An in-frame deletion in Kir6.2 (KCNJ11) causing neonatal diabetes reveals a site of interaction between Kir6.2 and SUR1

Abstract

Context: Activating mutations in genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the pancreatic ATP-sensitive K(+) channel are a common cause of permanent neonatal diabetes (PNDM). All Kir6.2 mutations identified to date are missense mutations. We describe here a novel in-frame deletion (residues 28-32) in Kir6.2 in a heterozygous patient with PNDM without neurological problems that are detectable by standard evaluation.

Objective: The aim of the study was to identify the mutation responsible for neonatal diabetes in this patient and characterize its functional effects.

Design: Wild-type and mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes.

Results: The Kir6.2-28Delta32 mutation produced a significant decrease in ATP inhibition and an increase in whole-cell K(ATP) currents, explaining the diabetes of the patient. Tolbutamide block was only slightly reduced in the simulated heterozygous state, suggesting that the patient should respond to sulfonylurea therapy. The mutation decreased ATP inhibition indirectly, by increasing the intrinsic (unliganded) channel open probability. Neither effect was observed when Kir6.2 was expressed in the absence of SUR1, suggesting that the mutation impairs coupling between SUR1 and Kir6.2. Coimmunoprecipitation studies further revealed that the mutation disrupted a physical interaction between Kir6.2 and residues 1-288 (but not residues 1-196) of SUR1.

Conclusions: We report a novel KCNJ11 mutation causing PNDM. Our results show that residues 28-32 in the N terminus of Kir6.2 interact both physically and functionally with SUR1 and suggest that residues 196-288 of SUR1 are important in this interaction.

Fig. 1

A, Representative whole-cell currents recorded from Xenopus oocytes coexpressing SUR1 and either Kir6.2, Kir6.2-28Δ32 (hom28Δ32), or both Kir6.2 and Kir6.2-28Δ32 (het28Δ32) in response to voltage steps of ± 20 mV from a holding potential of −10 mV. B, Mean steady-state whole-cell K_ATP currents recorded from oocytes coexpressing SUR1 and either wild-type or mutant Kir6.2, as indicated. Currents were evoked by a voltage step from −10 to −30 mV before (control, white bars) and after (black bars) application of 3 mM azide, and in the presence of 3 mM azide plus 0.5 mM tolbutamide (gray bars). The number of oocytes is indicated below the bars. *, P ≤ 0.05 against control (t test).

Fig. 2

A and B, Representative K_ATP currents recorded in response to successive voltage ramps from −110 to +100 mV in an inside-out patch excised from a Xenopus oocyte coexpressing the indicated homozygous K_ATP channels in the absence (A) and presence (B) of Mg²⁺. The dashed line indicates the zero current level. C and D, Mean relationship between [ATP] (C) or [MgATP] (D) and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc), for Kir6.2/SUR1 (WT, black filled circles) and homKir6.2-28Δ32/SUR1 (hom28Δ32, unfilled circles) channels. The lines are the best fit of the Hill equation to the mean data: panel C, WT, IC₅₀ = 5 μM, n = 9; hom28Δ32, IC₅₀ = 16 μM, n = 10. Panel D, WT, IC₅₀ = 13 μM, n = 6; hom28Δ32, IC₅₀ = 89 μM, n = 5. Figures are derived from the fit of the mean data.

Fig. 3

A, Representative K_ATP currents recorded in response to successive voltage ramps from −110 to +100 mV in an inside-out patch excised from a Xenopus oocyte expressing the indicated homozygous Kir6.2ΔC construct. The dashed line indicates the zero current level. B, Mean relationship between [ATP] and Kir6.2ΔC conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc), for Kir6.2ΔC (black filled circles) and homKir6.2ΔC- 28Δ32 (unfilled circles) channels. The lines are the best fit of the Hill equation to the mean data: WT, IC₅₀ = 99 μM, n = 7; hom28Δ32, IC₅₀ = 156 μM, n = 7. Figures are derived from the fit of the mean data.

Fig. 4

A, Schematic representation of the SUR1^(1-196) and SUR1^(1-288) constructs, showing transmembrane helices and an N-terminal FLAG tag. B and C, Representative K_ATP currents recorded in response to successive voltage ramps from −110 to +100 mV in an inside-out patch excised from a Xenopus oocyte coexpressing the indicated homozygous Kir6.2ΔC construct in the presence of SUR1^(1–196) (B) and/or SUR1^(1-288) (C). The dashed line indicates the zero current level. D and E, Mean relationship between [ATP] and Kir6.2ΔC conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc), for Kir6.2ΔC (black filled circles) and homKir6.2ΔC-28Δ32 (unfilled circles) channels in the presence of either SUR1^(1-196) (D) or SUR1^(1-288) (E). The lines are the best fit of the Hill equation to the mean data: D, WT, IC₅₀ = 311 μM, n = 6; hom28Δ32, IC₅₀ = 325 μM, n = 7. E, WT, IC₅₀ = 136 μM, n = 5; hom28Δ32, IC₅₀ = 202 μM, n = 5. Figures are derived from the fit of the mean data.

Fig. 5

Relative levels of surface expression in Xenopus oocytes of HA-tagged Kir6.2 (wild-type or mutant) coexpressed with or without SUR1, and HA-tagged Kir6.2ΔC (wild-type or mutant), as indicated. Data are the mean ± sem of 18–52 individually assayed oocytes from three different preparations. Control oocytes were uninjected. Surface expression is expressed as the relative light units (RLU) recorded in 15 sec from individual oocytes. **, P < 0.01 compared with wild-type for both Kir6.2-28Δ32 and Kir6.2ΔC-28Δ32.

Fig. 6

Coimmunoprecipitation assays performed on oocytes expressing FLAG-tagged SUR1^(1-196) and either HA-tagged Kir6.2ΔC or HA-tagged Kir6.2ΔC- 28Δ32. A, Top, Binding of Kir6.2ΔC and Kir6.2ΔC-28Δ32 to TMD0 [SUR1^(1-196), n = 6], expressed as a percentage of binding to Kir6.2ΔC. Bound protein levels were normalized to the expression levels of SUR1^(1-196). Bottom, upper panel, Representative anti-FLAG Western blots from individual experiments, showing SUR1^(1-196) binding to Kir6.2ΔC and Kir6.2ΔC-28Δ32 (corresponding to the graph above). Bottom, lower panel, Representative Western blots of the oocyte lysate inputs into the binding assay, showing expression levels of TMD0. B, Top, Binding of Kir6.2ΔC and Kir6.2ΔC-28Δ32 to SUR1^(1-288) (n = 4), expressed as a percentage of the binding of Kir6.2ΔC. **, P < 0.01 compared with Kir6.2ΔC. Bound protein levels were normalized to the precipitated levels of SUR1^(1-288) and expression levels of Kir6.2ΔC. Bottom, upper panel, Representative anti-HA Western blots from individual experiments, showing Kir6.2ΔC or Kir6.2ΔC- 28Δ32 binding to SUR1^(1-288), corresponding to the graph above. Bottom, lower panel, Representative Western blots of the oocyte lysate inputs into the binding assay, showing expression levels of Kir6.2ΔC or Kir6.2ΔC-28Δ32.