SK channels are unique as they sense changes in intracellular Ca2+ concentration and convert it to K+ conductance. In 2010, GWAS studies showed association with SK3 coding gene (KCNN3) with lone atrial fibrillation (AF) and in 2017 with KCNN2 gene. In pharmacological studies, blockade of SK channels has been efficacious in converting AF to sinus rhythm by prolonging the atrial effective refractory period (AERP) [1]. Being more prominent in atria than in ventricles elevates its importance as atrial-selective target for AF by reducing the risk of adverse toxicities of current anti-arrhythmic drugs; however, the molecular mechanism involved is not clear yet. PKA had shown to regulate surface expression of SK2 channel in hippocampal neurons [2]. This study aims to decipher PKA regulation on SK channel in AF. Mean± S.E.M. are stated and analysed by ANOVA. Whole-cell currents were acquired in HEK293-hKCa2.3 cells which showed that activation of PKA by forskolin (FSK) 10µM, an adenylyl cyclase activator inhibits SK3 current density and inhibition by PKA inhibitor, KT5720 0.1µM results in increased SK3 current density (-69±13,n=12 vs -298±31,n=16,p<0.0001). Immunofluorescence in HEK cells transfected with rat KCa2.3 showed that SK3 surface expression was reduced in FSK 10µM treated cells but KT5720 0.1µM increased it, which suggests role in SK channel trafficking. Langendorff experiments were performed using female Dunkin Hartley guinea pigs (350-450g) anesthetized with intraperitoneal injection of pentobarbital (200mg/ml), lidocaine hydrochloride (20mg/ml) at a dose of 0.1ml/100g body weight, after which tracheotomy was performed and the guinea pigs were ventilated using a rodent ventilator. The hearts were then excised and perfused via cannulated aorta in Langendorff retrograde setup (all groups n=6). All animal studies were performed in accordance with the Danish guidelines for animal experiments according to the European Commission Directive 86/609/EEC. Application of FSK 1µM increased the intrinsic rhythm (339±12 vs 216±18 bpm,p<0.01) compared to KT5720 0.1µM. Also FSK decreased atrioventricular Wenckebach point (100±2.7), whereas KT5720 showed vice-versa (126±3.3,p<0.01) with no significant change in AERP (56±1.7 vs 58±3.2,p>0.05) and rheobase (0.28±0.02 vs 0.18±0.02 p>0.05) between both groups. AF was induced by addition of 1μM acetylcholine with 40X burst pacing (50 Hz, 10 V) or until sustained AF of >3 min was observed. FSK showed protective role in duration and number of AF events compared to KT5720 that showed vulnerability to AF. Thus, in vitro studies show SK channel regulation mediated via PKA and ex vivo acute AF model shows activating PKA to be protective in acute AF and KT5720 inhibition of PKA making it more vulnerable to AF. Further studies are being carried out with SK blocker along with PKA regulators. This study provide evidence for the first time about PKA regulation in guinea pig explanted heart acute AF model.