A rare mutation in ABCC8/SUR1 leading to altered ATP-sensitive K+ channel activity and beta-cell glucose sensing is associated with type 2 diabetes in adults

Abstract

Objective: ATP-sensitive K(+) channels (K(ATP) channels) link glucose metabolism to the electrical activity of the pancreatic beta-cell to regulate insulin secretion. Mutations in either the Kir6.2 or sulfonylurea receptor (SUR) 1 subunit of the channel have previously been shown to cause neonatal diabetes. We describe here an activating mutation in the ABCC8 gene, encoding SUR1, that is associated with the development of type 2 diabetes only in adults.

Research design and methods: Recombinant K(ATP) channel subunits were expressed using pIRES2-based vectors in human embryonic kidney (HEK) 293 or INS1(832/13) cells and the subcellular distribution of c-myc-tagged SUR1 channels analyzed by confocal microscopy. K(ATP) channel activity was measured in inside-out patches and plasma membrane potential in perforated whole-cell patches. Cytoplasmic [Ca(2+)] was imaged using Fura-Red.

Results: A mutation in ABCC8/SUR1, leading to a Y356C substitution in the seventh membrane-spanning alpha-helix, was observed in a patient diagnosed with hyperglycemia at age 39 years and in two adult offspring with impaired insulin secretion. Single K(ATP) channels incorporating SUR1-Y356C displayed lower sensitivity to MgATP (IC(50) = 24 and 95 micromol/l for wild-type and mutant channels, respectively). Similar effects were observed in the absence of Mg(2+), suggesting an allosteric effect via associated Kir6.2 subunits. Overexpression of SUR1-Y356C in INS1(832/13) cells impaired glucose-induced cell depolarization and increased in intracellular free Ca(2+) concentration, albeit more weakly than neonatal diabetes-associated SUR1 mutants.

Conclusions: An ABCC8/SUR1 mutation with relatively minor effects on K(ATP) channel activity and beta-cell glucose sensing causes diabetes in adulthood. These data suggest a close correlation between altered SUR1 properties and clinical phenotype.

Figure 1. A: Schematic of K_ATP channel heterooctameric assembly B: Topological sketch of SUR1.

Mutations at the residues marked are associated with T2D (*), TND (**) and MODY (***). The residues are indicated according to (50) and homology modeling of TMD1 and TMD2 of SUR1 (see Supplementary Info). C: ATP concentration-inhibition curves for wild-type and mutant K_ATP channels measured in the presence of Mg²⁺ in the intracellular solution. The curves were fitted to equation 1. The mean parameters of ATP inhibition are given in the Supplementary Table 2.

Figure 2. Measurements of membrane currents.

A: Currents from inside-out patches excised from HEK293 cells overexpressing recombinant Kir6.2/SUR1-WT, Kir6.2/SUR1-Y356C and Kir6.2/SUR1-L582V, in Mg²⁺-containing (left) and Mg²⁺-free (right) solution. Addition of 100μM ATP (±Mg²⁺) is indicated. Stimulation protocol is given in the inset. B-E: ATP (±Mg²⁺) concentration-inhibition curves for wild-type (open circles) and ‘heterozygous’ (half-filled circles) and ‘homozygous’ (filled circles) mutant K_ATP channels. B: MgATP concentration-inhibition curves for wild type and Kir6.2/SUR1-Y356C K_ATP channels. C: ATP (Mg²⁺-free) concentration-inhibition curves for wild type and Kir6.2/SUR1-Y356C K_ATP channels. D: MgATP concentration-inhibition curves for wild type and Kir6.2/SUR1-L582V K_ATP channels. E: ATP (Mg²⁺-free) concentration-inhibition curves for wild type and Kir6.2/SUR1-L582V K_ATP channels.

Figure 3. Subcellular localisation of wild type and mutant K_ATP channels:

HEK cells were transfected either with c-myc-tagged SUR1 (wild type or mutant) subunit alone or together with Kir 6.2. A c-myc tag was inserted into the extracellular loop of the SUR1 subunit. Figures 2A, C and E show representative confocal images of cells expressing the SUR1 (Wt/Mut) subunit alone. Cells were fixed, permeabalised and stained with anti c-myc antibody. Figures 2B, D and F show representative images of cells expressing SUR1 (Wt/Mut) subunits together with Kir6.2. Cells were directly stained with anti c-myc antibody after fixation to detect surface channels.

Figure 4. Effect of extracellular glucose on membrane potential (A-C) and [Ca²⁺]_cyt (D-F) measured in β-cell lines transiently expressing wild-type and mutant K_ATP channels.

A: Representative recordings of membrane potential of INS1(823/13) cells expressing wild-type, homY356C and homL582V K_ATP channels. The addition of 10mmol/l glucose and 0.2mmol/l tolbutamide is indicated. B, C: dependence of the membrane potential on extracellular glucose for INS1(832/13) cells expressing wild-type (open circles) hetY356C (half-filled circles) and homY356C (closed circles) (B) or wild-type, hetL582V or homL582V (labels as above) (C). The wild-type data is given as open circles, the data from homomeric mutant is given in closed circles. The membrane potential at 0.2mM tolbutamide and 2μM FCCP is indicated with arrows. Statistical significance of differences between the sample and wild-type: P<0.05 (*) and P<0.01 (**). D, E: Representative recordings of Fura-Red fluorescence ratio (ex440/480)

Figure 5. Effect of SUR1 mutations on glucose-stimulated hGH release from INS-1(832/13) cells.

Cells were co-transfected with 0.5 µg of hGH-encoding plasmid pXGH5 together with 1 µg of SUR1 wt or mutant. hGH release was expressed as a percentage of the total hGH and was compared with values obtained under basal conditions (3mmol/l glucose).