Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.
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.
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