Regulation of Wild Type and CPVT-Linked Mutant Cardiac Ryanodine Receptors by Junctin and Calsequestrin

Abstract

The human cardiac ryanodine receptor (hRyR2) undergoes luminal regulation by accessory proteins including junctin (JUN) and calsequestrin (CSQ2). Furthermore, it has been suggested that defective luminal Ca2+ sensing by RyR2 is a candidate mechanism in the pathogenesis of catecholaminergic polymorphic ventricular tachycardia - an arrhythmogenic disorder caused by mutation of RyR2 or CSQ2. As such, we evaluated the effects of CSQ2 and JUN on mutant channel behaviour. [3H]-ryanodine binding was used to evaluate the cytosolic Ca2+ activation of wild-type (WT) or novel CPVT mutant (A4556T, positioned close to a JUN binding site) which were recombinantly (co)expressed in the presence/absence of CSQ2 and/or JUN in HEK293 cells, while store overload-induced Ca2+-release (SOICR) events were monitored to ascertain luminal Ca2+ effects. Co-immunoprecipitation experiments demonstrated that WT and mutant proteins showed equal association with both luminal accessory proteins. Co-expression of WT RyR2 with CSQ2 alone did not affect the amplitude, frequency or rate of release of SOICR events while preliminary experiments show that co-expression with JUN (alone and in combination with CSQ2) decreased the amplitude and increased the frequency of events, in line with observations that JUN both inhibits and activates RyR2 depending on SR Ca2+ levels. Co-expression of both accessory proteins significantly decreased the rate of release. Activation of WT RyR2 by cytosolic Ca2+ was not affected by the presence of either/both accessory proteins. A4556T showed enhanced activation in response to cytosolic Ca2+ that decreased to WT levels (at low Ca2+ concentrations only) in the presence of both JUN and CSQ2. This indicates that regulation by JUN and CSQ2 to some extent attenuates mutant dysfunction and will be further investigated by monitoring Ca2+ release events and single channel analysis. This work is supported by the BHF.