PROJECT SUMMARY Kir3 or GIRK (G protein gated inwardly rectifying K+) channel isoforms are expressed in multiple parts of the body, including the atria of the heart (GIRK1, 4) and platelets (GIRK1, 2, 4). In the heart, activation by the vagus nerve slows down heart rate. GIRKs are critical determinants of heart rate variability (HRV), an index of cardiac health, endowing the heart with the adaptability it needs to make rapid adjustments in heart rate. GIRK channels are attractive drug targets against atrial fibrillation (Afib), the most common arrhythmia. In the last cycle of this grant, we showed that increased Afib prevalence with age results from constitutive activation of PKC by the oxidative conditions of aging, which phosphorylates GIRK4 channels to increase their activity, leading to an increased risk of mortality, stroke and myocardial infarction. Additionally, the lack of specificity of the current antiarrhythmics used poses significant risk for ventricular side effects. This makes rather attractive targets expressed predominantly in the atria, like GIRK channels. Yet the lack of specific and partial inhibitors of GIRK activity in the atria that could reverse Afib, but not inhibit HRV, have been challenging to make. During vascular injury, P2Y12 receptor stimulation activates GIRKs to somehow result in generation of the lipid thromboxane A2 that amplifies platelet activation and recruitment of additional platelets to the site of injury to form clots and stop or prevent bleeding. Even though P2Y12R is the target of efficacious anti-thrombotic drugs, the lack of understanding of GIRK biology in platelet activation and the lack of specific GIRK inhibitors has stalled targeting them for anti-thrombotic effects. In Aim 1 of this proposal, we utilize state-of-the-art computational approaches to design drugs that target specific GIRK isoforms at either of two sites, transmembrane (TM) and cytoplasmic (CP) ones and couple preferentially to either of the two channel gates, the membrane (M) and the cytosolic (C). In Aim 2a, we use these powerful probes to find the balance between reversing Afib and maintaining HRV, thus aiming to address the unmet medical need for GIRK-isoform specific partial inhibitors. In Aim 2b, we also use them to decipher the involvement of GIRKs in platelet-mediated thrombosis. The complementary expertise of the three labs make this a unique team. The Logothetis lab has made seminal discoveries over the years on GIRK structure and function and more recently, drug discovery, while the Noujaim lab has great expertise in Afib and HRV animal models and together produced 5 high-profile manuscripts. The Kunapuli lab has great expertise in platelet-mediated thrombosis and pioneered discovery of GIRK involvement in this process. Specific inhibition of GIRK isoforms may unify targeting Afib and Stroke with a single approach.