Chandrashekar, A.; Xu, Y.; Ma, X.; Yauch, A. K.; Scholl, E.; Yang, Y.; Gada, K. D.; Kawano, T.; Cui, M.; Plant, L. D.

Background: Andersen-Tawil Syndrome type 1 (ATS1) is a multisystem channelopathy that predisposes patients to ventricular dysrhythmias and increases the risk of sudden cardiac death. ATS1 arises from loss-of-function mutations in Kir2.1, the inward rectifying potassium channel responsible for most of IK1 in ventricular cardiomyocytes. IK1 is suppressed by SUMOylation, a post-translational modification upregulated in hypoxia, a known proarrhythmic stimulus. We investigated whether current from the ATS1-linked variant Kir2.1-R67Q is inhibited by hypoxia and whether this suppression can be reversed by pharmacological inhibition of the SUMO pathway. Methods: We used patch-clamp recording to measure IK1 and Kir2.1 currents under acute hypoxia, with and without the SUMO pathway inhibitor TAK-981. To quantify SUMOylation stoichiometry, we applied single molecule photobleaching. A multidisciplinary approach combining electrophysiology, molecular modeling, and optogenetic phosphoinositide was used to measure the impact of Kir2.1-R67Q and SUMOylation on channel interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), a required gating cofactor. Results: Kir2.1 can be modified by up to two SUMO proteins attached to diagonally opposite subunits, with each SUMOylation event reducing current by ~20%. Heterozygouse channels containing two R67Q subunits were more susceptible to hypoxic suppression than wild type. TAK-981 blocked hypoxic inhibition of IK1 in ventricular cardiomyocytes and abolished Kir2.1 SUMOylation. In cells expressing Kir2.1-R67Q, TAK-981 significantly increased currents and mitigated hypoxic suppression. Computational modeling and optogenetic dephosphorylation revealed that both the R67Q mutation and converge to disrupt Kir2.1- PIP2 interactions, producing synergistic inhibition of channel function. Conclusions: Hypoxia-induced SUMOylation and the R67Q mutation synergistically suppress Kir2.1 activity by impairing channel-PIP2 interactions. TAK-981 restores IK1 by preventing SUMOylation under hypoxic conditions and enhancing current through Kir2.1-R67Q channels. These findings support a two-hit model of arrhythmogenesis in ATS1 and identify SUMO pathway inhibition as a potential therapeutic strategy to reduce arrhythmic risk in affected patients.