ABCMdb

ABCC8 p.Glu128Lys

Predicted by SNAP2:	A: D (66%), C: D (59%), D: D (85%), F: D (71%), G: D (85%), H: D (80%), I: D (75%), K: D (71%), L: D (75%), M: D (66%), N: D (85%), P: D (91%), Q: D (75%), R: D (80%), S: D (80%), T: D (80%), V: D (75%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Sulfonylurea receptor 1 mutations that cause oppos... J Biol Chem. 2009 Mar 20;284(12):7951-9. Epub 2009 Jan 16. Pratt EB, Yan FF, Gay JW, Stanley CA, Shyng SL
Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.
J Biol Chem. 2009 Mar 20;284(12):7951-9. Epub 2009 Jan 16., [PMID:19151370]

Abstract [show]
The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

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2 Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Login to comment
5 The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6and 12-fold lower than wild-type channels, respectively. Login to comment
42 Here, we report two mutants, R74W and E128K, which, upon rescue to the cell surface, surprisingly revealed reduced ATP sensitivity-gating defects. Login to comment
45 Interestingly, unlike previously reported ATP-insensitive mutants, which tend to have increased intrinsic Po, the R74W and E128K mutants showed reduced intrinsic Po. Login to comment
46 The finding suggests R74W and E128K diminish channel ATP sensitivity by a distinct mechanism that likely involves functional uncoupling between SUR1 and Kir6.2. Login to comment
106 RESULTS The R74W and E128K Mutations Reduce Channel Sensitivity to ATP-Previously, we reported several CHI-associated SUR1 mutations that reduce surface expression of KATP channels could be rescued to the cell surface efficiently by sulfonylureas such as glibenclamide and tolbutamide (13, 15). Login to comment
107 These mutations are all in the TMD0 of SUR1 (amino acids 1-196) and include G7R, N24K, F27S, R74W, A116P, E128K, and V187D. Login to comment
113 To our surprise, however, two of the mutants, R74W and E128K, exhibited significantly reduced ATP sensitivities (Fig. 2, A and B). Login to comment
117 The E128K mutation occurred in homozygous form in a child with diffuse hyperinsulinism, severe neonatal onset hypoglycemia, failed diazoxide therapy, and required near-total pancreatectomy for control of hypoglycemia (15). Login to comment
120 Although expression levels of R74W and E128K mutants were very low, sufficient currents in a small fraction of transfected cells (identified by co-transfected GFP) were detected. Login to comment
122 SUR1 Mutations, Hyperinsulinism, and Diabetes MARCH 20, 2009•VOLUME 284•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7953 Mechanisms of Reduced ATP Inhibition in R74W and E128K Channels-Several studies have shown that PNDM-causing SUR1 or Kir6.2 mutations can reduce channel ATP sensitivity by enhancing channel response to Mg-nucleotide stimulation (26, 27). Login to comment
123 In inside-out patches, MgADP stimulated channel activity in both R74W and E128K mutants (Fig. 3A); however, because the mutants were much less sensitive to nucleotide inhibition, it is difficult to directly compare their MgADP sensitivities to WT channels. Login to comment
124 We therefore tested the effect of R74W or E128K on channel ATP sensitivity in the background of SUR1-NBD mutations such as G1479D and G1479R in NBD2 and K719M in NBD1, which are known to abolish channel response to MgADP stimulation (28). Login to comment
125 In the E128K/G1479R double mutants, ATP sensitivity was as reduced as the E128K mutant; the R74W/G1479D double mutant also showed significantly reduced ATP sensitivity, although to a lesser degree than the R74W single mutant (Fig. 3, B and C). Login to comment
126 We also combined E128K with NBD1 mutation K719M (E128K/K719M), and the resulting channels were as insensitive to ATP as E128K (not shown). Login to comment
127 These results indicate that, even if the mutations increased Mg-nucleotide stimulation, this effect alone could not explain the reduced ATP sensitivity in R74W and E128K. Login to comment
130 To examinethispossibility,wefirstmeasuredthePo oftheR74Wand E128K channels expressed in COSm6 cells by single channel recording. Login to comment
132 Unexpectedly, however, the average Po values for R74W and E128K (0.35 Ϯ 0.08 and 0.18 Ϯ 0.06, respectively) were significantly lower than that of WT channels (0.63 Ϯ 0.06; Fig. 4B). Login to comment
133 Of note, 10 out of 11 patches of the E128K mutant had consistently lower Po with only one outlier showing a Po of 0.69 (Fig. 4B). Login to comment
146 R74W and E128K decrease channel sensitivity to ATP inhibition. Login to comment
148 Scale bars: WT: 500 pA, 5 s; R74W and E128K: 50 pA, 5 s. B, ATP dose-response relationships. Parameters describing best-fit curves to the Hill equation (Irel ϭ 1/(1 ϩ ([ATP]/ IC50)H )), including the [ATP] necessary for half-maximal inhibition (IC50) and Hill coefficient (H), are shown. Login to comment
152 Scale bars: WT: 500 pA, 10 s; R74W and E128K: 20 pA, 10 s. SUR1 Mutations, Hyperinsulinism, and Diabetes 7954 ranging from 0.01 to 0.88 (Fig. 4B). Login to comment
158 The Po values thus derived are 0.84 Ϯ 0.02 (n ϭ 10), 0.71 Ϯ 0.06 (n ϭ 9), and 0.35 Ϯ 0.05 (n ϭ 9) for WT, R74W, and E128K, respectively. Login to comment
159 These numbers, while all consid- erablyhigher,areneverthelessingeneralagreementwiththetrend observed in single channel recording experiments, with the Po of R74W and E128K significantly lower than that of WT (p Ͻ 0.05). Login to comment
161 Together, our data point to reduced intrinsic channel open probabilities for the R74W and the E128K mutant. Login to comment
164 Mutant Channel Biogenesis Defects and Correction by Sulfonylureas in Insulin-secreting Cells-The reduced ATP sensitivity of the R74W and E128K mutant channels predicts that, were the channels able to overcome their trafficking defect, they would be insensitive to metabolic stimuli and would cause beta-cell dysfunction resembling neonatal diabetes. Login to comment
166 Reduced ATP sensitivity in R74W and E128K is independent of MgADP stimulation. Login to comment
170 Scale bars: WT: 50 pA, 10 s; R74W: 50 pA, 10 s; E128K: 500 pA, 10 s. B and C, ATP sensitivity is still reduced in R74W and E128K containing SUR1-NBD mutations G1479D or G1479R. Login to comment
172 Scale bars: WT: 300 pA, 5 s; RW/GD (R74W/G1479D): 100 pA, 5 s; EK/GR (E128K/G1479R): 100 pA, 5 s. C, ATP dose-response relationships. Parameters describing best-fit curves are given as in Fig. 2B. Login to comment
175 R74W and E128K reduce channel intrinsic open probability. Login to comment
185 Western blots showed that, without glibenclamide treatment, the R74W and E128K mutant fSUR1 was seen only as a lower band corresponding to the core-glycosylated form in the ER, in contrast to WT fSUR1 seen as both a lower band and an upper band corresponding to the mature complex-glycosylated form found post medial-Golgi (35). Login to comment
188 By contrast, surface R74W- and E128K-fSUR1 was only easily detectable following overnight tolbutamide treatment. Login to comment
190 Following 24-h treatment with 5 ␮M glibenclamide, the expression level of R74W and E128K mutants increased from 9% to 90% and 9% to 80% that of WT, respectively (Fig. 5C). Login to comment
191 The trafficking and rescue characteristics of R74W and E128K were also confirmed in rat islets. Login to comment
195 Expression of R74W or E128K Mutant Alters INS-1 Cell Responses to Glucose Stimulation-Having established the sulfonylurea-dependent expression of mutant channels in INS-1 cells, we next determined if expression of the mutants at the cell surface alters membrane electrical properties. Login to comment
201 R74W and E128K have inappropriate channel openings in intact cells following high glucose stimulation. Login to comment
204 Representative current traces showing that both uninfected control and WT-infected cells had little or no channel activity; however, both R74W and E128K had robust channel openings. Login to comment
207 Scale bars: WT: 50 pA, 10 s; R74W: 100 pA, 10 s; E128K: 50 pA, 10 s. FIGURE 5. Login to comment
208 R74W and E128K surface expression was rescued by sulfonylurea treatment in insulin-secreting cells. Login to comment
212 The upper band is undetectable in untreated R74W- and E128K-infectedcells,indicatingdefectivechannelprocessingandtrafficking.Sulfonylurea treatment, however, restores upper band expression. Login to comment
214 B, surface immunostaining with FLAG-antibody of fSUR1 showed that R74W and E128K mutant channels are only detected at the cell surface following tolbutamide treatment.C,KATP surfaceexpressioninINS-1cellswasquantifiedusingchemilu- minescence assays. Login to comment
215 Under control conditions, R74W and E128K both express at 9% of WT. Login to comment
216 Sulfonylurea treatment greatly improves R74W and E128K expression to90and80%,respectively.ErrorbarsrepresentϮS.E.ofthreeexperiments.D, is- letsisolatedfromratpancreaswereculturedfor48handtheninfectedwithKATP subunit-encoding adenoviruses. Login to comment
219 In contrast, all cells infected with the R74W or E128K channel subunits and pretreated with tolbutamide had high on-cell activities (Fig. 6A). Login to comment
221 Cells that were infected with R74W or E128K but not rescued by tolbutamide were also tested; a small fraction of each mutant displayed some on-cell channel activity but less than that observed in tolbutamide-treated cells and decreased channel ATP sensitivity upon patch excision (supplemental Fig. S1), indicating some mutant channels were able to traffic to the plasma membrane without pharmacologic chaperone. Login to comment
230 In contrast, both R74W- and E128K-expressing cells receiving tolbutamide pretreatment were significantly more hyperpolarized at 12 mM glucose. Login to comment
231 The initial RMP was -33 Ϯ 3.8 mV for R74W and -54 Ϯ 4.0 mV for E128K. Login to comment
235 Taken together, these results are in line with the idea that expression of the R74W or E128K mutant channels at the INS-1 cell surface render the cell membrane potentials unable to depolarize in response to glucose stimulation. Login to comment
236 Lastly, we determined if rescue of the R74W or E128K mutant channels to the cell surface would cause defective insulin secretion in response to glucose stimulation. Login to comment
245 C, insulin secretion at basal (3 mM) and 12 mM glucose in uninfected controls and WT-, R74W-, or E128K-infected INS-1 cells. Login to comment
246 R74W- and E128K-infected cells pretreated with 300 ␮M tolbutamide for 4 h to rescue surface expression had significantly less insulin secretion relative to control or WT-infected cells. Login to comment
247 In R74W- or E128K-infected cells without tolbutamide rescue, insulin secretion was also reduced likely due to some leak expression of the mutants, although the extent of reduction was less than tolbutamide-rescued cells. Login to comment
254 These data led us to conclude that rescue of the CHI-causing R74W or E128K mutant KATP channels by sulfonylureas inverses the beta-cell dysfunction phenotype to diabetic. Login to comment
259 Mechanisms of Reduced ATP Sensitivities in the Mutant Channels-Our results indicate that the decreased ATP sensitivities of R74W and E128K are not due to enhanced MgADP stimulation, at least alone, because elimination of channel MgADP response by mutations in the nucleotide binding folds did not restore their ATP sensitivity to the level of WT channels (Fig. 3). Login to comment
260 Furthermore, we found that, rather than increasing channel intrinsic Po, the R74W and E128K mutations significantly lowered the average intrinsic Po (Fig. 4). Login to comment
261 These properties are unlike the previously reported ATP-insensitive mutants and place the R74W and E128K mutants in a distinct category in terms of the underlying mechanisms for loss of ATP sensitivity. Login to comment
264 That the E128K mutant has poor surface expression, lower Po, and reduced ATP sensitivity closer to those seen in Kir6.2⌬C channels suggests the mutation likely disrupts the functional coupling between TMD0-SUR1 and Kir6.2. Login to comment
270 In fact, the reduced ATP sensitivities observed in the R74W and E128K mutants indicate TMD0 is necessary for normal channel ATP sensitivity. Login to comment
272 If R74W and E128K cause functional uncoupling between TMD0-SUR1 and Kir6.2, one might ask if the mutations also result in reduced physical association between the two subunits. Login to comment
276 It would not be surprising if R74W and E128K do not affect the extent of co-immunoprecipitation between SUR1 and Kir6.2, because there are likely multiple chemical interactions retained (such as those mediated by Ala-116, Val-187, and Phe-132) to allow association of the two subunits. Login to comment
278 R74W and E128K, like all other neonatal diabetes-causing mutations, render KATP channels less sensitive to ATP inhibition during glucose stimulation, and yet they were identified in patients with severe hyperinsulinism, because they also prevent channels from being expressed at the cell membrane. Login to comment
281 The R74W was identified in one patient with diffuse disease who also carries an R1215Q mutation that reduces channel sensitivity to MgADP and two patients with focal disease (23-25), SUR1 Mutations, Hyperinsulinism, and Diabetes 7958 and E128K was identified as a disease-causing homozygous mutation in a patient with diffuse disease (15). Login to comment
157 The Po values thus derived are 0.84 afe; 0.02 (n afd; 10), 0.71 afe; 0.06 (n afd; 9), and 0.35 afe; 0.05 (n afd; 9) for WT, R74W, and E128K, respectively. Login to comment
160 Together, our data point to reduced intrinsic channel open probabilities for the R74W and the E128K mutant. Login to comment
163 Mutant Channel Biogenesis Defects and Correction by Sulfonylureas in Insulin-secreting Cells-The reduced ATP sensitivity of the R74W and E128K mutant channels predicts that, were the channels able to overcome their trafficking defect, they would be insensitive to metabolic stimuli and would cause beta-cell dysfunction resembling neonatal diabetes. Login to comment
165 Reduced ATP sensitivity in R74W and E128K is independent of MgADP stimulation. Login to comment
169 Scale bars: WT: 50 pA, 10 s; R74W: 50 pA, 10 s; E128K: 500 pA, 10 s. B and C, ATP sensitivity is still reduced in R74W and E128K containing SUR1-NBD mutations G1479D or G1479R. Login to comment
171 Scale bars: WT: 300 pA, 5 s; RW/GD (R74W/G1479D): 100 pA, 5 s; EK/GR (E128K/G1479R): 100 pA, 5 s. C, ATP dose-response relationships. Parameters describing best-fit curves are given as in Fig. 2B. All cells were pretreated with 300 òe;M tolbutamide to increase surface expression. Login to comment
173 R74W and E128K reduce channel intrinsic open probability. Login to comment
183 Western blots showed that, without glibenclamide treatment, the R74W and E128K mutant fSUR1 was seen only as a lower band corresponding to the core-glycosylated form in the ER, in contrast to WT fSUR1 seen as both a lower band and an upper band corresponding to the mature complex-glycosylated form found post medial-Golgi (35). Login to comment
186 By contrast, surface R74W- and E128K-fSUR1 was only easily detectable following overnight tolbutamide treatment. Login to comment
189 The trafficking and rescue characteristics of R74W and E128K were also confirmed in rat islets. Login to comment
193 Expression of R74W or E128K Mutant Alters INS-1 Cell Responses to Glucose Stimulation-Having established the sulfonylurea-dependent expression of mutant channels in INS-1 cells, we next determined if expression of the mutants at the cell surface alters membrane electrical properties. Login to comment
199 R74W and E128K have inappropriate channel openings in intact cells following high glucose stimulation. Login to comment
202 Representative current traces showing that both uninfected control and WT-infected cells had little or no channel activity; however, both R74W and E128K had robust channel openings. Login to comment
205 Scale bars: WT: 50 pA, 10 s; R74W: 100 pA, 10 s; E128K: 50 pA, 10 s. FIGURE 5. Login to comment
206 R74W and E128K surface expression was rescued by sulfonylurea treatment in insulin-secreting cells. Login to comment
210 The upper band is undetectable in untreated R74W- and E128K-infectedcells,indicatingdefectivechannelprocessingandtrafficking.Sulfonylurea treatment, however, restores upper band expression. Login to comment
213 Under control conditions, R74W and E128K both express at 9% of WT. Login to comment
217 In contrast, all cells infected with the R74W or E128K channel subunits and pretreated with tolbutamide had high on-cell activities (Fig. 6A). Login to comment
228 In contrast, both R74W- and E128K-expressing cells receiving tolbutamide pretreatment were significantly more hyperpolarized at 12 mM glucose. Login to comment
229 The initial RMP was afa;33 afe; 3.8 mV for R74W and afa;54 afe; 4.0 mV for E128K. Login to comment
233 Taken together, these results are in line with the idea that expression of the R74W or E128K mutant channels at the INS-1 cell surface render the cell membrane potentials unable to depolarize in response to glucose stimulation. Login to comment
234 Lastly, we determined if rescue of the R74W or E128K mutant channels to the cell surface would cause defective insulin secretion in response to glucose stimulation. Login to comment
244 C, insulin secretion at basal (3 mM) and 12 mM glucose in uninfected controls and WT-, R74W-, or E128K-infected INS-1 cells. Login to comment
253 These data led us to conclude that rescue of the CHI-causing R74W or E128K mutant KATP channels by sulfonylureas inverses the beta-cell dysfunction phenotype to diabetic. Login to comment
258 Mechanisms of Reduced ATP Sensitivities in the Mutant Channels-Our results indicate that the decreased ATP sensitivities of R74W and E128K are not due to enhanced MgADP stimulation, at least alone, because elimination of channel MgADP response by mutations in the nucleotide binding folds did not restore their ATP sensitivity to the level of WT channels (Fig. 3). Login to comment
263 That the E128K mutant has poor surface expression, lower Po, and reduced ATP sensitivity closer to those seen in Kir6.2èc;C channels suggests the mutation likely disrupts the functional coupling between TMD0-SUR1 and Kir6.2. Login to comment
269 In fact, the reduced ATP sensitivities observed in the R74W and E128K mutants indicate TMD0 is necessary for normal channel ATP sensitivity. Login to comment
271 If R74W and E128K cause functional uncoupling between TMD0-SUR1 and Kir6.2, one might ask if the mutations also result in reduced physical association between the two subunits. Login to comment
275 It would not be surprising if R74W and E128K do not affect the extent of co-immunoprecipitation between SUR1 and Kir6.2, because there are likely multiple chemical interactions retained (such as those mediated by Ala-116, Val-187, and Phe-132) to allow association of the two subunits. Login to comment
277 R74W and E128K, like all other neonatal diabetes-causing mutations, render KATP channels less sensitive to ATP inhibition during glucose stimulation, and yet they were identified in patients with severe hyperinsulinism, because they also prevent channels from being expressed at the cell membrane. Login to comment
280 The R74W was identified in one patient with diffuse disease who also carries an R1215Q mutation that reduces channel sensitivity to MgADP and two patients with focal disease (23-25), SUR1 Mutations, Hyperinsulinism, and Diabetes 7958 and E128K was identified as a disease-causing homozygous mutation in a patient with diffuse disease (15). Login to comment

[hide] N-terminal transmembrane domain of SUR1 controls g... J Gen Physiol. 2011 Mar;137(3):299-314. Epub 2011 Feb 14. Pratt EB, Tewson P, Bruederle CE, Skach WR, Shyng SL
N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2.
J Gen Physiol. 2011 Mar;137(3):299-314. Epub 2011 Feb 14., [PMID:21321069]

Abstract [show]
Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

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No. Sentence Comment
17 Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Login to comment
18 Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Login to comment
21 Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. Login to comment
30 Recently, we reported that two mutations (R74W and E128K) in the TMD0 domain of SUR1 identified in congenital hyperinsulinism cause loss of channel function by preventing channel trafficking to the cell surface. Login to comment
32 The R74W- and E128K-mutant channels exhibit reduced channel sensitivity to ATP inhibition. Login to comment
33 However, unlike most ATP-insensitive mutants in which an increased Po underlies the reduction in apparent ATP sensitivity by allosteric effects, the R74W and E128K mutants display decreased Po (Pratt et al., 2009). Login to comment
37 In contrast, E128K disrupts functional coupling between TMD0 and Kir6.2 by abrogating the effects of SUR1 on channel response to PIP2. Moreover, the mutation E128W leads to spontaneous current inactivation that can be prevented or reversed by PIP2. Login to comment
149 (E) Cells cotransfected with WT, R74W, R74K, or E128K fTMD0 and Kir6.2C36 were probed with -FLAG antibody 48 h after transfection to detect surface expression of mini-KATP channels. Login to comment
150 A rim of surface staining was detected in WTand E128K-transfected cells (green, inset images), but not in either R74W- or R74K-transfected cells. Login to comment
156 Also in contrast to R74X, E128K fTMD0 was detected at the cell surface of COSm6 cells when cotransfected with Kir6.2C36 (Fig. 3 E), indicating that the formation of mini-KATP channels is suitable for electrophysiological analysis. Login to comment
157 Previously, we reported that full-length KATP channels with the E128K mutation in SUR1 have decreased intrinsic Po (Po = 0.18 vs. 0.63 for WT; Pratt et al., 2009). Login to comment
158 We tested whether the same would be true for E128K mini-KATP channels, which would be a clear indication of each E128X mutant (Fig. S1), followed by a 2-h washout before recording. Login to comment
162 This suggests different roles of the two Figure 4. E128K mini-KATP channels have decreased intrinsic Po. Login to comment
163 (A) Representative traces from inside-out voltage clamp recordings made from COSm6 cells transfected with Kir6.2C36 (denoted as Kir6.2C) alone or with WT, E128K, or F132L fTMD0 to form mini-KATP channels. Login to comment
168 The average Po of E128K mini-KATP channels is significantly different from WT mini-KATP channels and Kir6.2C36 channels (*, P < 0.05; Student`s t test). Login to comment
173 As predicted, the E128K mini-channels were inhibited to a similar extent as Kir6.2C36 channels (85%), whereas the WT and F132L mini-channels were inhibited by only 52 and 15%, respectively (compare Fig. 4, B and C). Login to comment
174 This result provides further evidence that E128K directly uncouples functional interactions between TMD0 and Kir6.2, thus preventing the influence TMD0 has on the Po and ATP sensitivity of Kir6.2C36 channels. Login to comment
175 E128K attenuates the response of full-length channels to PIP2 The intrinsic Po of Kir channels is thought to be determined by channel interactions with membrane phosphoinositides, in particular, PIP2: the stronger the interactions, the higher the Po (Logothetis et al., 2007; Xie et al., 2007). Login to comment
178 In inside-out patches, the application of PIP2 to the bath solution rapidly increases WT channel that E128K disrupts functional coupling between TMD0 and Kir6.2. Login to comment
179 Single-channel recordings made from COSm6 cells cotransfected with E128K fTMD0 and Kir6.2C36 showed that the average intrinsic Po of E128K mini-KATP channels was, indeed, significantly lower than WT mini-channels (Po = 0.17 ± 0.04 for E128K vs. 0.60 ± 0.06 for WT; P < 0.05) (Fig. 4, A and B). Login to comment
180 Further, the E128K mini-channels exhibited short bursts of activity separated by relatively long closures, distinguishing them from WT mini-channels and channels composed of Kir6.2C36 alone. Login to comment
182 These results demonstrate that E128K in both full-length SUR1 and TMD0 alone reduces functional coupling to Kir6.2 with regard to intrinsic Po and single-channel kinetics. Login to comment
185 Because E128K abrogated the effect of TMD0 on the Po of Kir6.2, we predicted that mini-KATP channels with the E128K mutation will have an ATP sensitivity similar to Kir6.2C36 channels. Login to comment
186 WT, E128K, or F132L Figure 5. E128K full-length KATP channels have decreased PIP2 response. Login to comment
187 (A) Traces of inside-out voltage clamp recordings from COSm6 cells transfected with Kir6.2 and WT (top) or E128K (bottom) SUR1. Login to comment
189 Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 100 pA. Zero current is indicated by the dotted lines. Login to comment
191 Error bars represent SEM; n = 8 (WT) and 12 (E128K) patches (*, P < 0.05; Student`s t test). Login to comment
192 current increase upon PIP2 exposure in E128K is striking when one considers that the starting Po of E128K is so much lower than WT, such that WT Po has the potential to increase from 0.6 to 1.0, whereas E128K Po has the potential to increase from 0.2 to 1.0. Login to comment
193 To further illustrate the differential response in WT and E128K channels with respect to PIP2-induced current amplitude increase, we excised membrane patches into Kint plus 1 mM Mg2+ to induce rundown, such that WT channel Po before PIP2 exposure was substantially reduced (Fig. 6). Login to comment
195 In contrast, successive exposures of E128K channels to PIP2 after rundown also led to recovery of channel activity, but the current amplitude never reached that seen before rundown, such that the fold change in current amplitude relative to the initial current after successive 30-s PIP2 exposures was only 0.5 for E128K (0.4 ± 0.1, 0.5 ± 0.1, and 0.5 ± 0.1 after one, two, and three PIP2 exposures, respectively; Fig. 6 B). Login to comment
196 Interestingly, we noted that the E128K channels were activity to maximal Po and decreases channel sensitivity to ATP inhibition. Login to comment
198 We hypothesize that SUR1 modulates the Po of Kir6.2 via TMD0 by enhancing channel response to PIP2. Moreover, E128 is essential for this modulation, as shown by the diminished effect E128K TMD0 had on the Po of Kir6.2 (Fig. 4). Login to comment
199 Accordingly, we predicted that the E128K channel will have reduced sensitivity to PIP2. Login to comment
200 Indeed, we found that KATP channels harboring the E128K mutation were much less responsive to PIP2, both in terms of increase in current amplitude (i.e., Po) and decrease in ATP inhibition compared with WT. Login to comment
201 The effect of PIP2 on the current amplitude of WT and E128K channels was first examined using our standard recording protocol in which membrane patches were excised into Kint bath solution containing 1 mM EDTA, which prevents channel rundown (Lin et al., 2003). Login to comment
202 After 30 s of exposure to 5 µM PIP2, WT current increased 1.53 ± 0.12-fold relative to current before PIP2 exposure, whereas E128K channel current increased only 1.07 ± 0.10-fold (Fig. 5, A and B). Login to comment
204 The lack of Figure 6. Rundown induced by 1 mM Mg2+ enhances the difference between WT and E128K response to PIP2 stimulation. Login to comment
205 (A) Representative inside-out patch voltage clamp recordings from COSm6 cells transfected with WT (top) or E128K (bottom) KATP channels. Login to comment
206 Patches were pulled into Kint plus 1 mM MgCl2 for 1 min to induce rundown to accentuate the differential response between WT and E128K channels; EDTA was not included in any of the solutions. Login to comment
208 Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 100 pA. Zero current is indicated by the dotted lines. Login to comment
212 Error bars represent SEM. n = 5-8 for each data point (*, P < 0.05; Student`s t test comparing WT and E128K for each point analyzed). Login to comment
220 This effect was also abrogated in the E128K mutant. Login to comment
221 Patches containing WT or E128K channels were excised into Kint/EDTA solution and then exposed to differing concentrations of ATP (Fig. 7 A). Login to comment
223 However, in E128K channels, exposure to PIP2 had little effect on ATP inhibition (Fig. 7, A and C). Login to comment
224 Collectively, the reduced response to PIP2 in the E128K channels resembles that seen in Kir6.2C36 channels, providing evidence that E128K disrupts the ability of SUR1 to enhance channel response to PIP2. Login to comment
226 As mentioned above, the mutation of E128 to a trypto- Figure 7. Decrease of ATP sensitivity in response to PIP2 is abrogated by the E128K mutation. Login to comment
227 (A) Representative inside-out patch voltage clamp recordings from COSm6 cells transfected with WT (top) or E128K (bottom) KATP channels. Login to comment
228 Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 500 pA. Zero current is indicated by the dotted lines. Login to comment
229 (B and C) Current inhibition by ATP was assessed using several ATP concentrations (0.01, 0.1, and 1 mM) before PIP2 exposure (filled black symbols) and after three 30-s exposures to 5 µM ATP (open gray symbols) for WT (B) and E128K (C) channels. Login to comment
238 Mutations in TMD0 such as R74W and E128K offer potential for probing the molecular basis of TMD0-Kir6.2 signaling, as they cause dramatic impediments to channel trafficking and gating. Login to comment
239 Here, we studied full-length and mini-KATP channels to learn more about the functional roles of R74 and E128. We show that R74W and E128K disrupt channel function by different mechanisms. Login to comment
269 (A) Cartoon illustrating proposed physical relationships between SUR1, Kir6.2, and PIP2 in the WT, E128K mutant, and the Kir6.2C36 channels. Login to comment
275 Mini-KATP channels with the E128K mutation also follow this relationship; i.e., they show reduced Po and increased ATP inhibition relative to WT mini-channels (similar to that seen for Kir6.2C36). Login to comment
276 Although the inverse relationship between Po and ATP sensitivity might predict that in full-length channels the E128K mutant should show increased ATP sensitivity relative to WT, this is not the case. Login to comment
277 Rather, E128K full-length channels actually have reduced ATP inhibition. Login to comment
278 Thus, by abolishing the ability of TMD0 to enhance Kir6.2 response to PIP2, the E128K mutation abrogates the ability of structures downstream of TMD0 to confer increased ATP sensitivity to Kir6.2. Login to comment
279 Future identification of SUR1 residues responsible for increasing ATP sensitivity of the channel will be critical for understanding precisely how E128K uncouples SUR1 from Kir6.2 with respect to regulation by both PIP2 and ATP. Login to comment
290 Moreover, mutations at residue 128 have less effect on TMD0 protein levels, and E128K TMD0 can form mini-channels with Kir6.2C36 that traffic to the cell surface (Fig. 3). Login to comment
293 However, the E128K mutation in both full-length and mini-KATP channels shifts the Po and ATP sensitivity toward those seen in Kir6.2C36 channels, indicating that E128 contributes to functional coupling with Kir6.2. Login to comment
294 Strikingly, E128K renders full-length channels much less sensitive to PIP2 stimulation, resembling that seen in Kir6.2C36 channels. Login to comment
306 The detrimental effect of E128K on channel interaction with PIP2 therefore offers a clear explanation for how the mutation reduces Po in full-length and mini-KATP channels. Login to comment
307 However, the explanation for the effects of E128K on ATP sensitivity in full-length and mini-KATP channels is more complex. Login to comment

[hide] Impact of disease-causing SUR1 mutations on the KA... J Biol Chem. 2010 Jan 29;285(5):3084-91. Epub 2009 Nov 20. Hosy E, Dupuis JP, Vivaudou M
Impact of disease-causing SUR1 mutations on the KATP channel subunit interface probed with a rhodamine protection assay.
J Biol Chem. 2010 Jan 29;285(5):3084-91. Epub 2009 Nov 20., [PMID:19933268]

Abstract [show]
The function of the ATP-sensitive potassium (K(ATP)) channel relies on the proper coupling between its two subunits: the pore-forming Kir6.2 and the regulator SUR. The conformation of the interface between these two subunits can be monitored using a rhodamine 123 (Rho) protection assay because Rho blocks Kir6.2 with an efficiency that depends on the relative position of transmembrane domain (TMD) 0 of the associated SUR (Hosy, E., Derand, R., Revilloud, J., and Vivaudou, M. (2007) J. Physiol. 582, 27-39). Here we find that the natural and synthetic K(ATP) channel activators MgADP, zinc, and SR47063 induced a Rho-insensitive conformation. The activating mutation F132L in SUR1, which causes neonatal diabetes, also rendered the channel resistant to Rho block, suggesting that it stabilized an activated conformation by uncoupling TMD0 from the rest of SUR1. At a nearby residue, the SUR1 mutation E128K impairs trafficking, thereby reducing surface expression and causing hyperinsulinism. To augment channel density at the plasma membrane to investigate the effect of mutating this residue on channel function, we introduced the milder mutation E126A at the matching residue of SUR2A. Mutation E126A imposed a hypersensitive Rho phenotype indicative of a functional uncoupling between TMD0 and Kir6.2. These results suggest that the TMD0-Kir6.2 interface is mobile and that the gating modes of Kir6.2 correlate with distinct positions of TMD0. They further demonstrate that the second intracellular loop of SUR, which contains the two residues studied here, is a key structural element of the TMD0-Kir6.2 interface.

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5 At a nearby residue, the SUR1 mutation E128K impairs trafficking, thereby reducing surface expression and causing hyperinsulinism. Login to comment
124 Another residue of this short loop, Glu128 , also causes disease when mutated; mutation E128K is responsible for hyperinsulinism because it reduces KATP channel activity by interfering with proper trafficking of channels to the plasma membrane (16). Login to comment
125 Because of the opposite pathological consequences of F132L and E128K mutations despite their proximity, it was interesting to also subject E128K to the Rho protection assay. Login to comment
183 Structural Perturbations Induced by Disease-causing Mutations in the Second Intracellular Loop-Mutations F132L and E128K in short intracellular loop 2 located in TMD0 of SUR1 have opposite consequences. Login to comment
184 The neonatal diabetes F132L mutation reduces ATP sensitivity by augmenting open probability (23). Login to comment
186 The hyperinsulinism E128K mutation disrupts channel trafficking and reduces channel function through poor surface expression (16). Login to comment
187 After rescue to the cell surface by a chemical chaperone, the E128K mutant channels are hyperactive with a lower than normal ATP sensitivity like the F132L mutants, although they exhibit an abnormally low Po (25). Login to comment
188 The F132L and E128K mutations cause therefore an abnormally high activity through different mechanisms. Login to comment
189 Pratt et al. (25) speculated that E128K caused functional uncoupling between SUR1 and Kir6.2, thus removing the hypersensitization to ATP brought about by SUR1 (18). Login to comment
190 Based on co-immunoprecipitation of TMD0 alone and Kir6.2 showing that F132L impaired the association of TMD0 and Kir6.2, Proks et al. (23) assumed that TMD0 and SUR behaved identically and concluded that this mutation disconnects SUR from Kir6.2. Login to comment
216 Rho Protection Assay Unveils the Mechanism of Disease-causing Mutations-Even though the F132L and E128K mutations in SUR1 both tend to reduce channel sensitivity to ATP, their mechanisms of action obviously differ because F132L increases Po, and E128K decreases Po. Login to comment
217 Nonetheless, after detailed investigation, Proks et al. (23) working on F132L and Pratt et al. (25) working on E128K reached identical conclusions: the mutation disrupts the coupling/interaction between TMD0 and Kir6.2. Login to comment
218 The additional insight obtained by the Rho protection assay appears to resolve this discrepancy as it strengthens the proposed mechanism for E128K while questioning that for F132L. Login to comment
126 Another residue of this short loop, Glu128 , also causes disease when mutated; mutation E128K is responsible for hyperinsulinism because it reduces KATP channel activity by interfering with proper trafficking of channels to the plasma membrane (16). Login to comment
127 Because of the opposite pathological consequences of F132L and E128K mutations despite their proximity, it was interesting to also subject E128K to the Rho protection assay. Login to comment
185 Structural Perturbations Induced by Disease-causing Mutations in the Second Intracellular Loop-Mutations F132L and E128K in short intracellular loop 2 located in TMD0 of SUR1 have opposite consequences. Login to comment
191 Pratt et al. (25) speculated that E128K caused functional uncoupling between SUR1 and Kir6.2, thus removing the hypersensitization to ATP brought about by SUR1 (18). Login to comment
219 Nonetheless, after detailed investigation, Proks et al. (23) working on F132L and Pratt et al. (25) working on E128K reached identical conclusions: the mutation disrupts the coupling/interaction between TMD0 and Kir6.2. Login to comment
220 The additional insight obtained by the Rho protection assay appears to resolve this discrepancy as it strengthens the proposed mechanism for E128K while questioning that for F132L. Login to comment

[hide] Congenital hyperinsulinism associated ABCC8 mutati... Diabetes. 2007 Sep;56(9):2339-48. Epub 2007 Jun 15. Yan FF, Lin YW, MacMullen C, Ganguly A, Stanley CA, Shyng SL
Congenital hyperinsulinism associated ABCC8 mutations that cause defective trafficking of ATP-sensitive K+ channels: identification and rescue.
Diabetes. 2007 Sep;56(9):2339-48. Epub 2007 Jun 15., [PMID:17575084]

Abstract [show]
Congenital hyperinsulinism (CHI) is a disease characterized by persistent insulin secretion despite severe hypoglycemia. Mutations in the pancreatic ATP-sensitive K(+) (K(ATP)) channel proteins sulfonylurea receptor 1 (SUR1) and Kir6.2, encoded by ABCC8 and KCNJ11, respectively, is the most common cause of the disease. Many mutations in SUR1 render the channel unable to traffic to the cell surface, thereby reducing channel function. Previous studies have shown that for some SUR1 trafficking mutants, the defects could be corrected by treating cells with sulfonylureas or diazoxide. The purpose of this study is to identify additional mutations that cause channel biogenesis/trafficking defects and those that are amenable to rescue by pharmacological chaperones. Fifteen previously uncharacterized CHI-associated missense SUR1 mutations were examined for their biogenesis/trafficking defects and responses to pharmacological chaperones, using a combination of immunological and functional assays. Twelve of the 15 mutations analyzed cause reduction in cell surface expression of K(ATP) channels by >50%. Sulfonylureas rescued a subset of the trafficking mutants. By contrast, diazoxide failed to rescue any of the mutants. Strikingly, the mutations rescued by sulfonylureas are all located in the first transmembrane domain of SUR1, designated as TMD0. All TMD0 mutants rescued to the cell surface by the sulfonylurea tolbutamide could be subsequently activated by metabolic inhibition on tolbutamide removal. Our study identifies a group of CHI-causing SUR1 mutations for which the resulting K(ATP) channel trafficking and expression defects may be corrected pharmacologically to restore channel function.

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47 TABLE 1 Genetic and clinical information on patients carrying the CHI mutations Mutation Disease Haplotype Diazoxide response References G7R Focal G7R No 44 N24K Diffuse N24K/R1215W No Not reported F27S Focal F27S No 39 R74W Focal R74W/R1215Q No 39,45,46 E128K Diffuse E128K No Not reported R495Q Diffuse R495Q/R1215Q No 39 E501K Focal E501K No 39 L503P Focal L503P No 44 F686S Focal F686S No 39 G716V* Diffuse G716V/G716V No 47,48 K1337N Not done g3992-9a/K1337N Yes 39 L1350Q Focal L1350Q No 44 S1387F Diffuse S1387F/NA No 9,24 L1390P NA L1390P/NA No Not reported D1472H Diffuse ⌬F1388/D1472H No 39 *Patient was from consanguineous mating and therefore was homozygous for the G716V mutation (48). Login to comment
94 The first group, including G7R, N24K, F27S, R74W, and E128K, is located in the first transmembrane domain TMD0; the second group, including R495Q, E501K, L503P, F686S, and G716V, is located in the second transmembrane domain TMD1 extending through the first nucleotide binding domain; the third group, including K1337N, L1350Q, S1387F, L1390P, and D1472H, is clustered in the second nucleotide binding domain and the COOH terminus of the protein. Login to comment
102 The mutations that have not been previously reported in the literature include N24K, E128K, and L1390P. Login to comment
118 Results from this assay showed that F27S, R74W, E128K, R495Q, E501K, L503P, F686S, G716V, L1350Q, and D1472H mutant channels had greatly reduced surface expression (Ͻ20% of wild-type level)-whereas G7R and N24K mutant channels displayed modestly decreased surface expression level (Ͼ30% but Ͻ50% of wild-type level) and K1337N, S1378F, and L1390P exhibited normal or mildly reduced expression (Ͼ60% of wild-type level; Fig. 3A). Login to comment
130 In Western blots, several mutants, including G7R, N24K, F27S, R74W, and E128K, all located in TMD0, exhibited increased complex-glycosylated SUR1 in cells coexpressing Kir6.2 on overnight treatment with 1 ␮mol/l glibenclamide (Fig. 5A). Login to comment
48 TABLE 1 Genetic and clinical information on patients carrying the CHI mutations Mutation Disease Haplotype Diazoxide response References G7R Focal G7R No 44 N24K Diffuse N24K/R1215W No Not reported F27S Focal F27S No 39 R74W Focal R74W/R1215Q No 39,45,46 E128K Diffuse E128K No Not reported R495Q Diffuse R495Q/R1215Q No 39 E501K Focal E501K No 39 L503P Focal L503P No 44 F686S Focal F686S No 39 G716V* Diffuse G716V/G716V No 47,48 K1337N Not done g3992-9a/K1337N Yes 39 L1350Q Focal L1350Q No 44 S1387F Diffuse S1387F/NA No 9,24 L1390P NA L1390P/NA No Not reported D1472H Diffuse èc;F1388/D1472H No 39 *Patient was from consanguineous mating and therefore was homozygous for the G716V mutation (48). Login to comment
95 The first group, including G7R, N24K, F27S, R74W, and E128K, is located in the first transmembrane domain TMD0; the second group, including R495Q, E501K, L503P, F686S, and G716V, is located in the second transmembrane domain TMD1 extending through the first nucleotide binding domain; the third group, including K1337N, L1350Q, S1387F, L1390P, and D1472H, is clustered in the second nucleotide binding domain and the COOH terminus of the protein. Login to comment
103 The mutations that have not been previously reported in the literature include N24K, E128K, and L1390P. Login to comment
119 Results from this assay showed that F27S, R74W, E128K, R495Q, E501K, L503P, F686S, G716V, L1350Q, and D1472H mutant channels had greatly reduced surface expression (b0d;20% of wild-type level)-whereas G7R and N24K mutant channels displayed modestly decreased surface expression level (b0e;30% but b0d;50% of wild-type level) and K1337N, S1378F, and L1390P exhibited normal or mildly reduced expression (b0e;60% of wild-type level; Fig. 3A). Login to comment

[hide] Molecular and clinical analysis of Japanese patien... J Clin Endocrinol Metab. 2011 Jan;96(1):E141-5. Epub 2010 Oct 13. Yorifuji T, Kawakita R, Nagai S, Sugimine A, Doi H, Nomura A, Masue M, Nishibori H, Yoshizawa A, Okamoto S, Doi R, Uemoto S, Nagasaka H
Molecular and clinical analysis of Japanese patients with persistent congenital hyperinsulinism: predominance of paternally inherited monoallelic mutations in the KATP channel genes.
J Clin Endocrinol Metab. 2011 Jan;96(1):E141-5. Epub 2010 Oct 13., [PMID:20943781]

Abstract [show]
BACKGROUND: Preoperative identification of the focal form of congenital hyperinsulinism is important for avoiding unnecessary subtotal pancreatectomy. However, neither the incidence nor the histological spectrum of the disease is known for Japanese patients. AIMS: The aim of the study was to elucidate the molecular and histological spectrum of congenital hyperinsulinism in Japan. SUBJECTS: Thirty-six Japanese infants with persistent congenital hyperinsulinism were included in the study. METHODS: All exons of the ATP-sensitive potassium channel (K(ATP) channel) genes (KCNJ11 and ABCC8), the GCK gene, and exons 6 and 7 and 10-12 of the GLUD1 gene were amplified from genomic DNA and directly sequenced. In patients with K(ATP) channel mutations, the parental origin of each mutation was determined, and the results were compared with the histological findings of surgically treated patients. In one of the patients with scattered lesions, islets were sampled by laser capture microdissection for mutational analysis. RESULTS: Mutations were identified in 24 patients (66.7%): five in GLUD1 and 19 in the K(ATP) channel genes. Sixteen had a paternally derived, monoallelic K(ATP) channel mutation predictive of the focal form. In 10 patients who underwent pancreatectomy, the molecular diagnosis correctly predicted the histology, more accurately than [18F]-3,4-dihydroxyphenylalanine positron emission tomography scans. Three patients showed focal lesions that occupied larger areas of the pancreas. Preferential loss of the maternal allele was observed in these islets. CONCLUSION: The majority of the Japanese patients with K(ATP) channel hyperinsulinism (84.2%) demonstrated paternally inherited monoallelic mutations that accurately predicted the presence of the focal form.

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76 Parental origin 1 F 9 months 38 ͓2.1͔ 4.8 ͓33͔ 83 ͓49͔ GLUD1 c.661CϾT p.R221C yes ND F, D 2 M 7 months 30 ͓1.7͔ 3 ͓21͔ 132 ͓77͔ GLUD1 c.797AϾG p.Y266C yes ND F, D 3 F 3 months 29 ͓1.6͔ 4 ͓28͔ 246 ͓144͔ GLUD1 c.1336GϾA p.G446S Yes ND F, D 4 M 10 months Ͻ45 ͓2.5͔ 7.7 ͓53͔ 154 ͓90͔ GLUD1 c.1229AϾG p.N410S No ND F, D 5 M 0 d 10 ͓0.6͔ 10 ͓69͔ 250 ͓147͔ GLUD1 c.1229AϾC p.N410T Yes ND F, D 6a F 2 d 31 ͓1.7͔ 30.2 ͓210͔ 78 ͓46͔ ABCC8 c.382GϾA c.3748CϾT p.E128K p.R1250X Yes, Yes Biparental 7 M 2 d 5 ͓0.3͔ 7.5 ͓52͔ 131 ͓77͔ ABCC8 c.2506CϾT c.4575_4587del13 p.R836X p.M1524Mfs1539X Yes, No Biparental F, O 8 M 0 d Ͻ45 ͓2.5͔ 11 ͓76͔ 58 ͓34͔ ABCC8 c.4516GϾA p.E1506K Yes Mat F, D 9a F 1 month Ͻ20 ͓1.1͔ 42.4 ͓294͔ NA ABCC8 c.2506CϾT p.R836X Yes Pat 10a M 2 d 10 ͓0.56͔ 23.5 ͓163͔ NA ABCC8 c.4412-13GϾA - Yes Pat 11a F 0 d 33 ͓1.8͔ 46.6 ͓324͔ 79 ͓46͔ ABCC8 c.3745GϾT p.V1249F No Pat 12a F 3 months 20 ͓1.1͔ 5.16 ͓36͔ 78 ͓46͔ ABCC8 c.2992CϾT p.R998X Yes Pat 13a F 0 d 23 ͓1.3͔ 101 ͓701͔ 45 ͓24͔ ABCC8 c.4608 ϩ 1GϾA - No Pat 14a M 0 d 22 ͓1.2͔ 22.7 ͓158͔ 75 ͓44͔ ABCC8 c.2992CϾT p.R998X Yes Pat 15a M 5 months 33 ͓1.8͔ 5.42 ͓38͔ NA ABCC8 c.2992CϾT p.R998X Yes Pat 16a M 0 d 28 ͓1.6͔ 38.7 ͓269͔ 66 ͓39͔ ABCC8 c.331GϾA p.G111R Yes Pat 17 F 2 months 15 ͓0.8͔ 9.9 ͓69͔ 90 ͓53͔ ABCC8 c.61_62insG p.V21Gfs88X No Pat F, O 18 M 0 d 19.6 ͓1.1͔ 44 ͓306͔ 79 ͓46͔ ABCC8 c.2506CϾT p.R836X Yes Pat F, O 19 F 7 months 35 ͓1.9͔ 11.2 ͓78͔ 97 ͓57͔ ABCC8 c.2506CϾT p.R836X Yes Pat F, O 20 M 4 months Ͻ45 ͓2.5͔ 7.5 ͓52͔ 84 ͓49͔ ABCC8 c.3928_3929insG p.A1310Gfs1405X No Pat F, O 21 M 2 d 38 ͓2.1͔ 3.4 ͓24͔ 91 ͓53͔ ABCC8 c.4186GϾT p.D1396Y No Pat F 22 F 0 d 9 ͓0.5͔ 22 ͓153͔ NA ABCC8 c.2506CϾT p.R836X Yes Pat F, O 23 M 2 d 0 ͓0͔ 17.3 ͓120͔ 317 ͓186͔ ABCC8 c.4412-13GϾA - Yes Pat F, D 24a M 0 d 33 ͓1.8͔ 21.9 ͓152͔ 75 ͓44͔ KCNJ11 c.637GϾA p.A213T No Pat The clinical data are those at the initial presentation. Login to comment

[hide] Carbamazepine as a novel small molecule corrector ... J Biol Chem. 2013 Jul 19;288(29):20942-54. doi: 10.1074/jbc.M113.470948. Epub 2013 Jun 6. Chen PC, Olson EM, Zhou Q, Kryukova Y, Sampson HM, Thomas DY, Shyng SL
Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.
J Biol Chem. 2013 Jul 19;288(29):20942-54. doi: 10.1074/jbc.M113.470948. Epub 2013 Jun 6., [PMID:23744072]

Abstract [show]
ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to beta-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human beta-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

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125 At 10 òe;M, the F27S and E128K mutations exhibited the greatest improvement to nearly the level seen with 5 òe;M glibenclamide; R74W, A116P, and V187D showed moderate responses; whereas G7R and N24K, which have less severe processing defects (31), had weak responses (Fig. 1C). Login to comment
127 At 10 òe;M, the F27S and E128K mutations exhibited the greatest improvement to nearly the level seen with 5 òe;M glibenclamide; R74W, A116P, and V187D showed moderate responses; whereas G7R and N24K, which have less severe processing defects (31), had weak responses (Fig. 1C). Login to comment

[hide] ATP activates ATP-sensitive potassium channels com... Channels (Austin). 2011 Jul-Aug;5(4):314-9. doi: 10.4161/chan.5.4.16510. Epub 2011 Jul 1. Pratt EB, Shyng SL
ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity.
Channels (Austin). 2011 Jul-Aug;5(4):314-9. doi: 10.4161/chan.5.4.16510. Epub 2011 Jul 1., [PMID:21654216]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels are inhibited by ATP and activated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Both channel subunits Kir6.2 and sulfonylurea receptor 1 (SUR1) contribute to gating: while Kir6.2 interacts with ATP and PIP(2), SUR1 enhances sensitivity to both ligands. Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP(2) sensitivities resembling channels formed by Kir6.2 alone. We show here that when E128K SUR1 was co-expressed with Kir6.2 mutants known to disrupt PIP(2) gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. To explain this paradoxical gating behavior, we propose a model in which the open state of doubly mutant channels is highly unstable; ATP binding induces a conformational change in ATP-unbound closed channels that is conducive to brief opening when ATP unbinds, giving rise to the appearance of ATP-induced stimulation.

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4 Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP2 sensitivities resembling - channels formed by Kir6.2 alone. Login to comment
5 We show here that when E128K SUR1 was coexpressed with Kir6.2 mutants known to disrupt PIP2 gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. Login to comment
17 For two of these residues, R50 and R201, there is evidence that they are involved directly in ATP binding.16 Another mutant, R54E, had WT-like ATP-sensitivity as a single mutant and when it was co-expressed with E128K was still inhibited by 1 mM ATP although the currents were very small (smaller than E128K alone even with 300 bc;M tolbutamide pre-treatment) making it difficult to properly assess ATP sensitivity. Login to comment
18 The R54 residue has previously been implicated in PIP2 affinity or efficacy as mutation of this residue led to decreased channel open probability that could be rectified by exogenous PIP2 .17,18 Two mutants, R177E and R206D, had no detectable currents when co-expressed with either WT SUR1 or E128K SUR1 despite pre-treatment with 300 bc;M tolbutamide. Login to comment
19 Strikingly, three Kir6.2 mutants, R176E, R192E and R206E, when coexpressed with E128K SUR1 (denoted as R176E//E128K, R192E//E128K and R206E//E128K hereinafter) displayed increased activity with the application of ATP (Fig. 2A and B). Login to comment
20 The locations of the three Kir6.2 mutations that lead to ATP-activation when combined with E128K SUR1 are shown in a Kir6.2 homology model (Fig. 2C). Login to comment
21 All three residues have previously been implicated in PIP2 efficacy.7,8,19-21 Two of them, R176 and R206, lie near the plasma membrane on the cytoplasmic C-terminus of Kir6.2 and may be involved in interactions with the negatively charged phosphate groups in PIP2 .22 The R176E//E128K and R206E//E128K "ATP-activation" mutations showed no or small currents (usually distinguishable single channel openings with conductance consistent with KATP channels) in nucleotide-free Kint/EDTA solution. Login to comment
24 N*Po values in control and ATP solutions for R176E// E128K were 0.004 &#b1; 0.002 and 0.029 &#b1; 0.009, respectively, and 0 and 0.306 &#b1; 0.132 for R206E//E128K, respectively (Fig. 2B). Login to comment
25 Exposure of R206E//E128K to 5 bc;M PIP2 also caused a small increase in chosen based upon their placement on the tetrameric Kir6.2 structural model for likelihood to physically interact with E128, which is predicted to lie close to the plasma membrane. Login to comment
29 Next, COSm6 cells were co-transfected with mutant Kir6.2 and either WT or E128K SUR1 plasmids. Login to comment
31 The prediction is that a residue involved in direct electrostatic interaction with E128 of SUR1 would cause significant reduction in ATP-sensitivity when mutated to a negatively charged amino acid and co-expressed with WT SUR1, but would have WT-like ATP sensitivity when co-expressed with E128K SUR1 as the ionic interaction would be re-established. Login to comment
32 E128K mutation prevents efficient surface expression of the channel; we have previously shown that this defect is partially corrected by treating cells with a KATP channel antagonist, tolbutamide, which acts as a chemical chaperone.15 Cells expressing the E128K mutation were therefore pre-treated with 300 bc;M tolbutamide overnight to facilitate surface expression. Login to comment
36 Three of the mutations including K47E, R50E and R201E did indeed decrease ATP sensitivity when expressed with WT SUR1; but when co-expressed with E128K, even greater insensitivity to ATP inhibition was observed, indicating the Kir6.2 and SUR1 mutations possibly cause ATP- insensitivity through different, additive channels. Login to comment
38 Recently, we showed that a residue in the TMD0, E128, plays a critical role in the engagement between SUR1 and Kir6.2 by stabilizing Kir6.2 interactions with PIP2 .14 Mutation of this residue to an oppositely charged amino acid, E128K, disrupts functional coupling between the two subunits leading to decreased Po and also decreased PIP2 and ATP sensitivity in full-length channels. Login to comment
39 In mini-KATP channels, E128K also diminishes the Po and bursting behavior conferred by TMD0 and gives rise to an ATP-sensitivity approaching that of Kir6.2ƊC channels. Login to comment
45 Our simple hypothesis is that E128 interacts electrostatically with a positively charged Kir6.2 residue, which when mutated to a negatively-charged amino acid should suppress the defect brought about by the E128K mutation. Login to comment
46 Although we did not identify a residue that behaved as our hypothesis predicted in this study, we quite unexpectedly identified several mutations within Kir6.2 which, when coexpressed with E128K, were more active in the presence of ATP than in its absence. Login to comment
49 In the presence of Mg2+ , ATP stimulates channel activity by interacting with the nucleotide binding domains of SUR1.24 To exclude the possibility that the low level of stimulation by ATP is due to incomplete chelation of Mg2+ by EDTA, we further examined the effect of ATP on channels formed by one of the Kir6.2 mutants, R176E, and a SUR1 harboring both the subunit to stabilize - channel - activity.23 The R192E//E128K pair usually had current on isolation of the inside-out membrane patch but rapidly inactivated in nucleotide-free Kint/EDTA and only became active again in high concentrations of ATP (1 mM). Login to comment
51 R192E//E128K records were not channel opening (two patches were tested) but the extent of stimulation was far less than that seen with exposure to 5 mM ATP (Fig. 2A, bottom). Login to comment
57 Error bars represent SEM, except R50E//E128K 1 mM ATP which is the difference between the - individual values from the two patches tested; the number of patches tested (n) for each condition is given below the x-axis. Login to comment
58 WT: wild-type; EK: E128K. Login to comment
63 In mutant channels formed by E128K SUR1 and doubly mutant Kir6.2 containing R50E and R176E or R50E and R206E,nostimulatoryorinhibitoryeffects on the low spontaneous channel activity were observed with exposure to 1 mM ATP (data not shown). Login to comment
64 The results suggest that the stimulatory effect of ATP seen in the Kir6.2 R176E or R192E or R206E// E128K SUR1 mutant channels involves the ATP binding site that normally leads to inhibition of channel activity. Login to comment
65 E128K mutation and another mutation located in the second NBF, G1479R, that abolishes MgATP stimulation.25 The Kir6.2 R176E//SUR1 E128K-G1479R triple mutant channel was still more active in the presence of 1 mM ATP than in Kint/EDTA (data not shown), indicating that the ATP-activation effect observed is not due to MgATP stimulation via SUR1. Login to comment
68 ATP-activation observed with co-expression of E128K and Kir6.2 mutations that diminish PIP2 response. Login to comment
72 (B) Average currents in the absence and presence of ATP were quantified as N*Po values for R176E//E128K (0 vs. 1 mM ATP) and R206E//E128K (0 vs. 5 mM ATP); no activity was seen during control conditions (i.e., no ATP) in any R206E//E128K patch. Login to comment
85 That the E128K mutation in SUR1 causes the channel to become less sensitive to ATP inhibition-as a result of functional uncoupling between SUR1 and Kir6.2-helps unmask the temporary opening of the channel during the brief transitions between ATP-bound and ATP-unbound states. Login to comment
86 The ATP-induced conformational change referred to here is analogous to the previously reported effect of ATP on several mutations that cause spontaneous channel inactivation, including E128W in SUR1 and R192E, R301E and R314E in Kir6.2.14,23 In the inactivation mutants, exposure to high concentrations of ATP followed by subsequent washout to remove the inhibitory effect of ATP recovers channels from inactivation and allows channels to open briefly before they inactivate again (this inactivation phenomenon can be seen in the R192E// E128K mutant following removal of ATP, Fig. 2A, middle). Login to comment
89 In conclusion, the abnormal ATP- stimulation gating behavior we observed in the combined SUR1 E128K and Kir6.2 mutants that have severely impaired ability to interact with PIP2 has provided a glimpse of a gating step associated with ATP binding. Login to comment
91 Proposed mechanism by which ATP stimulates the activity of channels formed by E128K-SUR1 and R176E-, R192E- or R206E-Kir6.2. Login to comment
94 (B) In E128K-SUR1 + WT Kir6.2 channels, functional coupling between SUR1 and Kir6.2 is disrupted causing unstable Kir6.2-PIP2 interactions thereby reduced open probability. Login to comment
96 (C) In channels formed by E128K-SUR1 and Kir6.2 PIP2 mutants, most channels are unable to interact with PIP2 resulting in no channel activity in Kint/EDTA. Login to comment

[hide] Engineered interaction between SUR1 and Kir6.2 tha... J Gen Physiol. 2012 Aug;140(2):175-87. doi: 10.1085/jgp.201210803. Epub 2012 Jul 16. Pratt EB, Zhou Q, Gay JW, Shyng SL
Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels.
J Gen Physiol. 2012 Aug;140(2):175-87. doi: 10.1085/jgp.201210803. Epub 2012 Jul 16., [PMID:22802363]

Abstract [show]
The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity approximately 100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.

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45 When recording from cells expressing E128K mutant channels, the cells were pretreated with 300 &#b5;M tolbutamide overnight to augment expression, followed by a 2-h washout period. Login to comment
198 However, when coexpressed with E128K-SUR1, Q52R-Kir6.2 failed to confer increased channel interaction with PIP2. Login to comment
199 In fact, Q52R-Kir6.2//E128K-SUR1 channels were even more sensitive to neomycin than WT channels, consistent with reduced Po expected from the E128K-SUR1 mutation. Login to comment
200 As expected, addition of the E128K mutation in SUR1 also diminished the ability of Q52E-Kir6.2//E203K-SUR1 channels to respond to PIP2 stimulation (Fig. 7 B; also see Fig. 3 [A and B] for comparison). Login to comment
201 Thus, the E128K mutation in SUR1 exerts a dominant effect over the Q52 mutations with regard to channel Po and PIP2 response. Login to comment
202 Next, we measured ATP sensitivity of channels formed by coexpression of E128K-SUR1 and Q52R-Kir6.2. Login to comment
203 Rather than the very low ATP sensitivity seen in Q52R-Kir6.2//WT-SUR1 channels, Q52R-Kir6.2//E128K-SUR1 channels exhibited similar ATP sensitivity as WT-Kir6.2//E128K-SUR1 (Fig. 7, C and D). Login to comment
208 The effect of Q52-Kir6.2 mutation on ATP sensitivity is abrogated by a TMD0-SUR1 mutation E128K that uncouples SUR1 from Kir6.2 The profoundly reduced ATP sensitivity of the Q52R-Kir6.2//WT-SUR1 channel has been attributed to increased intrinsic channel open state stability (Proks et al., 2004). Login to comment
211 A mutation in the TMD0 of SUR1, E128K, uncouples SUR1 from Kir6.2, resulting in reduced Po and diminished PIP2 response that resemble those seen in channels formed by Kir6.2&#e044;C alone (Pratt et al., 2009, 2011). Login to comment
212 Interestingly, the WT-Kir6.2//E128K-SUR1 channels also have reduced ATP sensitivity close to that of Kir6.2&#e044;C channels, indicating that interactions between TMD0-SUR1 and Kir6.2 are required for full-length SUR1 to set the ATP sensitivity. Login to comment
213 We therefore tested how the E128K-SUR1 mutation affects the ability of Q52R-Kir6.2 to increase channel Po and reduce ATP sensitivity. Login to comment
214 Note because the E128K mutation impairs channel trafficking to the cell surface (Yan et al., 2007), in all experiments involving the use of E128K-SUR1, we treated cells with 300 &#b5;M tolbutamide (a KATP channel chaperone which binds reversibly to the channel) overnight to rescue the trafficking defect and increase surface expression of the mutant channels as we have shown previously (Pratt et al., 2009, Figure 6.ߓ Q52K-Kir6.2//WT(E203)-SUR1 channels do not have extremely increased ATP sensitivity. Login to comment
221 Error bars represent SEM, and some are smaller than the symbols. for Q52E-Kir6.2//E128K-SUR1 and Q52E-Kir6.2// E128K/E203K-SUR1 channels was closer to that of WT-Kir6.2//E128K-SUR1 (and Kir6.2&#e044;C channels [Pratt et al., 2009]) than that of either Q52E-Kir6.2//WT-SUR1 or Q52E-Kir6.2//E203K-SUR1 (Fig. 7, C and D). Login to comment
224 E128K-SUR1 also suppressed the effects of Q52E-Kir6.2 and Q52E-Kir6.2//E203K-SUR1 on ATP sensitivity. Login to comment
225 Current inhibition by ATP Figure 7.ߓ Phenotype of mutations at Q52 of Kir6.2 and/or E203 of SUR1 is abrogated by the E128K mutation in SUR1. Login to comment
226 (A) Q52R-Kir6.2 causes an increased apparent PIP2 affinity that is abrogated by E128K-SUR1. Login to comment
228 The increase in apparent PIP2 affinity (i.e., right-shifted neomycin dose-response) of Q52R-Kir6.2 relative to WT is abolished by the inclusion of E128K-SUR1. Login to comment
229 Dose-response curves (dotted lines) of currents inhibited by neomycin relative to control Kint/EDTA solution were calculated using the Hill equation (IC50 for WT, Q52R-Kir6.2, and Q52R-Kir6.2//E128K-SUR1 is 3.2 &#b1; 1.3 &#b5;M, 5,935 &#b1; 2,559 &#b5;M, and 0.9 &#b1; 0.5 &#b5;M, respectively). Login to comment
231 (B) E128K in SUR1 abolishes the ability of Q52E-Kir6.2//E203K-SUR1 channels to be stimulated by PIP2. Login to comment
235 (C and D) Inhibition by ATP was tested in channels composed of E128K-SUR1 with Q52E-Kir6.2, Q52R-Kir6.2, or Q52E-Kir6.2//E203K-SUR1. Login to comment
238 (D) Averaged inhibition by 1, 0.1, and 0.01 mM ATP for various channel types with and without the E128K-SUR1 mutation. Login to comment
239 For comparison, the degree of inhibition by 1 and 0.1 mM ATP for E128K-SUR1 is highlighted by gray dotted lines. Login to comment
243 Despite this widely accepted view, the molecular mechanism underlying the effect of SUR1 E128K mutation precludes the molecular interactions between SUR1 and Kir6.2 necessary to define channel ATP sensitivity. Login to comment
278 We recently showed that a naturally occurring mutation at this position, E128K, disrupts functional coupling between TMD0-SUR1 and Kir6.2, leading to channels that exhibit reduced Po and reduced PIP2 sensitivity as seen in channels formed by Kir6.2&#e044;C alone (Pratt et al., 2009, 2011). Login to comment
279 In addition, E128K reduces channel ATP sensitivity to that seen in Kir6.2&#e044;C, suggesting the mutation not only prevents functional coupling between TMD0 and Kir6.2 with respect to Po but also between SUR1 structures downstream of TMD0 and Kir6.2 that control ATP sensitivity. Login to comment
280 Our observations that when E128K-SUR1 was combined with Q52R-Kir6.2, Q52E-Kir6.2, or Q52E-Kir6.2//E203K-SUR1, the resulting channels all had PIP2 and ATP sensitivity closer to that of WT-Kir6.2//E128K-SUR1 (or Kir6.2&#e044;C) further support this notion. Login to comment

[hide] Engineered Kir6.2 mutations that correct the traff... Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21. Zhou Q, Pratt EB, Shyng SL
Engineered Kir6.2 mutations that correct the trafficking defect of K(ATP) channels caused by specific SUR1 mutations.
Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21., [PMID:23695995]

Abstract [show]
KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. The molecular interactions between SUR1 and Kir6.2 that govern channel gating and biogenesis are incompletely understood. In a recent study, we showed that a SUR1 and Kir6.2 mutation pair, E203K-SUR1 and Q52E-Kir6.2, at the SUR1/Kir6.2 interface near the plasma membrane increases the ATP-sensitivity of the channel by nearly 100-fold. Here, we report the finding that the same mutation pair also suppresses channel folding/trafficking defects caused by select SUR1 mutations in the first transmembrane domain of SUR1. Analysis of the contributions from individual mutations, however, revealed that the correction effect is attributed largely to Q52E-Kir6.2 alone. Moreover, the correction is dependent on the negative charge of the substituting amino acid at the Q52 position in Kir6.2. Our study demonstrates for the first time that engineered mutations in Kir6.2 can correct the biogenesis defect caused by specific mutations in the SUR1 subunit.

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16 Of the three TMD0 mutations tested, F27S and A116P showed a clear upper band in addition to the lower immature band in the E203K//Q52E background; by contrast, the same trafficking mutations placed in the background without the E203K//Q52E mutations only exhibited the lower band (Fig. 2), indicating the proteins were retained in the ER as reported previously.25,26 Another TMD0 mutation, E128K, as well as three other previously identified, congenital hyperinsulinism-causing SUR1 trafficking mutations outside of TMD0 (R495Q, F686S and L1350Q),25 however, showed no improvement in their processing efficiency when combined with E203K//Q52E (data not shown). Login to comment

[hide] Pharmacological rescue of trafficking-impaired ATP... Front Physiol. 2013 Dec 24;4:386. doi: 10.3389/fphys.2013.00386. Martin GM, Chen PC, Devaraneni P, Shyng SL
Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels.
Front Physiol. 2013 Dec 24;4:386. doi: 10.3389/fphys.2013.00386., [PMID:24399968]

Abstract [show]
ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic beta-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The beta-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed.

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218 Mutation Domain Rescue Rescue Gating References by SU by CBZ property SUR1 G7R TMD0 Yes Yes Normal Yan et al., 2007 N24K TMD0 Yes Yes Normal Yan et al., 2007 F27S TMD0 Yes Yes Normal Yan et al., 2007 R74W TMD0 Yes Yes ATP-insensitive Yan et al., 2007 A116P TMD0 Yes Yes Normal Yan et al., 2004 E128K TMD0 Yes Yes ATP-insensitive Yan et al., 2007 V187D TMD0 Yes Yes Normal Yan et al., 2004 R495Q TMD1 Yes Yes Unknown Yan et al., 2007 E501K TMD1 Yes Yes Unknown Yan et al., 2007 L503P TMD1 No No Unknown Yan et al., 2007 F686S NBD1 No No Unknown Yan et al., 2007 G716V NBD1 No No Unknown Yan et al., 2007 E1324K TMD2 N.D.3 N.D. Login to comment
342 INTERPLAY BETWEEN CHANNEL EXPRESSION AND GATING IN DISEASE MANIFESTATION Although some TMD0 mutations only impair channel trafficking such that pharmacological rescue of mutant channels to the cell surface is expected to partially or fully restore channel function, some impact both channel biogenesis and gating as exemplified by the R74W and E128K mutations (Pratt et al., 2009). Login to comment

[hide] Clinical and histological heterogeneity of congeni... Eur J Endocrinol. 2014 Dec;171(6):685-95. doi: 10.1530/EJE-14-0353. Epub 2014 Sep 8. Arya VB, Guemes M, Nessa A, Alam S, Shah P, Gilbert C, Senniappan S, Flanagan SE, Ellard S, Hussain K
Clinical and histological heterogeneity of congenital hyperinsulinism due to paternally inherited heterozygous ABCC8/KCNJ11 mutations.
Eur J Endocrinol. 2014 Dec;171(6):685-95. doi: 10.1530/EJE-14-0353. Epub 2014 Sep 8., [PMID:25201519]

Abstract [show]
CONTEXT: Congenital hyperinsulinism (CHI) has two main histological types: diffuse and focal. Heterozygous paternally inherited ABCC8/KCNJ11 mutations (depending upon whether recessive or dominant acting and occurrence of somatic maternal allele loss) can give rise to either phenotype. However, the relative proportion of these two phenotypes in a large cohort of CHI patients due to paternally inherited heterozygous ABCC8/KCNJ11 mutations has not been reported. OBJECTIVE: The purpose of this study is to highlight the variable clinical phenotype and to characterise the distribution of diffuse and focal disease in a large cohort of CHI patients due to paternally inherited heterozygous ABCC8/KCNJ11 mutations. DESIGN: A retrospective chart review of the CHI patients due to heterozygous paternally inherited ABCC8/KCNJ11 mutations from 2000 to 2013 was conducted. RESULTS: Paternally inherited heterozygous ABCC8/KCNJ11 mutations were identified in 53 CHI patients. Of these, 18 (34%) either responded to diazoxide or resolved spontaneously. Fluorine-18 l-3, 4-dihydroxyphenylalanine positron emission tomography computerised tomography 18F DOPA-PET CT) scanning in 3/18 children showed diffuse disease. The remaining 35 (66%) diazoxide-unresponsive children either had pancreatic venous sampling (n=8) or 18F DOPA-PET CT (n=27). Diffuse, indeterminate and focal disease was identified in 13, 1 and 21 patients respectively. Two patients with suspected diffuse disease were identified to have focal disease on histology. CONCLUSIONS: Paternally inherited heterozygous ABCC8/KCNJ11 mutations can manifest as a wide spectrum of CHI with variable 18F DOPA-PET CT/histological findings and clinical outcomes. Focal disease was histologically confirmed in 24/53 (45%) of CHI patients with paternally inherited heterozygous ABCC8/KCNJ11 mutations.

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63 Extracellular M1V S12X Q54X E128K R74W M429X H627fs D855E R934X K890fs E995X *A1185V D1194V *L1431F R1437Q *D1472N A1493T *A1508P *E1507K *R54H R136fs Kir6.2 SUR1 CL3 linker Walker A p.? Login to comment
74 Patient ID GA (weeks), birth weight (g) Gender Age at presentation (weeks) Blood glucose (mmol/l) Serum insulin (mU/l) Mutation protein description (DNA description) LOH Dzx Resp PET CT/PVS Outcome ABCC8 1 40, 3150 Male 52 2.4 4.3 L1431F/N (c.4291COT/N) Yes - On Dzx at 6.4 years 2 40, 4000 Male 2 2.4 1.9 p.?/N (c.2697C4AOT/N) Yes - Off Dzx at 2 years 3 40, 5010 Female !1 2.6 8.6 E1507K/N (c.4519GOA/N) Yes - On Dzx at 2.3 years 4 40, 5600 Male !1 2.0 7.5 A1508P/N (c.4522GOC/N) Yes - On Dzx at 12 years 5 37, 4820 Male !1 2.0 9.0 A1153T/N (c.3457GOA/N) Yes - On Dzx at 4 years 6 38, 3630 Female 72 3.1 !2 A1153T/N (c.3457GOA/N) Yes - On Dzx at 2 years 8 35, 2820 Male !1 1.2 12.9 A1185V/N (c.3554COT/N) Yes - Off Dzx at 8 months 9 36, 4450 Male !1 2 28.5 p.?/N (c.3992-9GOA/N) Yes - Off Dzx after 4.5 months 10 41, 2780 Female !1 1 9.3 D1472N/N (c.4414GOA/N) Yes - Off Dzx after 10 months 11 38, 3750 Male !1 2.6 5.90 V601I/N (c.1801GOA/N) Yes - Off Dzx after 14 months 13 40, 4160 Female !1 2.4 14.8 V185fs/N (c.554delT/N) No Diffuse Off octreotide at 5 years 14 37, 3090 Male !1 0.6 12.4 p.?/N (c.3992-9GOA/N) No Focal On octreotide at 9.5 years 15 40, 3600 Male !1 2.7 6.7 H627fs/N (c.1879delC/N) No Diffuse Off octreotide at 18 months 16 40, 4700 Female !1 2.0 !2 E1507K/N (c.4519GOA/N) NA - No treatment required 17 40, 4200 Male !1 1.0 !2 D1031N/N (c.3091GOA/N) NA - No treatment required 18 41, 4850 Male !1 1.2 10.1 M1V/N (c.1AOG/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 19 38, 2400 Male !1 2.1 16.3 D1194V; R1437Q/N (c.3581AOT; c.4310GOA/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 20 40, 4580 Female !1 1.1 103 A1493T/N (c.4477GOA/N) Yes a No Diffuse Near-total pancreatectomy (95%) 21 40, 4600 Male !1 1.2 22.5 K890fs/N (c.2669_2675del/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 22 40, 4335 Male !1 2.6 3.4 p.?/N (c.3992-9GOA/N) Yes No Focal Partial pancreatectomy 23 40, 3030 Male !1 1.8 15.6 p.?/N (c.3992-9GOA/N) Yes No Focal Partial pancreatectomy 24 40, 2770 Male !1 2.2 3.4 p.?/N (c.580-1GOC/N) No Indeterminate (PVS) Partial pancreatectomy - focal lesion on histology 25 41, 4290 Male 2 2.3 4.32 E128K/N (c.382GOA/N) No Focal Hypoglycaemia resolved after removal of focal lesion 27 40, 5095 Male !1 2.0 10.5 L1171X/N (c.3512delT/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 29 37, 3560 Male !1 1.6 21.8 p.?/N (c.1629-2AOC/N) No Focal Hypoglycaemia resolved after removal of focal lesion 30 40, 2750 Male !1 1.5 16.4 G111R/N (c.331GOA/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 31 37, 3340 Male !1 2.1 15 H627fs/N (c.1879delC/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 32 41, 4950 Male !1 1.2 11.95 R934X/N (c.2800COT/N) No Focal Hypoglycaemia resolved after removal of focal lesion 33 40, 3080 Female 12 1.5 4.6 S12X/N (c.35COA/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 34 39, 3600 Male !1 2.5 8 R1494W/N (c.4480COT/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 37 36, 3410 Male !1 0.9 17.42 R1494W/N (c.4480COT/N) No Focal Partial pancreatectomy Table 1 Continued Patient ID GA (weeks), birth weight (g) Gender Age at presentation (weeks) Blood glucose (mmol/l) Serum insulin (mU/l) Mutation protein description (DNA description) LOH Dzx Resp PET CT/PVS Outcome 38 39, 4900 Female !1 1.4 23.61 A113V/N (c.338COT/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 39 36, 3210 Male !1 0.6 114 Mosaic Q54X (c.160COT) No Diffuse Near-total pancreatectomy (95%) 40 41, 4300 Female !1 2.1 17 Q954X/N (c.2860COT/N) No Diffuse Near-total pancreatectomy (95%) 41 36, 2730 Male !1 ? Login to comment