Amino-terminus oligomerization regulates cardiac ryanodine receptor function

Abstract

SummaryThe ryanodine receptor (RyR) is an ion channel composed of four identical subunits mediating calcium efflux from the endo/ sarcoplasmic reticulum of excitable and non-excitable cells. We present several lines of evidence indicating that the RyR2 N-terminus is capable of self-association. A combination of yeast two-hybrid screens, co-immunoprecipitation analysis, chemical crosslinking and gel filtration assays collectively demonstrate that a RyR2 N-terminal fragment possesses the intrinsic ability to oligomerize, enabling apparent tetramer formation. Interestingly, N-terminus tetramerization mediated by endogenous disulfide bond formation occurs in native RyR2, but notably not in RyR1. Disruption of N-terminal inter-subunit interactions within RyR2 results in dysregulation of channel activation at diastolic Ca 2+ concentrations from ryanodine binding and single channel measurements. Our findings suggest that the N-terminus interactions mediating tetramer assembly are involved in RyR channel closure, identifying a crucial role for this structural association in the dynamic regulation of intracellular Ca 2+ release.

Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification

“…Ion channels are known to possess disulfide bonds to provide a structural scaffold or redox sensitivity. These include ion channels such as NMDA receptor, CLIC1, Kir2.2, cardiac ryanodine receptor, TWIK-1 channel, and TRPC5 (47)(48)(49)(50)(51)(52)(53). However, to our knowledge this is the first observation in which an ion channel uses the redox heterogeneity of a single specific Cys residue (Cys-258) to provide both oxidation sensitivity and protein stability.…”

N-terminus oligomerization is conserved in intracellular calcium release channels

“…The Y2H system, HEK (human embryonic kidney)-293 cell culture and transfection, chemical cross-linking, co-immunoprecipitation, SR preparation and Western blotting analysis were carried out as described previously [16–18]. Densitometry analysis was performed using a GS-700 scanner (Bio-Rad Laboratories) and Quantity One software (Bio-Rad Laboratories).…”

“…We have recently reported that the cardiac RyR2 N-terminus is found in tetrameric form, which helps regulate the closed channel conformation during the diastolic phase of cardiomyocyte contraction [16]. In the present study, we provide evidence that N-terminus tetramerization is a structural feature that is conserved across the three mammalian RyR isoforms and, significantly, this innate ability to oligomerize into tetramers is observed also in the N-terminus of the related IP 3 R1 intracellular Ca 2+ release channel.…”

Functional Impact of Ryanodine Receptor Oxidation on Intracellular Calcium Regulation in the Heart

“…In light of recent work, the susceptibility for intersubunit cross-linking appears to be increased for the cardiac isoform of RyR2 (Zissimopoulos et al 2013). Although some work has been done to examine the effects of intersubunit cross-linking on RyR2 function, no studies have examined its role in SR Ca 2+ cycling within the cellular environment.…”

“…Recently, a study done by Zissimopoulos et al provides some biochemical evidence to support this hypothesis for both RyR1 and RyR2. The major limitation to this study, however, was that full-length RyR was not used in the experimental approach (Zissimopoulos et al 2013). Their work shows that N-terminal fragments of RyR2 self-assemble into oligomers similar to that of RyR1.…”