Recent studies have shown that heterozygous mutations in KCNJ11 , which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K _ATP ) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg ²⁺ , to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic β-cells, K _ATP channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K _ATP channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg ²⁺ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K _ATP channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K _ATP current at physiological MgATP concentrations correlates with the severity of the clinical phenotype.