Endoplasmic reticulum Ca2+ measurements reveal that the cardiac ryanodine receptor mutations linked to cardiac arrhythmia and sudden death alter the threshold for store-overload-induced Ca2+ release

Abstract

A number of RyR2 (cardiac ryanodine receptor) mutations linked to ventricular arrhythmia and sudden death are located within the last C-terminal approximately 500 amino acid residues, which is believed to constitute the ion-conducting pore and gating domain of the channel. We have previously shown that mutations located near the C-terminal end of the predicted TM (transmembrane) segment 10, the inner pore helix, can either increase or decrease the propensity for SOICR (store-overload-induced Ca2+ release), also known as spontaneous Ca2+ release. In the present study, we have characterized an RyR2 mutation, V4653F, located in the loop between the predicted TM 6 and TM 7a, using an ER (endoplasmic reticulum)-targeted Ca2+-indicator protein (D1ER). We directly demonstrated that SOICR occurs at a reduced luminal Ca2+ threshold in HEK-293 cells (human embryonic kidney cells) expressing the V4653F mutant as compared with cells expressing the RyR2 wild-type. Single-channel analyses revealed that the V4653F mutation increased the sensitivity of RyR2 to activation by luminal Ca2+. In contrast with previous reports, the V4653 mutation did not alter FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide)-RyR2 interaction. Luminal Ca2+ measurements also showed that the mutations R176Q/T2504M, S2246L and Q4201R, located in different regions of the channel, reduced the threshold for SOICR, whereas the A4860G mutation, located within the inner pore helix, increased the SOICR threshold. We conclude that the cytosolic loop between TM 6 and TM 7a plays an important role in determining the SOICR threshold and that the alteration of the threshold for SOICR is a common mechanism for RyR2-associated ventricular arrhythmia.

The Cytoplasmic Region of Inner Helix S6 Is an Important Determinant of Cardiac Ryanodine Receptor Channel Gating

“…To assess whether the V4880A mutation affects the SOICR threshold, we measured the endoplasmic reticulum (ER) luminal Ca 2ϩ dynamics using the ER luminal Ca 2ϩ sensing protein D1ER (22,24). As shown in Fig.…”

“…The V4880A Mutation Reduces the Threshold for SOICR-SOICR is thought to occur when the store Ca 2ϩ load exceeds a threshold level (SOICR threshold) (17)(18)(19)(20)(21)(22)(23). To assess whether the V4880A mutation affects the SOICR threshold, we measured the endoplasmic reticulum (ER) luminal Ca 2ϩ dynamics using the ER luminal Ca 2ϩ sensing protein D1ER (22,24).…”

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

“…As shown in Fig. 7, increasing the extracellular Ca 2ϩ concentration from 0 to 2 mM induced spontaneous ER Ca 2ϩ oscillations in RyR2 WT-expressing HEK293 cells (shown as downward deflections of the FRET signal), as reported previously (26,28,29). SOICR occurred when the ER luminal Ca 2ϩ content reached a threshold level (F SOICR ) and terminated when the ER luminal Ca 2ϩ content decreased to another threshold level (F termi ) (Fig.…”

“…The SOICR activation and termination thresholds were assessed by monitoring the ER luminal Ca 2ϩ dynamics using a FRET-based ER luminal Ca 2ϩ -sensing protein, D1ER (25,26). As shown in Fig.…”

“…Single -sensitive chameleon protein D1ER, as described previously (25,26). The cells were grown to 95% confluence in a 75-cm 2 flask, dissociated with PBS, and plated in 100-mm-diameter tissue culture dishes at ϳ10% confluence 18 -20 h before transfection with D1ER cDNA using the Ca 2ϩ phosphate precipitation method.…”