Timothy Syndrome (TS) is a multi-symptom disorder characterized by arrhythmias and sudden cardiac death. TS is also associated with impaired cognitive function, and autism spectrum disorder. TS is the result of a de novo point mutation at the intracellular side of S6 in domain I of the L-type Ca2+ channel (Cav1.2). The G406R mutation is a gain of function mutation which interferes with normal inactivation of this Ca2+ channel. There are two forms of TS, with the identical mutation on two mutually exclusive exons, one on 8 (TS2) and the other on 8a (TS2). The TS2 mutation is lethal in mice. However, mice with the TS2 mutation and transcriptional interference with from a NEO promotor (TS2-NEO) survive to reproductive age. We crossed TS2-NEO with a mouse carrying a cardiac-specific tamoxifen-inducible CRE to develop a cardiac specific inducible form of TS. Exposure to tamoxifen upregulated expression of the exon with the TS2 mutation, and the CRE-TS2-NEO mouse had a noticeable phenotype compared to the mild phenotype of the TS2-NEO. Tamoxifen 10mg/mL suspended in corn oil was administered by intraperitoneal injection, 100ul (1mg/day) for 2 consecutive days. Tissue was harvested 4 days following the last injection. Mice were anaesthetized via injection of ketamine-xylazine (0.1ml/10g), and left ventricular myocytes dissociated using the Langendorff technique (1). Cells were voltage clamped or current clamped. Computer simulations were performed using our previously published model (1). All procedures were in accordance with institutional and government guidelines. In the computer model, complete elimination of both Ca2+ dependent and independent components of inactivation in 20% of L-type Ca2+ channels results in prolongation of the action potential (AP) by less than a millisecond. This was expected since inactivation of the Ca2+ current plays little role in repolarization, due to the short duration of the mouse AP. In contrast, experimental results show that there is a significant prolongation of the AP resulting from exposure to tamoxifen and removal of the NEO cassette. Injection with tamoxifen increased AP duration by 40.9 ± 15.8 ms in CRE-TS2-NEO mice vs. 1.9 ± 3.0 ms in control mice (n=5, p<0.01). Single cell measurements showed a similar increase in AP. This discordance between simulation and prolongation of repolarization is not unique to mice. AP simulations of TS in guinea pig models shows AP prolongation, but not the profound clinical AP prolongation associated with TS (2). Single cell Ca2+ currents suggest there is little change in inactivation of the L-type calcium channel. This was most evident at positive potentials where Ca2+ dependent inactivation is minimal. However, the changes in kinetics appeared mild compared to the simulation conditions. These data indicate that although TS arises from a point mutation in the L-type Ca2+ channel, there is substantial electrical remodeling in the heart that produce the extreme AP prolongation observed.