A novel cytoplasmic interaction between junctin and ryanodine receptor calcium release channels

Abstract

Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

Core skeletal muscle ryanodine receptor calcium release complex

“…Both insert into the SR membrane via a single alpha‐helix, with short cytoplasmic domains that interact with the cytoplasmic “foot” of RyR1 . In the SR lumen, basic residues in the longer C‐terminal domains bind to CSQ and to the minute luminal domain of RyR1 which is formed by the short luminal loops linking the transmembrane helices and contains <1.5% of the total protein mass. In bilayers, luminal addition of either protein increases purified RyR1 activity .…”

“…The luminal junctin‐binding sites are better resolved in RyR2 than RyR1. However junctin likely binds to homologous sites in RyR1 and RyR2 because junctin and its N‐ and C‐terminal components similarly alter RyR1 and RyR2 activity . Therefore the RyR2 findings are relevant to potential RyR1 sites.…”

“…The other region, RyR2‐D includes the RyR2‐equivalent of the Trisk95‐binding residues in RyR1. Within RyR1, Li et al . found that junctin's C‐terminal region (C‐jun, aa46‐210) binds weakly to an extended S1‐S2 linker region and binds more strongly to a construct containing the pore and S1‐S2 regions (Figure ).…”

PharmGKB summary

“…CSQ1 has no transmembrane domains but it remains close to the junctional face of RYR1 by anchoring to the integral membrane proteins junctin and triadin [12, 13]. Triadin binds to an intraluminal loop of RYR1 and is hypothesized to facilitate rapid release of calcium by maintaining CSQ1-bound calcium close to the RYR1 channel pore [12, 14] while junctin has been shown to be necessary for normal RYR1 function and may communicate signals between CSQ1 and RYR1 [15]. …”

Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain

“…Triadin and junctin interactions with RyR1 and RyR2 are also known to be direct, with the RyR2–junctin interaction being Ca 2+ -independent (Guo and Campbell, 1995; Zhang et al, 1997). The triadin KEKE motif interacts with negatively charged residues within the second luminal loop of RyR1 (Goonasekera et al, 2007; Lee et al, 2004), whereas junctin association with the RyR1 or RyR2 involves both luminal and cytoplasmic sites (Altschafl et al, 2011; Li et al, 2015). Evidence for direct RyR–CSQ association is very scarce.…”