Background: Alternating hemiplegia of childhood (AHC) is a rare de novo syndrome that manifests with episodic hemiplegia, seizures, dystonia, and, notably, sudden unexplained death. Gene positive patients carry a pathogenic variant in the ATP1A3-encoded Na/K ATPase alpha 3 isoform (ATP1A3), and the variant D801N is the most common pathogenic variant. Our group recently found that AHC patients with ATP1A3-D801N missense have short QTc and are at risk of ventricular fibrillation.Hypothesis: We hypothesized that the D801N variant results in reduced ATPase function leading to Ca overload resulting in shortened repolarization time and increased arrhythmogenic risk.Methods: We differentiated iPSCs into cardiomyocytes from an AHC patient hosting the ATP1A3-D801N variant (iPSC-CM), and an ostensibly healthy individual (iPSC-CM). Patch clamp was conducted to determine APD90, and the ratio of cells with delayed after depolarization events (DADs). iPSC-CM were perfused with 10M ouabain, an ATP1A3 inhibitor, and APD90 and DADs were measured. SR store ([Ca]SR) was measured with Cal-520 dye under 10 mM caffeine. Resting [Ca]i was measured with Fura-2 dye, and Na/Ca exchanger (NCX) peak current was measured as Ni sensitive current in a stepwise voltage-clamp protocol.Results: iPSC-CM APD90 was shorter 40% than iPSC-CM. IPSC-CM treated with ouabain had 17% shorter APD90 than vehicle. iPSC-CM had DADs (3/11 cells) when iPSC-CM (0/24) did not, and 6/12 ouabain treated iPSC-CM had DADs vs vehicle treated (0/18). Post caffeine, iPSC-CM had higher [Ca]SR than iPSC-CM. Finally, iPSC-CM had higher resting [Ca]i than iPSC-CM and iPSC-CM had higher peak NCX current density at +80mV but no difference at RMP.Conclusions: Our results show that both iPSC-CM and ouabain-treated iPSC-CM, have a shorter APD compared to WT controls and exhibit DADs. This is associated with higher NCX-mediated influx of Ca and increased intracellular Ca. Our findings highlight the emerging role of ATP1A3-D801N in creating a myocardium vulnerable to arrhythmias.