BPS2026 – Cav1.2 EF-hand and IQ domain variants of uncertain significance (VUS) affect Ca2+ current and Ca2+ homeostasis differently

Calcium conducted by voltage-gated calcium channels (Ca v 1.2) shapes the duration of the cardiac action potential (AP) and initiates calcium-induced calcium-release and contraction. Therefore, precise regulation of calcium conducted by Ca v 1.2 is crucial for cardiac function. Negative feedback mechanisms that control channel inactivation reside in the C terminus, but the structure of this region is not yet fully understood. To investigate implications of the structure of the EF-hand and IQ domains on channel function, we performed electrophysiological and fluorescent characterization of variants of uncertain significance (VUS), L1520I-, L1521I-, and R1586Q-Cav1.2, each reported independently in patients following syncope or cardiac arrest. Patch-clamp technique was used to record calcium (I Ca ) and barium currents as well as APs in wt and mutated Ca v 1.2 expressed in doxycycline-inducible Flp-In HEK293 cell lines and human iPSC-derived cardiomyocytes (iPSC-CM), respectively. Spontaneous changes in cytosolic calcium concentrations were recorded in fluorescently Fluo4-labeled iPSC-CM monolayers. Prediction of functional effects of VUS was performed using bioinformatic tools. Homology-based models of the mutant version of the EF-hand and IQ domains were prepared. We find that the peak I Ca is diminished in L1521I-Ca v 1.2, V 1/2 is right-shifted, and slope of normalized fractional activation is increased. No changes in the macroscopic I Ca properties were found for the other mutations. Window current parameters were assessed as well. Calcium-dependent inactivation (CDI) was accelerated for L1520I-Ca v 1.2 and delayed for L1521- and R1586Q-. The frequency of spontaneous Ca 2+ transients of iPSC-CM was increased for L1521I-Ca v 1.2; however, the AP duration was unchanged. To date, our data suggest that L1520I, L1521I, and R1586Q alter Ca v 1.2 CDI. We speculate that altered CDI is sufficient to modify the frequency of spontaneous Ca 2+ transients and may be proarrhythmogenic.