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The complex disorder Cantu syndrome (CS) arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which KATP function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (86Rb+) efflux assays and patch-clamp electrophysiology. Our results indicate that D207E increases KATP channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS–associated SUR2 mutations result in KATP gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS.
ATP-sensitive potassium (KATP) channels are potassium-selective ion channels formed by obligate co-assembly of pore-forming Kir6.x subunits and regulatory sulfonylurea receptors (SURx)
). Channel opening is dynamically regulated by intracellular nucleotides and membrane phospholipids and thereby couples the membrane potential of excitable cells to their metabolic state (
). By binding to, and stabilizing, closed states of the Kir6.x subunit, ATP decreases channel open probability, whereas magnesium-nucleotide complexes (MgADP and MgATP) bind to the nucleotide-binding domains (NBDs) of SURx subunits to activate the channel (
In cardiac, smooth, and skeletal muscle, SUR2 subunits (of which there are two main splice variants, SUR2A and SUR2B) co-assemble variously with Kir6.1 (as in vascular smooth muscle) or Kir6.2 (as in ventricular and skeletal muscle) (
). In the heart, Kir6.2/SUR2A KATP channels have been proposed to be critical for ischemic pre-conditioning, whereas in skeletal muscle Kir6.2/SUR2A channels may provide a “brake” to hyperpolarize the membrane potential despite elevations in intracellular calcium during periods of exercise and increased metabolism (
). In smooth muscle, Kir6.1/SUR2B KATP activity is a key determinant of electrical excitability and consequent contractility in blood and lymphatic vessels, as well as in bladder and uterine muscle (
There have now been multiple reports of mutations in the ABCC9 and KCNJ8 genes (which encode SUR2 and Kir6.1, respectively) associated with the complex heritable disorder, Cantu syndrome (CS) (
). CS patients exhibit diverse cardiovascular features, including dilated and tortuous vessels, cardiomegaly, electrophysiological alterations in the cardiac conduction system, decreased neurovascular coupling, and persistence of fetal circulation (
). An emerging model for the molecular basis of CS is that missense mutations in ABCC9 or KCNJ8 result in increased KATP channel activity and consequently reduced smooth muscle excitability and contractility (
). CS–associated mutations in SUR2 have previously been shown to result in KATP channel gain-of-function (GoF) by distinct mechanisms, including enhanced Mg2+-nucleotide activation and increased intrinsic open probability with consequent decreases in ATP inhibition (
). Here, we examined the functional effects of previously uncharacterized CS mutations that are predicted to cluster together at the link between NBD1 and TMD2: Y981S (human Y985S), G985E (G989E), and M1056I (M1060I) (Fig. 1), and we compared the molecular consequences to those of D207E, located in the intracellular L0-linker, between TMD0 and TMD1 (Fig. 1). In addition, the sensitivity of mutant channels to the sulfonylurea KATP-inhibitor glibenclamide was tested. Glibenclamide holds promise as a potential treatment for CS, although numerous Kir6.2- and SUR1-dependent KATP GoF mutations, which reduce sulfonylurea sensitivity, have previously been reported (
Conserved functional consequences of disease-associated mutations in the slide-helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel.
). Therefore, determining sulfonylurea sensitivity for specific mutations may be required for future individualized therapy. The results are interpreted alongside structural insights from recently reported high resolution cryo-EM structures of KATP channel complexes (
) to provide further detail of the molecular basis of KATP channel GoF in CS.
Figure 1Structure of KATP channels.A, KATP channels form as hetero-octamers of four pore-forming Kir6.x subunits each associated with a SUR subunit (two SUR subunits omitted from figure). B, schematic representation of the position of D207E, Y981S, G985E, M1056I, and R1150Q/R1150W in the linear sequence of SUR2. C, expected positions of D207E, Y981S, G985E, M1056I, and R1150Q/R1150W mapped onto the pancreatic KATP (Kir6.2/SUR1; Protein Data Bank code 5WUA) structure (
). The residues shown are the analogous positions in hamster SUR1 (Asp-209, Tyr-1004, Ala-1008, Thr-1089, and Arg-1183, respectively; there is 70% sequence identity between hSUR1 and rSUR2A, and structural the domains are expected to be conserved). ATP is modeled in the Kir6.2-binding site.
The subject is the fourth child of healthy, unrelated Caucasian parents, with no family history of relevance to her condition. The pregnancy was complicated with raised nuchal translucency at 12 weeks and polyhydramnios at 32 weeks of gestation. At 38 weeks of gestation, labor was induced, with uncomplicated vaginal delivery. Birth weight was 5.3 kg (>99 centile). There was no significant delay in early development, but language skills developed slowly. At birth, hypertrichosis was evident, with a full head of dark hair with low anterior hairline; shoulders, arms, legs, and back were covered with long, thick, and dark hair. At 3 years of age, facial features were rather coarse, with mild epicanthic folds and down-slanting palpable fissures with full lips and a broad face. The forehead was extremely low with fine hair in front of the ears, extending over her chin, and hypertrichosis over her neck and chest. The heart was slightly enlarged, but there was no overt evidence of cardiomyopathy.
At age 5, the subject presented with recurrent respiratory infections and required hospital admission for pneumonia, leading to tonsillectomy and adenoidectomy, which improved severe snoring and obstructive sleep apnea. Height was on the 50th centile, weight on the 91st centile, and her head circumference was on the 98th centile. Facial features remained coarse with down-slanting palpable fissures, full cheeks, broad tip to the nose with mild thickening of the alae nasae, and a low columella. Significant joint laxity was evident in the hands, with deep palmar creases and soft skin on the palms and generous fetal finger pads.
This subject thus exhibited most of the features typically found in individuals with CS (
). Sequencing of ABCC9-coding regions revealed a heterozygous mutation (c.2954A→C, p.Y985S) that was absent in genomic DNA from either parent. Heterozygous de novo mutations (p.G989E; p.M1060I) were also identified in two additional diagnosed CS subjects, for whom clinical details are not available. The three mutated residues are predicted to cluster in a similar location within the SUR2 protein (Fig. 1). We therefore analyzed the molecular consequences of these mutations and compared them with the consequences of the most common CS mutation (p.R1154Q), and another uncharacterized CS mutation p.D207E (
Cantu syndrome mutations result in gain-of-function of KATP channel in intact cells
To determine the effect of mutations on KATP channel function, SUR2A constructs were co-expressed with Kir6.2 in Cosm6 cells, and channel activity was assessed using a radioactive 86Rb+ flux assay. First, basal KATP activity under quasi-physiological regulation by intracellular nucleotides in intact cells was determined by measuring 86Rb+ efflux from cells bathed in Ringer's solution. As shown in Fig. 2, A and B, the KATP-dependent 86Rb+ efflux rate in these conditions was significantly increased by the D207E, G985E, and M1056I substitutions, although a trend toward an increase was observed for Y981S. Efflux rates in basal conditions are a function of both channel activity and surface membrane expression. We assessed efflux rates in cells subjected to metabolic inhibition (MI) to decrease intracellular ATP synthesis and relieve channels of nucleotide regulation. Assuming that the single channel conductance is unaffected and that MI maximally activates available channels, the comparison of the MI efflux rate between constructs provides an estimation of the relative number of channels in the membrane. As shown in Fig. 2, C and D, there was a statistically insignificant decrease in Y981S efflux rate, and a statistically insignificant increase in MI efflux rate was observed for D207E, implying that these mutations result in only a small decrease or increase, respectively, in channel number. Otherwise, the maximum flux under these conditions was not markedly different between constructs, suggesting that the number of active channels was similar.
Figure 2D207E, G985E, and M1056I significantly increase basal KATP channel activity in intact cells.A, cumulative 86Rb+ efflux was measured from Cosm6 cells transfected either with GFP alone or with Kir6.2 plus WT or mutant SUR2A. Efflux as a function of time was first recorded in basal conditions (cells incubated in Ringer’s solution) (A), and the KATP-dependent efflux rate was attained from exponential fits to efflux time data (B). Second, efflux was measured from cells subjected to MI (induced by incubation in Ringer’s solution with 2.5 mg/ml oligomycin and 1 mm 2-deoxy-d-glucose from 10 min prior to commencing the flux assay) (C), and the rate constant for KATP-dependent efflux in MI was calculated from exponential fits to the early time points (2.5, 5, and 12.5 min) (inset, C and D). Efflux-time data are shown as mean ± S.E., and KATP-dependent efflux rate scatter plots show mean ± S.D. from 3 to 5 independent experiments. Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann-Whitney U test.
All known CS patients are heterozygous, and we modeled heterozygous conditions by co-expressing Kir6.2 together with WT SUR2A and mutant SUR2A subunits at a 1:1 ratio. The resultant channels were assayed by monitoring 86Rb+ efflux. Only very minor increases in basal efflux rate were observed for D207E, G985E, and M1056I, although a moderate, statistically significant increase was observed for Y981S channels (Fig. 3). Taken together, these data demonstrate that whereas all tested mutations result in KATP gain-of-function, in heterozygous conditions the effect is subtle under basal conditions.
Figure 3Y981S confers gain-of-function when co-expressed with WT subunits in intact cells whereas only minor effects are observed for other mutations. Cumulative 86Rb+ efflux was measured from Cosm6 cells transfected either with GFP alone or with Kir6.2 alongside SUR2A-WT alone or SUR2A-WT with mutant SUR2A at a 1:1 ratio. Basal efflux shown as function of time (A), and KATP-dependent efflux rate constants were calculated (B). Efflux rate was measured in MI conditions (induced by incubation in Ringer’s solution with 2.5 mg/ml oligomycin and 1 mm 2-deoxy-d-glucose from 10 min prior to commencing the flux assay) (C), and rate constants were calculated from early time points (2.5, 5, and 12.5 min) (inset, C and D). Efflux-time data are shown as mean ± S.E., and KATP-dependent efflux rate scatter plots show mean ± S.D. from 3 to 5 independent experiments. Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann-Whitney U test.
D207E in the L0-linker increases open-state stability and decreases ATP inhibition
Increased basal KATP channel activity could arise from changes in multiple distinct nucleotide-sensing mechanisms, including increased Mg-nucleotide activation or decreased ATP inhibition, either as a result of decreased binding affinity or decreased efficacy due to increase in intrinsic open-state stability (
). To investigate the effects of the above mutations on nucleotide sensitivity, we used inside-out patch-clamp recordings. As shown in Fig. 4, channels comprising Kir6.2 and WT SUR2A were inhibited by ATP in the absence of Mg2+ with an IC50 of ∼15 μm. In the presence of Mg2+, the IC50 was ∼20 μm. The presence of Mg2+ results in increased channel activity for a given ATP concentration, resulting from mixed effects of the MgATP activation and magnesium-independent ATP inhibition, and is reflected in a right-shift in the MgATP dose-response curve, relative to Mg2+-free ATP. The sensitivity of D207E-containing channels to both MgATP and Mg2+-free ATP was reduced ∼3-fold in each case, with IC50 in Mg2+-free ATP of ∼40 and ∼55 μm, respectively (Fig. 4).
Figure 4D207E provokes decreases in ATP inhibition in the presence and absence of Mg2+. Inside-out patch-clamp recordings were made from Cosm6 cells transfected with Kir6.2 plus either SUR2A-WT or D207E. The response to MgATP was determined from voltage-clamped patches (−50 mV), as shown in representative traces (scale bars denote 500 pA/5 s unless otherwise stated) (A). ATP inhibition in the absence of Mg2+ was also determined from voltage-clamped patches (−50 mV), as shown in representative traces (B). Kir6.2/SUR2A-WT channels were inhibited by ATP in the presence of Mg2+ with an IC50 of 21.3 ± 3.0 μm (Hill coefficient 1.3 ± 0.1; n = 16) and by ATP in the absence of Mg2+ with an IC50 of 14.4 ± 2.3 μm (Hill coefficient 1.2 ± 0.1; n = 14). This was increased in both cases by the D207E mutation; here, the IC50 for ATP in the presence of Mg2+ was 55.8 ± 11.9 μm (Hill coefficient 1.2 ± 0.1; n = 9), and by ATP in the absence of Mg2+ with an IC50 of 39.9 ± 6.0 μm (Hill coefficient 1.2 ± 0.1; n = 9) (C). Scatter plots show data from individual experiments with mean IC50 ± S.D. D and E, notably, the ratio of the IC50 for ATP in the absence and presence of Mg2+ was unchanged by the D207E mutation, indicating the mutation had little effect on Mg2+-nucleotide activation (F). Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann-Whitney U test.
The relationship between the IC50 in ATP in the presence and absence of Mg2+ (IC50[MgATP]/IC50[ATP]) can provide an indirect measure of the extent of Mg2+-nucleotide activation. For D207E and WT SUR2A, the IC50[MgATP]/IC50[ATP] was very similar (∼1.5), indicating that sensitivity to Mg2+ activation was unaffected by this mutation (Fig. 4F) and suggesting that GoF results from decreased sensitivity to inhibitory ATP itself. Considering the location of this residue, predicted to be in close proximity to the ATP-binding site on Kir6.1 in the octameric KATP complex (
), this could conceivably arise from altered binding affinity. Alternatively, decreased ATP sensitivity could be the result of enhanced open-state stability of channels (
). To test the latter directly, we measured the response of channels to PIP2 perfused onto the intracellular surface of excised membrane patches (Fig. 5). PIP2 increases open probability (Po) to ∼1, and the ratio of initial current levels to the activated level in PIP2 provides an estimate of the initial “intrinsic” Po (
). As shown in Fig. 5, the intrinsic Po was increased from ∼0.4 in WT to ∼0.7 in D207E channels. Therefore, the D207E mutation within the L0-linker results in KATP gain-of-function by increasing the intrinsic open probability of channels, rather than by decreasing inhibitory ATP binding affinity.
Figure 5D207E increases intrinsic open-state stability. Inside-out patch-clamp recordings were made from Cosm6 cells transfected with Kir6.2 alongside either SUR2A-WT or D207E. Patches were administered 3 mm ATP followed by washout and administration of 5 μg/ml PIP2 to maximally increase channel activity, as indicated on representative traces (A). The stable peak current observed in PIP2 was divided by the current observed in the absence of nucleotides/PIP2 to determine the level of basal channel activity. D207E channels displayed increased basal activity (I[basal]/I[PIP2] was 0.44 ± 0.08 for SUR2A-WT and 0.73 ± 0.06 for D207E; scatter plot shows data from individual experiments with mean ± S.D. from seven patches) (B). Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann Whitney U test.
Mutations within the Y981/G985/M1056 cluster increase Mg2+-nucleotide activation
The disease-associated mutations Y981S, G985E, and M1056I are all clustered together on transmembrane helices 12 and 13 in TMD2 (Fig. 1). In comparison with WT SUR2A, the IC50 for ATP inhibition in the presence of Mg2+ was significantly increased by each of these mutations; however, in contrast to D207E, there was no effect on ATP sensitivity in the absence of Mg2+ (Fig. 6). This is further demonstrated by the increase in IC50[MgATP]/IC50[ATP] for all mutants (Fig. 6G), indicating that the mutations in this cluster of residues linking NBD1 to TMD2 increase channel activity by enhancing Mg2+-nucleotide activation.
Figure 6Y981S, G985E, and M1056I cause KATP GoF by enhancing Mg2+-nucleotide activation. Inside-out patch-clamp recordings were made from Cosm6 cells transfected with Kir6.2 alongside mutant SUR2A. The response to ATP in the presence (left) and absence (right) of Mg2+ was determined from voltage-clamped patches (−50 mV) of cells expressing either Y981S (A), G985E (B), or M1056I (C), as shown in representative traces (scale bars denote 500 pA/5 s unless otherwise stated). Analysis of dose-response experiments showed that each mutation increased the IC50 for MgATP compared with WT (MgATP IC50 for Y981S was 39.6 ± 7.3 μm, Hill coefficient 1.2 ± 0.1, n = 6; MgATP IC50 for G985E was 48.4 ± 8.7 μm, Hill coefficient 1.1 ± 0.1, n = 11; MgATP IC50 for M1056I was 91.9 ± 14.4 μm, Hill coefficient 1.7 ± 0.2, n = 6), with little effect on ATP inhibition in the absence of Mg2+ (ATP IC50 for Y981S was 11.8 ± 2.2 μm, Hill coefficient 1.0 ± 0.1, n = 4; ATP IC50 for G985E was 17.5 ± 4.7 μm, Hill coefficient 1.1 ± 0.1, n = 6; ATP IC50 for M1056I was 10.0 ± 5.6 μm, Hill coefficient 0.9 ± 0.1, n = 3) (D). Scatter plots show data from individual experiments with mean IC50 ± S.D. (E and F). Decreased ATP inhibition in the presence of Mg2+ only is demonstrated by the ratio of the IC50 for ATP in the presence and absence of Mg2+, which is markedly increased for Y981S, G985E, and M1056I (G). Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann Whitney U test.
R1150Q and R1150W in TMD2 also enhance Mg2+-nucleotide activation
Having established that the TMD2 Y981S, G985E, and M1056I mutations enhance Mg2+-nucleotide activation, we sought to test whether this mechanism was conserved for other TMD2 mutations, and the most common CS–associated mutations are R1150Q and R1150W. In agreement with a previous report (
), we show that R1150Q causes a large increase in MgATP IC50, whereas R1150W has a more modest effect (Fig. 7). In contrast, R1150Q and R1150W caused only slight increases in ATP IC50 (Fig. 7), again reflected in increased IC50[MgATP]/IC50[ATP] for R1150Q and R1150W (Fig. 7F), and thus both R1150Q and R1150W cause gain-of-function predominantly by enhancing Mg2+-nucleotide sensitivity.
Figure 7R1150Q and R1150W also cause KATP GoF by enhancing Mg2+-nucleotide activation. The response to MgATP (A) and ATP in the absence of Mg2+ (B) was determined for channels expressing Kir6.2 with R1150Q or R1150W, by inside-out patch-clamp recordings from Cosm6 cells as shown in representative traces (scale bars denote 500 pA/5 s unless otherwise stated). Dose-response analysis (C) demonstrated that both mutations markedly increased the IC50 for ATP in the presence of Mg2+, compared with WT (MgATP IC50 for R1150Q was 163.7 ± 36.8 μm, Hill coefficient 2.3 ± 0.3, n = 6; MgATP IC50 for R1150W was 46.0 ± 7.8 μm, Hill coefficient 1.2 ± 0.2, n = 6). The effect on ATP sensitivity in the absence of Mg2+ was more modest for both mutations (ATP IC50 for R1150Q was 17.3 ± 4.3 μm, Hill coefficient 1.2 ± 0.2, n = 8; ATP IC50 for R1150W was 20.6 ± 3.4 μm, Hill coefficient 1.3 ± 0.2, n = 10). Scatter plots show data from individual experiments with mean IC50 ± S.D. (D and E). An increase in the ratio of the IC50 for ATP in the presence over the IC50 for ATP in the absence of Mg2+ indicates that these mutations also predominantly confer GoF by augmentation of Mg2+-nucleotide activation (F). Statistical significance is denoted by asterisk and defined as p < 0.05 according to Mann-Whitney U test.
Effect of CS GoF mutations on glibenclamide sensitivity
Glibenclamide (glyburide) inhibits KATP channels in a biphasic manner, with high-affinity inhibition arising from interaction with the SUR subunit occurring at nanomolar to micromolar concentrations and low-affinity inhibition due to interaction with the Kir6.x subunit (
). To specifically measure high-affinity inhibition, we applied glibenclamide up to 10 μm. Glibenclamide inhibited WT SUR2A KATP currents (in the absence of nucleotides), as well as D207E, Y981S, G985E, and M1056I (Fig. 8), with maximal inhibition of ∼70% and IC50 values ranging from ∼15 to 45 nm. In contrast, mutations at residue 1150, in particular R1150W, resulted in a significant decrease in glibenclamide potency (Fig. 9).
Figure 8D207E, Y981S, G985E, and M1506I have no significant effect on glibenclamide sensitivity. Inside-out patch-clamp recordings were made from Cosm6 cells transfected with Kir6.2 alongside WT or mutant SUR2A. Reversible inhibition was observed following administration of increasing glibenclamide concentrations, as shown in representative traces (scale bars denote 500 pA/5 s unless otherwise stated) (A). Dose-response analysis demonstrated that only minor, non-statistically significant variations in glibenclamide IC50 values were observed for these mutations (IC50 for SUR2A-WT was 29.5 ± 11.1 nm, Hill coefficient 1.0 ± 0.1, n = 11; IC50 for D207E was 42.8 ± 11.4 nm, Hill coefficient 0.7 ± 0.1, n = 4; IC50 for Y981S 13.1 ± 5.9 nm, Hill coefficient 0.9 ± 0.1, n = 3; IC50 for G985E 39.2 ± 9.8 nm, Hill coefficient 1.0 ± 0.2, n = 4; IC50 for M1056I 47.3 ± 13.8 nm, Hill coefficient 1.0 ± 0.4, n = 4) (B). Scatter plots show data from individual experiments with mean IC50 ± S.D. (C). Statistical significance was determined using Mann-Whitney U tests.
Figure 9R1150Q and R1150W decrease glibenclamide sensitivity. Glibenclamide sensitivity was measured from inside-out patch-clamp recordings from Cosm6 cells expressing Kir6.2 with either SUR2A-WT, R1150Q, or R1150W as shown in representative traces (scale bars denote 500 pA/5 s unless otherwise stated) (A). Dose-response analysis shows that both R1150Q and R1150W induce a statistically significant decrease in glibenclamide sensitivity (IC50 for R1150Q was 86.3 ± 23.4 nm, Hill coefficient 0.8 ± 0.3, n = 5; IC50 for R1150W was 303.5 ± 91.1 nm, Hill coefficient 0.8 ± 0.1, n = 5) (B). Scatter plots show data from individual experiments with mean IC50 ± S.D. (C). Statistical significance was determined using Mann-Whitney U tests; *, p < 0.05.
Cantu syndrome–associated ABCC9 mutations all cause KATP GoF
To date, the few analyzed Cantu syndrome–associated mutations in ABCC9 (SUR2), have been shown to result in gain-of-function of KATP channels in the presence of Mg2+-nucleotides, which can arise either from decreased sensitivity to inhibitory ATP, or augmented activation by Mg2+-nucleotides (
). In this study, we have demonstrated that four previously uncharacterized mutations in SUR2 (D207E (human D207E), Y981S (Y985S), G985E (G989E), and M1056I (M1060I)) lead to increased channel activity in the presence of regulatory nucleotides via diverse molecular mechanisms. As in previous reports, we used Kir6.2/SUR2A channels for analysis due to the difficulty of recording Kir6.1/SUR2 currents, although it is expected that the mechanism of SUR2 mutations will be conserved irrespective of the pore-forming subunit. In addition, although the dependence of channel activity on intracellular nucleotides differs quantitatively between the two major SUR2 splice variants (SUR2A and SUR2B, which differ only in their C-terminal exon) (
), it is anticipated that, qualitatively, the changes observed for mutant SUR2A-containing channels will be common for SUR2B-containing channels. Interestingly, many CS features such as vascular dilatation and lymphedema likely arise from smooth muscle dysfunction, whereas the effects on cardiac electrophysiology and skeletal muscle are less obvious (
). This may suggest that the biophysical effects of mutations are more severe in SUR2B-containing channels than in SUR2A-containing channels. Alternatively, it is possible that the predominantly smooth muscle consequences of CS arise due to the physiological context of KATP function in smooth muscle rather than unique biophysical effects on SUR2B compared with SUR2A.
Mechanistic basis of GoF varies between mutations
Here, we compared the sensitivity of wildtype and mutant channels to ATP in the absence and presence of Mg2+ to separate the Mg2+-independent inhibitory effect of ATP from the activating effect of MgATP. This analysis shows that the D207E mutation reduces ATP inhibition itself, whereas the Y981S/G985E/M1056I mutations all increase activity by enhancing MgATP activation. The D207E mutation is found in the intracellular L0-linker between the two transmembrane domains TMD0 and TMD1 (Fig. 1). A critical role of the L0-linker in determining channel-gating properties has been suggested for both SUR1 and SUR2 (
). Recent high-resolution structures of the related Kir6.2/SUR1 KATP channel complex confirm earlier predictions that the L0-linker is closely apposed to the intracellular domains of the Kir6 subunit, and likely it provides a structural interaction between the subunits that is involved in the transduction of functional signals. Interestingly, multiple mutations in the L0-linker of SUR1 have been reported in neonatal diabetes patients, including the analogous mutation to D207E (
). Functional characterization of L0-linker mutations in SUR1 show that channel activity is increased due to elevations in the intrinsic channel open probability, arising from decreased occupation of long-lived, ATP-accessible closed states (
). Consistent with this finding, we show that enhanced basal open probability of D207E-containing channels, reflected in decreased PIP2 activation, underlies decreased sensitivity to inhibitory ATP (Figure 4, Figure 5).
In contrast, we show that the Y981S, G985E, and M1056I mutations all act by increasing KATP channel activation by MgATP (Fig. 6). These residues are all predicted to lie in close proximity to each other in a cluster within TMD2; Tyr-981 and Gly-985 are found at the N-terminal end of TM12, immediately following the NBD1-TMD2 linker, whereas M1056I is situated on the opposing TM13 (Fig. 1). The location of the Tyr-981/Gly-985/Met-1056 cluster, at the link between the NBDs and the TM domains (Fig. 1), is appropriate for transduction of movements between the intracellular and transmembrane domains of SUR2. Biochemical analyses of SUR and related ABC proteins indicate that MgADP or MgATP binding to the NBDs of SUR may act to stabilize dimerization of NBD1 and NBD2 (
). As free Mg2+ levels are tightly regulated in most cells, the physiological effect of CS mutations that alter Mg2+ nucleotide sensitivity will be altered responses to nucleotide concentrations (in the presence of Mg2+), not to changing Mg2+ concentrations (
In addition, we show that the previously reported common CS mutations R1150Q and R1150W (located in TM15 of TMD2) also enhance MgATP activation (Fig. 7), demonstrating that multiple transmembrane regions of TMD2 are involved in the conformational changes associated with Mg2+-nucleotide activation.
Notably, the gain-of-function induced by each mutation is quite subtle when mutant and WT SUR2A subunits are co-expressed to mimic the clinically relevant heterozygosity (Fig. 2). Recent reports of GoF mutations in Kir6.2 and SUR1 that underlie neonatal diabetes demonstrate that even very subtle biophysical effects can result in disease (
), suggesting that dramatic changes may not be necessary. In contrast, because SUR2B may be the more pathologically relevant splice variant, it is possible that these mutations will have a greater effect on channels containing SUR2B. In addition, the channel activity measured in 86Rb+ experiments under basal conditions may not fully recapitulate the metabolic and physiological context for KATP channels in muscle or other differentiated cell types, and so we cannot rule out a more significant activating effect under other conditions.
Consequences for sulfonylurea sensitivity
Previous studies have demonstrated that second generation sulfonylureas such as glibenclamide inhibit SUR2-containing KATP channels, albeit with lower potency than SUR1-containing channels (
). As such, glibenclamide, or other sulfonylureas, represents a potential pharmacotherapy for CS. However, there are multiple reports of neonatal diabetes mutations in the Kir6.2/SUR1 KATP subunits that reduce sulfonylurea sensitivity (
) and, as we have recently demonstrated, the CS–associated mutation V65M in Kir6.1 profoundly reduces glibenclamide inhibition of recombinant channels (
Conserved functional consequences of disease-associated mutations in the slide-helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel.
). Therefore, it is important to assess the effect of SUR2 CS mutations on inhibitor sensitivity. Here, we show that the D207E, Y981S, G985E, and M1056I mutations do not obviously affect glibenclamide sensitivity (Fig. 8). It has been reported that sulfonylurea inhibition of SUR2-containing channels is affected by nucleotide regulation (
), and so it is possible that these mutations may alter SU sensitivity under more complex physiological regulation, but this remains to be established.
A decrease in glibenclamide potency was observed in both the R1150Q and R1150W mutations (Fig. 9). Interestingly, Arg-1150 lies in TM15 (Fig. 1), and previous studies have demonstrated that TMs 14–16 are critical for high-affinity SU binding to SUR subunits (
Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.
). Indeed, serine to tyrosine substitution of a single residue in TM16 (predicted to lie within ∼15 Å of Arg-1150 on the cytoplasmic extensions of the TM helix) is sufficient to confer SUR2-like sensitivity to SUR1, and vice versa (
Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.
). This raises the possibility that the Arg-1150 mutations may directly decrease glibenclamide sensitivity via disruption of the drug-binding site. The R1150W mutation exhibited a more pronounced effect than the glutamine mutation at the same site, perhaps due to a greater steric effect of the bulky tryptophan side chain. Regarding the relevance to treatability of disease, it is important to note that glibenclamide sensitivity was evaluated in a “homozygous” context where all SUR2 subunits were mutated, but all CS patients identified so far are heterozygous. The data highlight the relatively minor effect of most mutations on drug sensitivity, although they suggest that the Arg-1150 mutation may reduce SU sensitivity in patients. Potentially, studies of native channels in human-induced pluripotent stem cell-derived cardiovascular cells may allow more accurate assessment of the potential impact of CS mutations on the pharmacological response.
Taken together, these results provide further evidence for KATP gain-of-function consequences of SUR2 mutations in Cantu syndrome. For several mutations clustered in TM12–13, the results illustrate a common mechanism (enhanced MgATP activation) without marked effect on sulfonylurea sensitivity. The results provide novel insights into the function of KATP channel complexes, can be useful for linking CS genotype to phenotype in this complex disorder, and will inform the consideration of therapeutic approaches to CS.
Experimental procedures
Molecular biology and cell culture
Mutations were introduced into a rat SUR2A (pCMV_rSUR2A; GenBankTM accession no. D83598.1) cDNA construct using site-directed mutagenesis and verified by direct Sanger sequencing. The residue numbering refers to the rSUR2A clone, which shares 97% sequence identity with the human sequence and was used to allow for comparison with previous reports of the effects of other Cantu syndrome mutations that also used rSUR2A. Cosm6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and transfected using FuGENE 6 (Roche Applied Science) with wildtype pcDNA3.1_mKir6.2 (0.6 μg; GenBankTM accession no. D50581.1) and wildtype or mutant pCMV_rSUR2A constructs (1 μg) in addition to 0.2 μg of pcDNA3.1_eGFP for visual detection of successful transfection. To model heterozygous expression of mutant subunits, cells were transfected with WT Kir6.2 along with a 1:1 ratio of WT and mutant rSUR2A (0.5:0.5 μg). Radioactive rubidium efflux experiments were performed 36 h post-transfection, while the excised patch-clamp recordings were made 48–72 h post-transfection.
86Rb+ efflux assay
Transfected Cosm6 cells were plated in 12-well plates to reach 70–80% confluence on the day of experimenting. Prior to commencing the efflux assay, the culture medium was replaced by DMEM supplemented with 1 μCi/ml 86RbCl (PerkinElmer Life Sciences) and incubated for >6 h (37 °C/5% CO2) to load the cells with the 86Rb+ isotope. After the loading incubation, cells were washed with Ringer’s solution containing (in mm) 118 NaCl, 10 HEPES, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, and 1.2 MgSO4 either alone or supplemented with 2.5 mg/ml oligomycin and 1 mm 2-deoxy-d-glucose to induce metabolic inhibition (MI) and incubated at room temperature for a further 10 min. Cells were then washed three times with Ringer’s solution (either with or without MI supplements) before the experiment was commenced. Ringer’s solution was added to each well, collected, and replaced at the defined time points (2.5, 5, 12.5, 22.5, and 37.5 min). After the experiment, cells were lysed with 2% SDS to attain the remaining intracellular 86Rb+, and sample radioactivity was determined by scintillation counting.
The cumulative 86Rb+ efflux at each time point was calculated from the total counts from each sample (including the 86Rb+ remaining post-cell lysis). Apparent KATP-independent efflux rate constants (k1) were obtained from GFP-transfected cells using Equation 1,
(1)
and KATP-dependent efflux rate constant (k2) was obtained from KATP-transfected cells using Equation 2,
(2)
where k1 was obtained from GFP-transfected cells (Equation 1). The number of active channels was assumed to be proportional to k2. In MI conditions a time-dependent divergence from a mono-exponential efflux is observed. This is attributed to inactivation of background efflux mechanisms over time; therefore, in this condition rate constants were derived from exponential functions fit to early time points only (2.5–12.5 min). Efflux-time data shown represents the mean ± S.E. from at least three independent experiments each with multiple replicates (N ≥ 3, n ≥ 4). KATP-dependent flux rate data is shown as mean ± S.D. Statistical significance was determined using Mann-Whitney U tests with a p value <0.05 deemed statistically significant.
Inside-out excised patch-clamp recordings
Pipettes were made from soda lime glass microhematocrit tubes (Kimble) and had a resistance of 1–2 megohms when filled with pipette solution. The bath and pipette solutions (KINT) contained (in mm): 140 KCl, 10 HEPES, 1 EGTA (pH 7.4 with KOH). Currents were recorded at a constant holding potential of −50 mV in the absence and presence of nucleotides as indicated. Where included, free Mg2+ concentrations were maintained at 0.5 mm by supplementation of MgCl2 as calculated using CaBuf (Katholieke Universiteit Leuven). Where stated, porcine brain PIP2 (Avanti Polar Lipids) was applied at 5 μg/ml. Rapid solution exchange was attained using a Dynaflow Resolve perfusion chip (Cellectricon). Experiments were performed at 20–22 °C. KATP channel currents in solutions of varying nucleotide concentrations were normalized to the basal current in the absence of nucleotides, and dose-response data were fit with a four-parameter Hill fit according to Equation 3, using the Data Solver Function in Microsoft Excel,
(3)
where the current in KINT = Imax = 1; Imin is the normalized minimum current observed in ATP/MgATP/glibenclamide; [X] refers to the concentration of ATP/MgATP/glibenclamide; IC50 is the concentration of half-maximal inhibition; and H denotes the Hill coefficient.
Data were tested for statistical significance using the Mann Whitney U test, and presented as mean ± S.E. in dose-response plots, and as mean ± S.D. in scatter plots showing IC50 values from individual experiments.
Author contributions
C. M., G. v. H., and C. G. N. conceptualization; C. M., A. H., M. S.-R., H. I. R., D. J., D. K. G., and G. v. H. data curation; C. M., A. H., M. S.-R., and C. G. N. formal analysis; C. M. investigation; C. M., G. v. H., and C. G. N. methodology; G. v. H. and C. G. N. funding acquisition.
Acknowledgments
We thank Risha Shah (Washington University) for help with 86Rb+ efflux experiments.
References
Shyng S.
Nichols C.G.
Octameric stoichiometry of the KATP channel complex.
Conserved functional consequences of disease-associated mutations in the slide-helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel.
Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.
This work was supported in part by National Institutes of Health Grant HL140024 from NHLBI (to C. G. N.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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