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KCNN4 (IK) is a calcium activated potassium (K) channel. However, its role in diarrhea and regulation by Epac1 in cAMP stimulation is unknown. By RT-PCR and Western blot, we dentified KCNN4 expression in mouse intestine and human intestinal cell line. We hypothesized that apical KCNN4 was involved in diarrhea and regulated by Epac1 associated signaling. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor significantly abolished cAMP stimulated Cl secretion and apical K conductance IK(ap) in T84 cells. We studied the current-voltage relationships in basolaterally permeabilized monolayers treated with 8-pCPT-2'-O-Me-cAMP in a symmetric K ion concentration. The presence of an inwardly rectified, Ca-activated K channel was evident in T84WT cells, whereas 8-pCPT-2'-O-Me-cAMP was not able to activate this inwardly rectified current in Epac1 knock down (Epac1KDT84) cells. Furthermore, addition of KCNN4 channel specific activator, 1-EBIO to intact wild type (T84WT) cells resulted 44±5 µA/cm2 rise in Isc while in Epac1KDT84 cells and T84WT + TRAM 34, 1-EBIO enhanced Isc was only 5±1 µA/cm2 and 4 ± 3 µA/cm2 respectively. Reconstructed confocal image in the X-Z plane of T84Epac1KD monolayers revealed redistribution of KCNN4c proteins into sub-apical intracellular compartment. We compared the surface amount of KCNN4c in these cells that Epac1 depleted cells have ~83% lower surface membrane expression of KCNN4c compared with T84WT cells. The role of Epac1-Rap1 signaling in endogenous regulation of KCNN4 was further assessed in T84 cells by Rap1 activation assays. FSK and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1 in T84WT cells. To better understand the specific involvement of Rho-A and Rho-associated kinase ROCK in KCNN4 regulation in intestinal Cl secretion, Isc and IK(ap) was measured in T84WT cells. Finding that both Rho-A and ROCK kinase inhibitors significantly reduced FSK stimulated Isc as well as IK(ap). To explore the therapeutic potential of Epac1 regulated signaling of KCNN4c channels, we tested the effect of Rap1a inhibitor GGTI-298, ROCK inhibitor H1152 or KCNN4c inhibitor TRAM-34, in a closed loop mouse model. Our result demonstrated Epac1-Rap1-Rho-A-ROCK signaling lead to the alleviation of diarrhea by recruiting KCNN4c to the apical membrane which has important therapeutic value in diarrhea [ABSTRACT FROM AUTHOR]