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 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.

Bridging the myoplasmic gap II: more recent advances in skeletal muscle excitation–contraction coupling

“…Following closely on the heels of the generation of the β 1 null mouse, a series of studies employing expression of β 1a deletion mutants and β 1a -β 2a chimeras in null mouse myotubes demonstrated that the distal carboxyl terminus of β 1a was a critical component for the maintenance of skeletal-type EC coupling (Beurg et al, 1999;Sheridan et al, 2003aSheridan et al, ,b, 2004. In broad agreement with these data, a carboxyterminal fragment of β 1a corresponding to its final 35 residues (V490-M524) was shown to stick to RyR1 in vitro and to promote channel opening in planar lipid bilayers (Rebbeck et al, 2011). A similar, but shorter peptide (V490-M508) activated RyR1 in a later study (Hernández-Ochoa et al, 2014).…”

Calcium signaling at ER membrane contact sites

“…In cardiac muscle, upon excitation, cytosolic Ca 2+ is rapidly increased by coordinated DHPR and RyR channel opening that couples Ca 2+ influx from the extracellular space with release from SR stores, in a process of Ca 2+ induced Ca 2+ release (CICR). In skeletal muscle, a voltage-dependent conformational change in DHPR causes opening of RyRs through Ca 2+ -independent mechanical coupling of DHPRs with the cytoplasmic domain of the RyRs at MCSs, in a process termed voltage-initiated Ca 2+ release (VICR) [3,51].…”

Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor

“…The disruption of this region not only prevents ␤ 1a -RyR1 interactions in vitro but also weakens electrically evoked Ca 2ϩ release signals in cultured myotubes (41). Interestingly, ␤ 1a peptides from the same C-terminal region identified in our study were found to activate native and purified RyR1 channels fused into lipid bilayers (20,21). Moreover, microinjection of these peptides into flexor digitorum brevis fibers was reported to specifically enhance depolarization-induced Ca 2ϩ release (42), further supporting the idea that the C-terminal domain of ␤ 1a subunit directly interacts with RyR1 and that this interaction is involved in EC coupling signaling.…”

“…However, because no structural information was obtained in these studies, it is unclear whether the effects of ␤ 1a in EC coupling are simply due to an effect on DHPR positioning. Recent in vitro studies have shown that peptides fragments from the C-terminal domain of ␤ 1a modulate RyR1 channel function, giving support to the idea of a direct functional interaction between ␤ 1a and RyR1 (20). Mutational analysis of these peptides identified the critical motif responsible for RyR1 activation in a hydrophobic pocket formed by amino acid residues Leu 496 -Leu 500 -Trp 503 (21).…”