ATP-sensitive K⁺ (K_ATP) channels control electrical signalling in diverse cell types by coupling metabolism to transmembrane K⁺ fluxes. They comprise pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits. Metabolic regulation is mediated by changes in intracellular adenine nucleotides: ATP binding to Kir6.2 inhibits, and interaction of MgATP with SUR, increases, channel activity. In pancreatic β-cells, K_ATP channels mediate glucose-stimulated insulin secretion. Heterozygous (het) mutations in Kir6.2 (KCNJ11) cause permanent neonatal diabetes alone (PNDM; R201H/C) or in association with developmental delay, muscle weakness, and epilepsy (DEND syndrome; Q52R, V59G, I296L). Functional analysis in the absence of Mg²⁺, to isolate the effects of ATP on Kir6.2, showed both types of mutation reduce channel inhibition by ATP (Gloyn et al., 2004; Proks et al., 2004). However, in cells, K_ATP channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by SUR. We therefore studied the MgATP sensitivity of Kir6.2 mutant channels. K_ATP currents were recorded from Xenopus laevis oocytes coinjected with wild-type (wt) or mutant Kir6.2 and SUR1 mRNAs. To simulate the het state, we used a 1:1 mixture of wt and mutant Kir6.2 mRNAs. Macroscopic (or single-channel) currents were recorded by patch-clamping inside-out patches. The pipette solution contained (mM): 140 KCl, 1.2 MgCl₂, 2.6 CaCl₂, 10 HEPES (pH 7.4). The Mg free internal (bath) solution contained (mM): 107 KCl, 1 K₂SO₄, 10 EGTA, 10 HEPES (pH 7.2). The Mg-containing internal solution consisted of Mg-free solution without K₂SO₄ plus 2mM MgCl₂ and MgATP (instead of ATP). Mg²⁺ caused a small increase (1.9±0.4-fold; n=6) in the IC₅₀ (13±1 μM) for ATP inhibition of wt channels. In contrast, the IC₅₀ for homozygous R201C and R201H mutant channels was dramatically increased: IC₅₀ = 2.4±0.2 mM (n=5) and 2.0±0.2 mM (n=6) (23.5±2.9-fold and 6.7±0.8-fold increase) respectively. Mg²⁺ dramatically altered the shape of the dose-response curve of homozygous Q52R, V59G and I296L mutant channels, resulting in a substantial fraction (35-87%) of unblocked current at high MgATP. The fraction of unblocked current in 3 mM MgATP for het channels was correlated with disease severity, with V59G(40%)>I296L(32%)>Q52R(27%)>R201C(15%)> R201H(8%). This supports the idea that neonatal diabetes results from an increased K_ATP current in β-cells, which reduces electrical activity and insulin secretion. Larger increases in K_ATP current may be required to influence electrical activity in other cell types and cause the neorological symptoms associated with DEND syndrome.