involved in binding, did not contribute to RyR1 activation.
Conclusions:
- Specific hydrophobic surface residues within the beta-1a C-terminus directly bind to RyR1.
- This binding interaction increases RyR1 channel activity, suggesting a key role in skeletal muscle excitation-contraction coupling.