A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Linwei Li,Shamaruh Mirza,Chris Thekkedam,Spencer J Richardson,Esther M Gallant,Angela F Dulhunty,Dan Liu,Francesco Zorzatto,Nicole A Beard,Suzy M Pace

doi:10.1242/jcs.160689

Abstract

ABSTRACTJunctin, 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.

Highlights

Contraction in the heart and skeletal muscle depends on the release of Ca2+ from the intracellular sarcoplasmic reticulum (SR) Ca2+ store
The efficacy of Ca2+ release depends an influence of the ‘Ca2+ load’ in the SR on the activity of ryanodine receptor (RyR) Ca2+ release channels, which is facilitated by a Ca2+dependent interaction between CSQ proteins and the RyR through the ‘anchoring’ protein junctin, which is a non-catalytic splice variant encoded by the aspartate-bhydroxylase (Asph) gene
Full-length junctin, and its C-terminal and N-terminal domains bind to RyR1 and RyR2 Co-immunoprecipitation and affinity chromatography were used to determine the molecular nature of the association between junctin and RyRs

Summary

Introduction

Contraction in the heart and skeletal muscle depends on the release of Ca2+ from the intracellular sarcoplasmic reticulum (SR) Ca2+ store. The efficacy of Ca2+ release depends an influence of the ‘Ca2+ load’ in the SR on the activity of ryanodine receptor (RyR) Ca2+ release channels, which is facilitated by a Ca2+dependent interaction between CSQ proteins ( known as CASQ) and the RyR through the ‘anchoring’ protein junctin, which is a non-catalytic splice variant encoded by the aspartate-bhydroxylase (Asph) gene Different isoforms of the RyR, CSQ and triadin are expressed in cardiac and skeletal muscle, whereas the same isoform of junctin is expressed in both muscle types

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Discussion

Conclusion