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. 2004 Dec 14;101(50):17539-44.
doi: 10.1073/pnas.0404756101. Epub 2004 Dec 6.

Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features

Peter Proks  1 , Jennifer F Antcliff, Jon Lippiat, Anna L Gloyn, Andrew T Hattersley, Frances M Ashcroft
Affiliations

Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features

Peter Proks et al. Proc Natl Acad Sci U S A. .

Abstract

Inwardly rectifying potassium channels (Kir channels) control cell membrane K(+) fluxes and electrical signaling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus (PNDM). For some mutations, PNDM is accompanied by marked developmental delay, muscle weakness, and epilepsy (severe disease). To determine the molecular basis of these different phenotypes, we expressed wild-type or mutant (R201C, Q52R, or V59G) Kir6.2/sulfonylurea receptor 1 channels in Xenopus oocytes. All mutations increased resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, but, in the simulated heterozygous state, mutations causing PNDM alone (R201C) produced smaller K(ATP) currents and less change in ATP sensitivity than mutations associated with severe disease (Q52R and V59G). This finding suggests that increased K(ATP) currents hyperpolarize pancreatic beta cells and impair insulin secretion, whereas larger K(ATP) currents are required to influence extrapancreatic cell function. We found that mutations causing PNDM alone impair ATP sensitivity directly (at the binding site), whereas those associated with severe disease act indirectly by biasing the channel conformation toward the open state. The effect of the mutation on ATP sensitivity in the heterozygous state reflects the different contributions of a single subunit in the Kir6.2 tetramer to ATP inhibition and to the energy of the open state. Our results also show that mutations in the slide helix of Kir6.2 (V59G) influence the channel kinetics, providing evidence that this domain is involved in Kir channel gating, and suggest that the efficacy of sulfonylurea therapy in PNDM may vary with genotype.

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Figures

Fig. 1.
Fig. 1.
Functional role of KATP channels and location of mutations. (A) Schematic of how metabolic regulation of KATP channel activity sets the cell membrane potential. (B) Homology model of Kir6.2 (7), indicating the location of residues R201, Q52R, and V59G associated with neonatal diabetes. For clarity, only two subunits are shown, and the intracellular and transmembrane domains are from separate subunits. ATP (black) is shown in its binding site. (C) Schematic of the mixture of channels with the different subunit compositions expected when wild-type and mutant Kir6.2 are coexpressed (as in the heterozygous state). The relative numbers of the channel types expected if wild-type and mutant subunits segregate independently (i.e., follow a binomial distribution) are indicated above the figure.
Fig. 2.
Fig. 2.
Effects of mutations on whole-cell KATP currents. (A) Whole-cell currents recorded from Xenopus oocytes coexpressing SUR1 and either wild-type or mutant Kir6.2, as indicated, in response to voltage steps of ±20 mV from a holding potential of –10 mV in 90 mM K+. The start of azide application is indicated by arrows. The initial decrease in KATP current is due to a small block by azide that is mediated by an interaction with SUR1. (B) Mean steady-state whole-cell currents evoked by a voltage step from –10 to –30 mV before (control) and after application of 3 mM azide. The number of oocytes is 8 –12 in each case.
Fig. 3.
Fig. 3.
Effects of mutations on KATP channel ATP sensitivity. (A) KATP current recorded in response a succession of voltage ramps from –110 to +100 mV to an inside-out patch excised from a Xenopus oocyte coexpressing SUR1 and either wild-type or mutant Kir6.2, as indicated. The dotted line indicates the zero current level. ATP was applied to the intracellular membrane face as indicated. (B) (Upper Left) Mean relationship between [ATP] and KATP conductance, G, expressed relative to the conductance in the absence of nucleotide, Gc, for Kir6.2/SUR1 (open blue circles, n = 6), and heterozygous (filled red circles, n = 6) and homomeric (filled black squares, n = 6) Kir6.2-R201C/SUR1 channels. The smooth curves are the best fit to the Hill equation. For wild-type, IC50 = 6.6 μM and h = 1.1. For heterozygous R201C, IC50 = 10.4 μM and h = 1.0. For homomeric R201C, IC50 = 102 μM and h = 1.3. (Upper Right) Mean relationship between [ATP] and KATP conductance expressed relative to the conductance in the absence of nucleotide for Kir6.2/SUR1 (open blue circles, n = 6) and heterozygous (red filled circles, n = 6) and homomeric (black filled squares, n = 6) Kir6.2-V59G/SUR1 channels. The smooth curves are the best fit to a modified Hill equation containing 1/16 of homomeric channels. For wild-type, IC50 = 6.6 μM and h = 1.1. For heterozygous V59G, IC50 = 26 μM and h =1.18. For homomeric V59G, IC50 = 8.1 mM and h = 0.75. (Lower Left) Mean relationship between [ATP] and KATP conductance expressed relative to the conductance in the absence of nucleotide for Kir6.2/SUR1 (open blue circles, n = 6) and heterozygous (filled red circles, n = 6) and homomeric (filled black squares, n = 6) Kir6.2-Q52R/SUR1 channels. The smooth curves are the best fit to the Hill equation. For wild-type, IC50 = 6.6 μM and h = 1.1. For heterozygous Q52, IC50 = 21 μM and h = 1.2. For homomeric Q52R, IC50 = 83 μM and h = 1.7. (Lower Right) Mean relationship between [ATP] and KATP conductance expressed relative to the conductance in the absence of nucleotide for wild type (open blue circles) and hetR201C (filled red circles), hetQ52R (filled black squares), and hetV59G (filled green hexagons) channels.
Fig. 4.
Fig. 4.
Effects of mutations on single-channel currents. Single KATP channel currents were recorded at –60mV from inside-out patches excised from oocytes coinjected with mRNAs encoding SUR1 plus either wild-type or mutant Kir6.2, as indicated.
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