A variably spliced region in the type 1 ryanodine receptor may participate in an inter-domain interaction

Abstract

The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.

Null mutations causing depletion of the type 1 ryanodine receptor (RYR1) are commonly associated with recessive structural congenital myopathies with cores

“…The p.Ala2421Pro and p.Met2423Lys mapped to exon 45 in the MH2 domain of the protein [Monnier et al, 2005;Robinson et al, 2006]. The p.Arg3539His variant is localized within a protein domain that has been involved in protein-protein interaction Dulhunty et al, 2002;Kimura et al, 2007] and lies in the vicinity of the p.Pro3527Ser mutation, a recessive MmD mutation [Ferreiro et al, 2002b] associated with a decreased release of Ca 21 upon activation of RYR1 when expressed at a homozygous level [Ducreux et al, 2006]. Both p.Val4842Met and p.Val4849Ile variants substituted very wellconserved amino acids and mapped to the M8 trans-membrane fragment of the calcium channel [Du et al, 2002].…”

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

“…[ 3 H]Ryanodine Binding and Single Channel Recording of Purifi ed RyRs WT and ∆M 1,2,3 RyR1 constructs were expressed and purifi ed from HEK293 cells as previously described (Kimura et al, 2005;Kimura et al, 2007). [ 3 H]Ryanodine binding was performed as previously described (Kimura et al, 2005), except that 5mg of purifi ed RyR1 protein was used in all experiments and in some experiments preincubation with triadin (5μg/ml) was done for 30min on ice, before initiating [ 3 H]Ryanodine binding.…”

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition

“…The splice variants differ in only five residues in >5000 and would not be separated on SDS-PAGE. In addition, the ASI region in the RyR1 protein is inaccessible to an antibody that detects a peptide corresponding to ASI(+) and surrounding residues [20]. This is likely because the ASI site is located in the closely packed helical domain 2 of RyR1 which is also partly occluded by the SPRY and P2 domains [36].…”

“…A decrease in overall RyR1 levels (determined by 3[H]Ryanodine binding) with age has been reported in mixed and fast-twitch fibers of rats and mice [28,29]. However, other 3 [H]Ryanodine binding and western blot studies report no change in RyR1 expression in fast-and slow-twitch muscle from aged rats [8,20,26]. We find that there is no correlation between age and RyR1 expression in the human muscles investigated in this study (Online resource 4).…”