A new familial form of a late-onset, persistent hyperinsulinemic hypoglycemia of infancy caused by a novel mutation in KCNJ11

Abstract

The ATP-sensitive potassium channel (K_ATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic β-cells. The pancreatic K_ATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that K_ATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.

Keywords: ATP-Sensitive Potassium Channel (KATP); Inwardly Rectifying Potassium Channel 6.2 (Kir6.2); Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI); sulphonylurea Receptor 1 (SUR1).

Figure 1.

(A) The family pedigree of the index patient. Each family member that carries the A28V hKir6.2 mutation exhibits some degree of PHHI. (B to D) Whole-cell recording of K_ATP currents in HEK cells. (B) A representative wild-type K_ATP current (blue trace) was elicited by a voltage ramp pulse (0.5V/s). As predicted, the K_ATP current was augmented by 300 µM K_ATP channel opener diazoxide (red trace) and inhibited by 300 µM K_ATP channel blocker tolbutamide (black trace). (C) In contrast, HEK cells transfected with A28V hKir6.2 exhibited minuscule K_ATP current (blue trace) and neither 300 µM K_ATP channel opener diazoxide (red trace) or 300 µM K_ATP channel blocker tolbutamide (black trace) had an effect on the A28V K_ATP currents. (D) Summary of wild-type and A28V K_ATP currents in HEK cells. Wild-type K_ATP currents (n = 5) were significantly larger than A28V K_ATP currents (n = 9), as determined by Mann-Whitney U-test (*p < 0.05).

Figure 2.

Surface expression of K_ATP channels in HEK cells. An HA-tag was inserted into the extracellular domain on rSUR1 and then co-transfected with either wild-type hKir6.2 (A) or A28V hKir6.2. As Kir6.2 must be co-assembled with SUR1 for correct trafficking, the subcellular distribution of HA staining should faithfully represent the K_ATP channel distribution. K_ATP channels located on the cell surface were labeled green. Total K_ATP channels were labeled red, and cell nuclei were counterstained with DAPI (blue). The wild-type K_ATP channels were clearly visible on the cell surface (A), but A28V hKir6.2-containing channels were not readily observed on the cell surface (C). (B&D): Cross-sectional staining intensity profiles of HEK cells expressing wild-type hKir6.2 (C) and A28V hKir6.2 (D). The profiles were determined from cross-sections indicated by the white lines in A and C. Surface staining signals are clearly visible in HEK cells transfected with wild-type hKir6.2, as the green line shows distinct peaks at the cell boundary. By contrast, in HEK cells transfected with A28V hKir6.2, the cell surface boundary is not clearly demarcated (green line, D). (E) Quantitative analysis of K_ATP channel surface staining signals. K_ATP channels containing A28V hKir6.2 had greatly reduced surface staining signals compared to wild-type hKir6.2 containing K_ATP channels (***p < 0.0005, Mann-Whitney U-test n = 11 for each group).

Figure 3.

hKir6.2(A28V) has a dominant-negative effect on K_ATP channel surface expression in HEK cells. (A) An HA-tag was inserted into the extracellular domain of rSUR1 and then co-transfected with one of the hKir6.2 tandem constructs that are linked by a self-cleaving P2A linker. Only cells transfected with the wt-P2A-wt hKir6.2 construct showed strong surface staining (top row). Surface expression was not apparent in HEK cells expressing K_ATP channels that contain wt-P2A-A28V, A28V-P2A-wt or A28V-P2A-A28V hKir6.2. (B) Quantitative analysis of the surface signal from cells transfected with various hKir6.2 tandem constructs. K_ATP channels formed from wt-P2A-wt tandem construct expression had much stronger surface staining signals compared to any other hKir6.2 tandem constructs that contain the A28V Kir6.2 mutation. (**p = 0.001, ***p < 0.0005, one-way ANOVA with posthoc multiple comparison test, n = 8 for wt-P2A-wt, 8 for A28V-P2A-A28V, 9 for A28V-P2A-wt, and 14 for wt-P2A-A28V).

Figure 4.

Two-electrode voltage clamp recording of K_ATP currents in the Xenopus oocytes. (A) A representative wild-type K_ATP current (red trace) was elicited by a voltage ramp pulse (0.5V/s). As predicted, the K_ATP current was inhibited by 200 µM K_ATP channel blocker, tolbutamide (blue trace). Shifting extracellular potassium concentration from 2 mM to 100 mM caused a shift of reversal potential, as predicted by the potassium equilibrium potential. (B) A representative A28V hKir6.2 K_ATP current (red trace) was elicited by a voltage ramp pulse (0.5V/s). This A28V hKir6.2 K_ATP current was relatively smaller than the wild-type K_ATP current, but it could be inhibited by 200 µM tolbutamide (blue trace). The K_ATP channels containing A28V hKir6.2 were still potassium selective, as the reversal potential followed the potassium equilibrium potential (black trace). (C) A representative recording trace from a Xenopus oocyte injected with an equimolar ratio of wt and A28V hKir6.2 mRNA. K_ATP current (red trace) was elicited by a voltage ramp pulse (0.5V/s). This wt/A28V K_ATP current size was in between wild-type and A28V hKir6.2 K_ATP current, and was inhibited by 200 µM tolbutamide (blue trace). The wt/A28V K_ATP currents were also potassium selective, as the reversal potential followed the potassium equilibrium potential (black trace). (D) Summary of wt, A28V and wt/A28V K_ATP currents in the Xenopus oocytes. wt K_ATP currents (n = 7) were significantly larger than A28V K_ATP currents (n = 6) and wt/A28V K_ATP currents (n = 8), as determined by one-way ANOVA with posthoc multiple comparison test, (**p = 0.001, ***p < 0.0005). (E) Summary of the reversal potentials of wt, A28V, and wt/A28V K_ATP currents. In all three groups, the reversal potentials followed closely with the extracellular potassium concentration, indicating this hKir6.2(A28V) mutant is still potassium selective.

Figure 5.

Predicted protein structures of wild-type (A to C) and A28V (D to F) hKir6.2. The first and second transmembrane segments are labeled as M1 and M2, respectively. The selective filter and the position 28 are marked with blank and filled arrowhead, respectively. The N and C represent the N- and C- terminus of the hKir6.2 protein (A and D). (G to K) A superimposed image of the predicted wild-type and A28V hKir6.2. The side chain of position 28 (alanine in wild-type and valine in the mutant) are shown as cyan balls for wild-type and orange balls for the mutant (H and I). Di-acidic motifs of wild-type and A28V hKir6.2 (D280 and E282) are shown as cyan and orange sticks, respectively. (J) The A28V mutation caused a clockwise rearrangement of the RKR motif. (L to N) The fully assembled K_ATP channel complex (PDB: 5WUA).18 The solid and dashed circles represent the presumptive N-terminal region containing the 28th alanine residue and C- terminal region containing the RKR motif, respectively. (O) A plausible molecular mechanism of the A28V mutation on K_ATP channel trafficking. In K_ATP channel formed by the wild-type Kir6.2, the RTR motif is hinged on the neighboring SUR1, and the chaperone may dock onto the K_ATP channel complex to facilitate the assembled channel complex exiting the ER. In K_ATP channel formed by the A28V Kir6.2, the C-terminus is distorted and the RKR motif is no longer hidden. The exposed RKR motif may cause a hindrance for the chaperone docking and hence, prevent the forward trafficking of the mutated K_ATP channel.