In vitro exploration of interactions between junctin and the Ryanodine receptor from skeletal and cardiac muscle

Abstract

Muscle contraction is dependent on a large Ca2+ release from its intracellular Ca2+ store, the Sarcoplasmic Reticulum (SR), through Ryanodine receptor Ca2+ release channels (RyRs). In the resting state, striated muscle requires a tight control of Ca2+ release through RyRs. Another SR bound protein, junctin, is thought to facilitate this. Located in both skeletal and cardiac muscle, junctin has a short N-terminal domain (Njun) and bulky, highly charged C-terminal domain (Cjun), that are exposed to the cytoplasm and SR lumen respectively. Although originally thought to function as a linker between the SR calcium binding protein (calsequestrin) and RyRs, recent studies suggest the junctin may have a more complex role in regulating RyR activity and SR Ca2+ handling. In vitro studies have shown that addition of full-length junctin (FLjun) to the luminal domain of RyR directly activates RyR in lipid bilayers. However, the molecular mechanism by which junctin regulates RyR activity has been unclear. To explore the specific regions of junctin that regulate RyR, constructs corresponding to Njun, Cjun and FLjun were produced. Modified co-immunoprecipitation and affinity chromatography were employed to evaluate the direct interactions between junctin constructs and purified RyR. Both Cjun and Njun bound to the full-length RyRs. The effect of the junctin constructs on RyR1 (skeletal isoform) or RyR2 (cardiac isoform) activity was tested in lipid bilayer experiments with 1 mM luminal [Ca2+] to replicate resting conditions. As found previously, addition of FLjun to the luminal solution increased both RyR1 and RyR2 activity by ~2-3-fold. Curiously, luminal addition of Cjun strongly inhibited both RyR1 and RyR2, whereas cytoplasmic addition of Njun significantly increased RyR1 and RyR2 activity by ~5-6-fold. This was unexpected as the RyR-junctin interaction was assumed to occur only luminally. Neither luminal addition of Njun nor cytoplasmic addition of the scrambled Njun sequence altered channel activity, suggesting a specific cytoplasmic effect of Njun. This suggestion was further substantiated as cytoplasmic Njun did not change the activity of native RyRs that retained endogenous junctin. Sequential addition of Njun to the cytoplasmic solution and Cjun to the luminal solution resulted in channel activity increasing by ~2-3-fold, as observed with luminal addition of FLjun. On the other hand, the RyR inhibition induced by luminal addition of Cjun was not altered by subsequent addition of Njun to the cytoplasmic solution. The functional region of Cjun was localized to residues 85-106 that contain a KEKE motif, as luminal addition of a peptide corresponding to this region replicated the inhibitory effect of Cjun. In conclusion, the study demonstrates that junctin regulates RyR1 and RyR2 via interactions in both the cytoplasm and SR lumen, the combined actions of Njun and Cjun indicate that the novel cytoplasmic Njun-RyR interaction must be established before the luminal Cjun-RyR interaction to replicate the FLjun modulation of RyR1 and RyR2 activity. Show more