Cantu syndrome–associated SUR2[H60Y] mutation confers selective gain of function on Kir6.1 ATP-sensitive potassium channels

Figures

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  Figure 1. H60Y induces cell hyperpolarization when associated with Kir6.1 but not with Kir6.2.A, representatives of images of stable cell lines incubated with 3 μM DiBAC(4)3 without any other drug treatment. Row 1: phage contrast; row 2: mCherry fluorescence; and row 3: DiBAC fluorescence. The length of the scale bar is 150 μm. B, summary of DiBAC(4)3 fluorescence of stable cell lines in the absence or presence of pinacidil (3 μM PIN) or glibenclamide (1 μM GBM). The data points represent individual biological replicates. Two-way ANOVA with Dunnett’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001 with comparison as noted. ##p < 0.01, ###p < 0.001 compared with the corresponding data under basal conditions. C, representative traces of currents recorded by inside–out patch-clamp assay in the presence of different concentrations of ATP in the presence of 0.5 mM free Mg2+. D, summary data of the dose-dependent relationship and the IC50 values of recordings in (C). Unpaired Student's t test. ns p ≥ 0.05. ns, nonsignificant.

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  Figure 2. Two or more sequence elements are responsible for the effect of H60Y on Kir6.1.A, scheme of chimera sequences engineered between Kir6.1 and Kir6.2. Chimeras with sequence from Kir6.1 (red) and Kir6.2 (blue) are shown aligned with the Kir6.1 linear structure. B, DiBAC(4)3 fluorescence of untransfected cells and cells expressing Kir6.1, Kir6.2, or their chimeras associated with SUR2B[H60Y] in the absence or the presence of pinacidil (3 μM PIN) or glibenclamide (1 μM GBM). The data points represent individual biological replicates. Two-way ANOVA with Dunnett’s multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001 with comparison as noted. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the corresponding data under basal condition.

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  Figure 3. N and C termini of Kir6.1 are primarily responsible for transducing the activation effect of H60Y.A, scheme of chimera sequences engineered between Kir6.1 and Kir6.2. Chimeras with sequence from Kir6.1 (red) and Kir6.2 (blue) are shown aligned with the Kir6.1 linear structure. B, DiBAC(4)3 fluorescence of untransfected cells and cells expressing Kir6.1, Kir6.2, or their chimeras associated with SUR2B[H60Y] under the basal condition. The data points represent individual biological replicates. One-way ANOVA with Dunnett’s multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001 with comparison as noted.

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  Figure 4. Further localization of key sequence elements for the H60Y effect.A, C, and E, scheme of chimera sequences engineered between Kir6.1 and Kir6.2. Chimeras with sequence from Kir6.1 (red) and Kir6.2 (blue) are shown aligned with the Kir6.1 linear structure. B, D, and F, DiBAC(4)3 assay data of stable cell lines expressing SUR2B[H60Y]-associated channels under the basal condition, with chimeras explained in (A), (C), and (E), respectively. The data points represent individual biological replicates. One-way ANOVA with Dunnett’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 with comparison as noted.

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  Figure 5. R325V converts Kir6.2 into an H60Y-sensitive pore subunit.A, amino acid sequence alignment of Kir6.1 and Kir6.2 in the corresponding region of D(1). B, normalized DiBAC(4)3 data for indicated WT or mutant channel–expressing cell lines under basal conditions or treatment with 3 μM pinacidil. C, normalized DiBAC(4)3 data for indicated WT, Kir6.2[R325V], or Kir6.1[V334R] channel–expressing cell lines under basal condition or treatment with 3 μM pinacidil. The data points represent individual biological replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001 with comparison as noted. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the corresponding data under basal condition.

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  Figure 6. Further subdivision of the N terminus generates a confounding GOF effect.A and C, scheme of chimera sequences engineered between Kir6.1 and Kir6.2. Chimeras with sequence from Kir6.1 (red) and Kir6.2 (blue) are shown aligned with the Kir6.1 linear structure. B and D, DiBAC(4)3 assay data of stable cell lines under the basal condition, with chimeras explained in (A, C), respectively. The data points represent individual biological replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗∗p < 0.01, ∗p < 0.05, ∗∗∗p < 0.001, ns p ≥ 0.05 with comparison as noted. GOF, gain of function; ns, not significant.

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  Figure 7. Mutation R325V couples the gain-of-function effect of SUR2B[H60Y] in Kir6.2-A(1) chimera.A, representative inside–out patch-clamp recording traces of Kir6.2-A(1) or Kir6.2-A(1)[R325V] associated with WTSUR2B or SUR2B[H60Y] in the presence of different concentrations of ATP in the presence of 0.5 mM free Mg2+. B, summary data of the dose-dependent relationship and the IC50 values of recordings in (A). The data points represent individual biological replicates. One-way ANOVA with Games–Howell’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01.

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  Figure 8. Localization of His60 in SUR2B and Val334 in Kir6.1. Structures of Kir6.1–SUR2B in propeller (A) and quatrefoil conformations (B) (4). Residues of His60 in SUR2 and Val334 in Kir6.1 are shown as red and green spheres, respectively, with the Cα–Cα distances noted for propeller (C) and quatrefoil (D) conformations. The N terminus of Kir6.1 is shown as a yellow ribbon. Val334, valine 334.