The Kv7 family of voltage gated potassium channels are widely expressed with key roles in neuronal, cardiac and vascular systems (1). In the vasculature, the Kv7.4 isoform has been shown to be of particular importance for maintaining vascular tone, as a reduction in Kv7.4 protein and impaired relaxations to Kv7 activators is associated with hypertension, whilst siRNA directed knockdown of Kv7.4 results in impaired vasodilatory responses (2,3). Whilst Kv7 channel activation is involved in mediating endogenous vasodilator responses coupled to Gαs/cyclic AMP-linked G-protein coupled receptors (GPCR) the underlying signalling mechanisms coupling receptor to channel remain unclear. This study focused on one aspect of GPCR signal cascade, namely to define what effects βγ subunit signalling have on Kv7.4 currents.Using a HEK293 cell line that stably express Kv7.4, electrophysiological experiments show that intracellular perfusion of βγ subunits (250ng/ml) significantly increased currents (62.8 ± 5.5 pA/pF at 20mV, n=17) compared with control (47.3 ± 5.4 pA/pF at 20mV, n=23, p<0.001) and increases the rate of activation of currents (from 556.7 ± 42.9 to 390.8 ± 29.7 ms, p<0.05). Similarly in rat renal artery myocytes from male Wistars (200-225g), perfusion of βγ subunits significantly increased the linopiridine sensitive current (from 1.87 ± 0.19 to 2.51 ± 0.24 pA/pF, n=10, p<0.05) with a concomitant increase in the rate of activation (from 53.6±3.6 to 15.8 ± 2.3 ms at 20mV, n=10, p<0.001). In HEK293 Kv7.4 cells addition of the small molecule βγ subunit inhibitor gallein (100μM), or perfusion of either the βγ subunit inhibitor Grk2i (10μM) or a G-protein β-subunit antibody (1:200), resulted in complete ablation of currents (amplitude at 20 mV was 3.95 ± 0.8 (n=4), 8.55 ± 1.4 (n=7) and 4.95 ± 1.4 (n=5) pA/pF, respectively, all p<0.001). All statistics represent mean ± SEM, significance was determined by two way ANOVA.These data implicate a G-protein βγ subunit involvement in activation of Kv7.4 currents and suggests they are also a constitutive requirement for Kv7.4 activity. This data shows for the first time that βγ subunits have a crucial role in the regulation of Kv7.4 channels, which may underlie GPCR mediated activation of Kv7 channels.