Acid-sensitive background two-pore potassium channels (K_2P) are important contributors to the maintenance of cellular resting membrane potential and cellular excitability. Membrane expression of acid-sensitive K_2P channels, K_2P3.1, K_2P9.1 and K_2P15.1 is mediated by phosphorylation dependent trafficking by cytosolic protein 14-3-3. Loss of 14-3-3 interaction results in impaired membrane expression that could result in disrupted cellular excitability. We used two-electrode voltage-clamp to measure the functional activity of the acid-sensitive K_2P channels. Xenopus laevis oocytes expressing K_2P3.1 or K_2P9.1 exhibited a pH-dependent current, typical of acid-sensitive K_2P channels, however no current was seen in oocytes expressing K_2P15.1. K_2P3.1ΔV₄₁₁ and K_2P9.1ΔI₃₉₆ mutants, which have disrupted 14-3-3 binding sites, displayed significantly reduced current. K_2P3.1ΔV₄₁₁ tagged with green fluorescent protein (eGFP) was transfected into COS-7 cells and imaged using immunofluorescence microscopy. Co-staining transfected cells with markers for the ERGIC and golgi using ERGIC-53 and GM-130 respectively did not show co-localisation in the majority of cells. K_2P3.1ΔV₄₁₁ was shown to be localised to the endoplasmic reticulum (ER) by co-staining for the ER marker protein disulphide isomerase. The mutant K_2P3.1ΔV₄₁₁ was able to overcome ER retention by co-expressing with wildtype K_2P3.1, K_2P9.1 or K_2P15.1. This data suggests that when 14-3-3 cannot bind K_2P3.1 is retained in the ER, but can be rescued by co-expression with wildtype channel and hence suggests the stoichiometry of interaction of 14-3-3. Furthermore this also demonstrates heterodimerisation of K_2P3.1 and K_2P15.1. Physiological significance of these results are far reaching as K_2P3.1 and K_2P15.1 demonstrate co-expression in an array of tissues such as the pancreas, placenta, kidney and lung.