An α‐helical C‐terminal tail segment of the skeletal L‐type Ca ²⁺ channel β _1a subunit activates ryanodine receptor type 1 via a hydrophobic surface

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.

Domain cooperativity in the β1asubunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle

“…According to our model, β 3 is able to reconstitute proper conformation of the intracellular α 1S loops enabling RyR1 anchoring, but not of the hydrophobic core region, which is required for charge movement function. Additional putative β-RyR1 interaction sites (green arrows) (29,30) have no direct relevance for our model. (C) Replacement of either the SH3 domain or the C terminus of β 3 (orange) by corresponding β 1a sequence (blue) leads to a partial restoration (+/−) of charge movement and thus partial EC coupling, measured as intracellular Ca 2+ release.…”

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

“…As noted above, β 1a residues V490-M508 have been identified as being critical for biochemical interactions with RyR1, the activation of RyR1 in lipid bilayers and the potentiation of EC coupling and L-type Ca 2+ current in FDB fibers (Karunasekara et al, 2012;Hernández-Ochoa et al, 2014). A hydrophobic pocket formed by residues L496/L500/W503 was shown to support these actions of the V490-M508 peptide, but the critical importance of these residues was challenged in an even more recent study in which the authors stably expressed a β 1a L496A/L500A/W503A mutant in a β 1 null cell line (Eltit et al, 2014).…”

“…The results of this study have since been challenged as these combined mutations failed to affect Ca V 1.1 targeting, tetrad formation, charge movement or EC coupling in β 1 null (relaxed) zebrafish myotubes . In addition, short peptides mimicking the β 1a carboxyl terminus with alanine substitutions at positions corresponding to L478, V485, V492 failed to increase RyR1 activity in bilayers (Karunasekara et al, 2012). These challenges have cast grave doubt on the importance of the heptad repeat, but a conclusive test is still lacking.…”

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

“…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). However, it is currently unknown whether the alleged ␤ 1a -RyR1 interaction mediated by this motif plays any role either in the bidirectional signaling between RyR1 and DHPR or in the DHPR/RyR1 physical linkage that supports DHPR tetrad arrays.…”

“…Fitting parameters are summarized in Table 1. , and Trp 503 of the ␤ 1a C terminus region has been recently identified in in vitro studies as a putative region of interaction with the RyR1 (21). To test whether the absence of residues Leu 496 -Leu 500 -Trp 503 in our construct ␤ Ϫ36 is directly responsible for the failure of this truncated subunit to restore EC coupling, we designed the construct ␤ LLW (Fig.…”