The β₁ₐ Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

Abstract

Although it has been suggested that the C-terminal tail of the β₁ₐ subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca²⁺ 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 β₁ₐ bound to RyR1 in affinity chromatography. The full-length β₁ₐ subunit and the C-terminal peptide increased [³H]ryanodine binding and RyR1 channel activity with an AC₅₀ of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca²⁺, ATP, and Mg²⁺ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg²⁺ inhibition or addition of 100 nM Ca²⁺ (without ATP). Maximum increases were seen with 1-10 μM Ca²⁺, in the absence of Mg²⁺ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [³H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β₁ₐ subunit and RyR1 may support an in vivo function of β₁ₐ during voltage-activated Ca²⁺ release.