The Ser96Ala (S96A) mutation within the histidine rich Ca(2+) binding protein 
(HRC) has recently been linked to cardiac arrhythmias in idiopathic dilated 
cardiomyopathy patients, potentially attributable to an increase in spontaneous 
Ca(2+) release events. However, the molecular mechanism connecting the S96A 
mutation of HRC to increased Ca(2+) release events remains unclear. Previous 
findings by our group indicate that these spontaneous Ca(2+) release events may 
be linked to store overload induced Ca(2+) release (SOICR) via the cardiac 
ryanodine receptor (RyR2). Therefore, in the present study we sought to 
determine whether HRC wild type (HRC WT) and S96A mutant (HRC S96A) expression 
has a direct effect on SOICR. Using both cytosolic and intra-Ca(2+) store 
measurements in human embryonic kidney cells expressing RyR2, we found that HRC 
WT significantly inhibited the propensity for SOICR by buffering store free 
Ca(2+) and inhibiting store Ca(2+) uptake. In contrast, HRC S96A exhibited a 
markedly suppressed inhibitory effect on SOICR, which was attributed to an 
impaired ability to buffer store Ca(2+) and reduce store Ca(2+) uptake. In 
addition to impairing the ability of HRC to regulate bulk store Ca(2+), a 
proximity ligation assay demonstrated that the S96A mutation also disrupts the 
Ca(2+) microdomain around the RyR2, as it alters the Ca(2+) dependent 
association of RyR2 and HRC. Importantly, in contrast to previous reports, the 
absence of triadin in our experimental model illustrates that the S96A mutation 
in HRC can alter the propensity for SOICR without any interaction with triadin. 
Collectively, our results demonstrate that the human HRC mutation S96A leads to 
an increase in spontaneous Ca(2+) release and ultimately arrhythmias by 
disrupting the regulation of intra-store free Ca(2+). This is primarily due to 
an impaired ability to act as an effective bulk and local microdomain store 
Ca(2+) buffer.