G-protein βγ subunits have recently been shown to be positive regulators of KCNQ4-encoded voltage-gated potassium channels, Kv 7.4 (PNAS, 112: 6497-6502, 2015). The aim of the present work was investigate whether there was an interplay between Gβγ subunits and phosphatidylinositol 4,5-bisphosphate, (PIP2) an established positive modulator of Kv 7.4 (J.Gen.Physiol. 140: 41-53, 2012). Electrophysiological recordings were performed using standard perforated-, whole-cell, cell-attached and inside-out configurations of patch-clamp technique on HEK293 cells heterologously expressing Kv 7.4 (Am.J.Physiol. 280: C859-C866, 2001). PIP2 levels under different conditions were estimated using an In-cell Western Blot technique (Biochem.Biophys.Res.Commun. 452: 852-857, 2014). Data are presented as mean ± S.E.M. Bath application of PIP2 (1-300 µM) to inside-out patches increased apparent NPo of Kv 7.4 in dose-dependent manner with a EC50 of 117.4 ± 54 µM (n = 4-11). Similarly, application of Gβγ subunits (0.4-50 ng/ml) dose-dependently increased NPo and a notable increase was observed at concentrations higher than 2 ng/ml. After PIP2 depletion by wortmannin (an inhibitor of PIP2 re-synthesis), in combination with a short stimulation of G-protein-coupled proteinase-activated receptors or purinoceptors, currents through Kv 7.4 channels were decreased by 95% in perforated-, cell-attached and inside-out patches. Bath application of PIP2 re-established channel activity of inside-out patches, whereas Gβγ subunits had no effect. Inhibitors of Gβγ subunits (gallein, M201or GRK2i) also depressed Kv 7.4 currents, but PIP2 was not able to enhance channel activity in this case. Application of a low concentration of Gβγ subunits (1 ng/ml) to inside-out patches was augmented the action of PIP2. Thus, 3 µM of PIP2 increased NPo to 0.077 ± 0.017 (n = 8) alone and to 0.255 ± 0.073 (n = 6) in the presence of Gβγ subunits. This study shows that Kv 7.4 channels are regulated by an interplay of PIP2 and Gβγ subunits