Abstract
Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR)
low-affinity Ca-binding protein, with mutations that are associated
with catecholamine-induced polymorphic ventricular tachycardia (CPVT).
To better understand how CASQ2 mutants cause CPVT, we expressed two
CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2(DEL))
or CASQ2 containing a point mutation (CASQ2(R33Q)), in canine ventricular
myocytes and assessed their effects on Ca handling. We also measured
CASQ2-CASQ2 variant interactions using fluorescence resonance transfer
in a heterologous expression system, and evaluated CASQ2 interaction
with triadin. We found that expression of CASQ2(DEL) or CASQ2(R33Q)
altered myocyte Ca signaling through two different mechanisms. Overexpressing
CASQ2(DEL) disrupted the CASQ2 polymerization required for high capacity
Ca binding, whereas CASQ2(R33Q) compromised the ability of CASQ2
to control ryanodine receptor (RyR2) channel activity. Despite profound
differences in SR Ca buffering strengths, local Ca release terminated
at the same free luminal Ca in control cells, cells overexpressing
wild-type CASQ2 and CASQ2(DEL)-expressing myocytes, suggesting that
a decline in Ca(SR) is a signal for RyR2 closure. Importantly,
disrupting interactions between the RyR2 channel and CASQ2 by expressing
CASQ2(R33Q) markedly lowered the Ca(SR) threshold for Ca release
termination. We conclude that CASQ2 in the SR determines the magnitude
and duration of Ca release from each SR terminal by providing both
a local source of releasable Ca and by effects on luminal Ca-dependent
RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which
cause mechanistically different defects in CASQ2 and RyR2 function,
lead to increased diastolic SR Ca release events and exhibit a similar
CPVT disease phenotype.
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