At the molecular level, K_ATP is an octameric protein complex, composed of an inwardly rectifying K⁺ channel subunit (Kir6.0) which forms the channel pore and a regulatory sulphonylurea receptor subunit (SUR). By sensing intracellular nucleotide concentrations, K_ATP channels couple the membrane potassium conductance of a cell to its metabolic state. To understand the molecular basis of metabolic regulation of the K_NDP/K_ATP channels Kir6.1/SUR2B and Kir6.2/SUR2A, the cell-based Rubidium-86 (⁸⁶Rb⁺) efflux assay was employed, using HEK293 cells as the expression system. After channel activation the ⁸⁶Rb⁺ distribution between intracellular and extracellular space was determined via the measurement of Cherenkov radiation, the relative amount of ⁸⁶Rb⁺ in the cell supernatant being a direct measure of channel activity. ⁸⁶Rb⁺ efflux was activated on the addition of 10µM levcromakilim, 10µM diazoxide and metabolic poisoning (induced by 20mM 2-deoxyglucose and 2.5mM sodium cyanide), and inhibited by 10µM glibenclamide. Co-expression of SUR and Kir6.0 is necessary to shield their respective RKR retention motifs, generate plasmalemmal currents and allow cell surface expression of the channel. Kir6.2ΔC26 has previously been shown to form ATP-sensitive channels in the absence of SUR1 (1), and deletion of the N-terminus of Kir6.0 has been found to increase basal activity (2). Kir6.1 C-and N-terminal truncations were made to delete the RKR motif, enabling understanding of the role of each subunit pharmacologically in channel gating. Immunofluorescent staining using an anti-HA-fluorescein conjugated antibody confirmed that removal of the RKR motif allows surface expression. Kir6.1/SUR2B and Kir6.2/SUR2A were initially characterised to determine the time at which the linear phase of efflux was achieved after incubation with agonist, upon which Kir6.1ΔN and ΔC were screened to determine functionality. Kir6.1Δ48C/ΔN13 was found to be constitutively active without SUR2B. When expressed with SUR2B glibenclamide was able to reverse metabolic inhibition, suggesting that the SUR was responsible for channel regulation under this condition. However, when mutations were introduced into nucleotide binding domain 1 (NBD) or NBD2 (K708A and K1349M respectively) of SUR2B and expressed with Kir6.1, normal pharmacology was observed, suggesting that neither NBD is independently involved in metabolic regulation. Metabolic inhibition was induced when Kir6.2ΔC26 was expressed without the SUR subunit, which was reversed via BaCl₂ (a pore blocker). Since Kir6.1Δ48C was not found to be metabolically sensitive when expressed alone, this further suggests that Kir6.1 is not involved in metabolic regulation, but that Kir6.2 is. Overall, this highlights a difference in metabolic regulation between Kir6.1 and Kir6.2 forming channels.