Interactions between Dihydropyridine β1A Subunit and Ryanodine Receptor Isoforms

Abstract

Skeletal excitation contraction (EC) coupling requires a physical interaction between the L-type voltage gated dihydropyridine receptor (DHPR) in the transverse tubule membrane and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum membrane. The C-terminus of the DHPR β1a subunit influences EC coupling in skeletal myotubes (Beurg et al., Biophys J. 1999;77:2953-67, Sheridan et al., Biophys J. 2003;84:220-37, Sheridan et al., Biophys J. 2004;87:929-42). This may be through a direct interaction with RyR1, as we identified a hydrophobic interaction with L496, L500 and W503 in the last 35 residues of β1a that increases RyR1 channel activity in phospholipid bilayers (Rebbeck et al., Biophys J. 2011;100;922-30, Karunasekara et al., FASEB J. 2012). Additionally, the K3495KKRR_ _R3502 motif in a RyR1 fragment (M3201-W3661) pulls down β1a and facilitates EC coupling (Cheng et al., PNAS USA. 2005;102:19225-30). Our preliminary data indicate that substitution of these 6 basic residues with glutamines, abolishes the effect of β1a on the full length RyR1. We also show that β1a increases RyR2 activity in a similar manner to RyR1 except for significantly less activation of RyR2 by 10nM β1a (10 and 100nM β1a subunit increased RyR2 activity by 1.8- and 2.8-fold, in contrast to 2.6- and 2.8-fold with RyR1). Curiously, this reduced activation of RyR2 by 10nM β1a is similar to lesser activation of the embryonic alternative spliced (ASI(-)) RyR1 isoform by 10nM β1a, that lacks residues A3481-Q3485, compared with activation of adult (ASI(+)) RyR1 isoform by 10nM β1a. Notably, as rabbit RyR2 lacks 4 of the 5 ASI residues. We conclude that β1a may bind to a hydrophobic pocket conserved in the RyR1 and RyR2 and that this region is influenced by the presence of the alternatively spiced ASI residues and the polybasic K3495-R3502 motif.