The Kv7.4 channel, from the Kv7 family of voltage gated potassium channels, is important in the regulation of vascular smooth muscle tone. Recently it was shown that Kv7.4 channels and native vascular Kv7 channels are positively regulated by G protein βγ subunits, and that these subunits are essential for the basal activity of these channels (1). However, there are 5 different Gβ and 12 Gγ subunits and it is increasingly clear that in the regulation of other Gβγ effectors individual isoform combinations can perform specific functions. Which subunits are important in the regulation of Kv7.4 channels is unclear, so here we investigate the effect of Gβ1-5 subunits on Kv7.4 whole cell currents Chinese Hamster Ovary cells were transfected with the relevant plasmids using Lipofectamine 2000/ optiMEM for 24 hours. A total of 3µg was transfected in a 1:1 ratio of Kv7.4:EV (Empty Vector) or Kv7.4:Gβ1-5. Whole cell K+ currents were studied by ruptured patch electrophysiology. Proximity Ligation Assay was performed to detect protein-protein interactions between Kv7.4 and Gβ subunits. Rat renal artery myocytes were isolated from freshly dissected arteries of male wistar rats (175-225g). Animals were culled by cervical dislocation, in accordance with the Animals (Scientific Procedures) Act 1986. Currents recorded from cells expressing Kv7.4 and either Gβ1, Gβ3 or Gβ5 were significantly greater that those expressing Kv7.4 alone (Kv7.4 alone = 13.5±2.1pA/pF (n=24), with Gβ1= 35.8±12.5pA/pF (n=7), with Gβ3 = 22.7±5.9pA/pF (n=5), or with Gβ5 = 23.4±5.4pA/pF (n=6), as measured at +40mV). Conversely, co-expression of Gβ2 or Gβ4 with Kv7.4 resulted in significantly decreased currents than in control (with Gβ2 = 5.8±1.4pA/pF (n=7) and with Gβ4 = 5.9±2.1 (n=8)). In rat renal artery myocytes proximity ligation assay detected interactions between Kv7.4 and Gβ1 (12.7±1.4 puncta/cell (N=1, n=7)) and Gβ3 (27.6 ±5.7 puncta/cell (N=1, n=6)) which were reduced when cells were treated with the general G protein βγ subunit inhibitor, gallein (Gβ1 – 3.2±1.4 puncta/cell (N=1, n=5) and Gβ3 – 3.3±1.4 puncta/cell (N=1, n=5)). These results show that specific Gβ subunits have divergent role in positively (Gβ 1, 3 and 5) or negatively (Gβ 2 and 4) regulating Kv7.4 channels, which could have important implications in the regulation of vascular tone.