Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels, found in smooth muscle cells and cardiomyocytes. In the latter, the channel forms the KCNQ1/KCNE1 complex, mediating the slow delayed-rectifier currents (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause Long QT Syndrome (LQT), which increases the risk of sudden cardiac death and ventricular arrhythmia. One possible therapeutic intervention would be based on specifically targeting IKs currents to restore the channel function and the physiological QT interval. PUFAs are ideal candidates as they activate IKs current by shifting the voltage dependence of opening toward negative voltages and by increasing the maximum conductance of the channel (Gmax). PUFAs mechanism of action rely on two different binding sites, one at the voltage sensor domain - which shifts the voltage dependence - and the other at the pore region of the channel - which increases the Gmax. However, the mechanism by which PUFAs increase the Gmax of the channels is still poorly understood. In this study, we propose that the increase in the channel currents is caused by a complex mechanism of several residues in the external mouth of the pore, including K326, P320, and the two tryptophans of the conserved aromatic ring cuff. Our investigations showed that by mutating some of these residues, the effect of PUFAs on Gmax is reduced, describing a complex mechanism involving a wide network of residues in the Gmax effect of PUFAs.