ATP-sensitive potassium (K ATP ) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K ATP channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 Å resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATPbinding cassette proteins related to SUR1, and supports Rb þ fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

MthK is a calcium-gated, inwardly rectifying, prokaryotic potassium channel. Although little functional information is available for MthK, its high-resolution structure is used as a model for eukaryotic Ca2+-dependent potassium channels. Here we characterize in detail the main gating characteristics of MthK at the single-channel level with special focus on the mechanism of Ca2+ activation. MthK has two distinct gating modes: slow gating affected mainly by Ca2+ and fast gating affected by voltage. Millimolar Ca2+ increases MthK open probability over 100-fold by mainly increasing the frequency of channel opening while leaving the opening durations unchanged. The Ca2+ dose–response curve displays an unusually high Hill coefficient (n = ∼8), suggesting strong coupling between Ca2+ binding and channel opening. Depolarization affects both the fast gate by dramatically reducing the fast flickers, and to a lesser extent, the slow gate, by increasing MthK open probability. We were able to capture the mechanistic features of MthK with a modified MWC model.

KATP channels regulate insulin secretion from pancreatic β-cells. Loss- and gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause hyperinsulinism of infancy and neonatal diabetes, respectively. We report two novel mutations in the gating loop of Kir6.2 which cause neonatal diabetes with developmental delay (T293N) and hyperinsulinism (T294M). These mutations increase (T293N) or decrease (T294M) whole-cell KATP currents, accounting for the different clinical phenotypes. The T293N mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly decreasing channel inhibition by ATP and increasing whole-cell currents. T294M channels exhibit a dramatically reduced Po(0) in the homozygous but not in the pseudo-heterozygous state. Unlike wild-type channels, hetT294M channels were activated by MgADP in the absence but not in the presence of MgATP; however, they are activated by MgGDP in both the absence and presence of MgGTP. These mutations demonstrate the importance of the gating loop of Kir channels in regulating Po(0) and further suggest that Mg-nucleotide interaction with SUR1 may reduce ATP inhibition at Kir6.2.

Aims/hypothesisHeterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects.MethodsThe patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg−1 day−1). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes.ResultsHeterozygous (het) and homozygous L164P KATP channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients.Conclusions/interpretationOur results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.