ABCMdb

ABCC8 p.Arg74Trp

Predicted by SNAP2:	A: N (53%), C: D (63%), D: D (80%), E: N (53%), F: D (63%), G: D (95%), H: N (82%), I: N (57%), K: N (87%), L: N (78%), M: N (57%), N: N (61%), P: N (53%), Q: N (66%), S: N (61%), T: N (61%), V: N (57%), W: D (91%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: 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
114 The R74W mutation has been reported in two focal cases (disease causing a paternally derived mutation that was expressed due to loss of heterozygosity for the maternal allele) (24). Login to comment
116 R74W was also reported as a compound heterozygous mutation with another mutation, R1215Q, in a patient with diffuse HI who failed medical therapy with diazoxide and required a near-total pancreatectomy (15, 25). 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
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
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
134 The R74W mutant exhibited more variable Po FIGURE1.NucleotidesensitivitiesofTMD0mutantsG7R,N24K,andF27S. 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
153 Two recordings representing both ends of the R74W Po spectrum are shown in Fig. 4A. 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
265 The R74W mutation might also disrupt functional interactions between TMD0-SUR1 and Kir6.2; however, this disruption would be predicted to be less pronounced as the mutation only caused mild reduction in ATP sensitivity and Po. 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
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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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
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
36 We show that R74W reduces the stability of TMD0 protein and thus physical coupling between TMD0 and Kir6.2. Login to comment
100 Because of significantly reduced surface expression in most mutants, cells were pretreated with 300 µM tolbutamide overnight, which partially corrects the trafficking defect caused by R74W, as reported previously (Yan et al., 2004, 2007; Pratt et al., 2009), as well as all other R74 mutations, with the exception of R74D (Fig. S1). Login to comment
122 Next, we examined the surface expression of R74W and R74K fTMD0 when coexpressed with Kir6.2C36 using immunofluorescent obtain the IC50 for ATP inhibition (Fig. 2 B). Login to comment
142 Consistently, in single-channel recordings from cells coexpressing R74K or R74W fTMD0 with Kir6.2C36, no channel kinetics distinct from those characteristic of channels formed by Kir6.2C36 alone were observed (unpublished data). 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
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

[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
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
203 For example, both R74W and R495Q are compound heterozygous mutations with R1215Q. Login to comment
204 Based on in vitro functional phenotypes of mutant hamster SUR1/rat Kir6.2 channels expressed in COS cells, one would expect that in patients, in addition to the expression defects caused by the R74W or R495Q mutation, the R1215Q mutation would also reduce channel function by reducing channel response to MgADP (28). Login to comment
209 This was particularly the case in a patient with compound heterozygous R74W/R1215Q mutations who had a greater than normal insulin response to tolbutamide. 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
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
202 For example, both R74W and R495Q are compound heterozygous mutations with R1215Q. Login to comment
207 This was particularly the case in a patient with compound heterozygous R74W/R1215Q mutations who had a greater than normal insulin response to tolbutamide. Login to comment

[hide] Histopathology of congenital hyperinsulinism: retr... Pediatr Dev Pathol. 2003 Jul-Aug;6(4):322-33. Suchi M, MacMullen C, Thornton PS, Ganguly A, Stanley CA, Ruchelli ED
Histopathology of congenital hyperinsulinism: retrospective study with genotype correlations.
Pediatr Dev Pathol. 2003 Jul-Aug;6(4):322-33., [PMID:14692646]

Abstract [show]
The majority of the most severe cases of congenital hyperinsulinism (HI) are caused by defects in the beta-cell adenosine triphosphate (ATP)-sensitive potassium channel and usually require pancreatectomy to control blood sugar levels. In contrast to the recent advances in understanding the pathophysiology and genetic bases of HI, the histologic classification of this condition remains controversial. A recent proposal to classify the HI pancreata into diffuse and focal forms has drawn much interest because of its relative simplicity and its good correlation with the genetic abnormalities. We undertook a retrospective study to determine whether this classification scheme could be applied to 38 pancreata resected for HI at our institution. We also obtained leukocyte genomic DNA from 29 cases and screened the exons of ABCC8 and KCNJ11 genes for the presence of mutations. Nineteen cases (50.0%) were histologically classified as diffuse HI and 14 cases (36.8%) were categorized as focal form. The mutational analysis revealed that 14 of the 16 diffuse cases analyzed had either homozygous or compound heterozygous mutations of ABCC8 or KCNJ11 and 7 of 10 focal cases had only the paternally inherited mutations, consistent with the previous observations. Two patients (5.3%) had normal pancreatic histology but had persistent hypoglycemia postoperatively, leaving the possibility of residual focal lesion. Three of 38 cases (7.9%) did not fit well into either diffuse or focal category. Two cases differed from the described pattern for the diffuse form in that the nuclear enlargement was confined to a single area of the pancreas. The other case had a focal lesion but beta-cell nuclear enlargement was present in nonadjacent areas. Mutations for typical diffuse or focal HI were not identified in two of these three equivocal cases. We conclude from this study that nearly 90% of HI cases can be classified into either a diffuse or a focal form. However, a small percentage of cases represented a diagnostic challenge.

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148 Clinical features, histologic diagnoses, and genotypes of HI patients Case no. Sex Age at surgery Type of pancreatectomy Immediate outcome Histology ABCC8 and KCNJ11 mutations (paternal/ maternala ) 1 F Newborn Near total Hypoglycemia Diffuse 3992-9 g to a/delF1388 2 F 5 m, 28 d Near total Hypoglycemia Diffuse ND 3 M 1 m, 14 d Near total Hypoglycemia Diffuse 3992-9 g to a/R1215Q 4 F 1 m, 6 d Near total Hypoglycemia Diffuse delF1388/3992-9 g to a 5 F 0 m, 28 d Near total Hypoglycemia Diffuse 3992-9 g to a/3992-9 g to a 6 M 0 m, 14 d Near total Hypoglycemia Diffuse 3992-9 g to a/delF1388 7 M 0 m, 17 d Near total Diabetes mellitus Diffuse 3992-9 g to a/3992-9 g to a 8 M 1 y, 11 m Near total Hypoglycemia Diffuse -/- 9 M 1 y, 2 m Near total Hypoglycemia Diffuse 3992-9 g to a/3992-9 g to a 10 M 1 m, 17 d Near total Hypoglycemia Diffuse ND 11 M 0 m, 27 d Near total Hypoglycemia Diffuse 3992-9 g to a/3992-9 g to a 12 F 1 m, 10 d Near total Hypoglycemia Diffuse ND 13 M 1 m, 3 d Partial Hypoglycemia Diffuse delF1388/3992-9 g to a 2 m, 24 d Partial Hypoglycemia Diffuse 2 y, 6 m Near total Hypoglycemia Diffuse 14 F 1 m, 7 d Near total Hypoglycemia Diffuse delF1388/3992-9 g to a 15 M 0 m, 16 d Near total Hypoglycemia Diffuse 3992-9 g to a/3992-9 g to a 16 F 1 y, 8 m Near total Hypoglycemia Diffuse -/- 17 F 4 m, 19 d Near total Diabetes mellitus Diffuse * G134Ab /* P266Lb 18 M 1 m, 5 d Near total Diabetes mellitus Diffuse * R74W/R1215Q 19 F 3 m, 27 d Near total Hypoglycemia Diffuse * R999X/* R598X 20 F Infant Near total Cure Focal ND 21 F 1 m, 16 d Near total Cure Focal ND 22 M 0 m, 13 d Near total Cure Focal ND 23 M 1 m, 7 d Near total Hypoglycemia Focal * 2116 ϩ 1 g to t (not maternal) 24 M 1 m, 26 d Near total Cure Focal ND 25 M 0 m, 15 d Near total Cure Focal 3992-9 g to a/- 26 M 1 m, 30 d Near total Cure Focal R1494Q/- 27 F 7 m, 24 d Near total Cure Focal -/- 28 M 0 m, 27 d 95% Hypoglycemia Normal 3992-9 g to a/- 1 m, 18 d Near total Hypoglycemia Focal 29 M 1 m, 27 d Near total Hypoglycemia Focal * 3576del g/- 2 m, 0 d Near total Hypoglycemia 2 m, 18 d Total Hypoglycemia 30 M 2 m, 28 d Near total Cure Focal * R74W/- 31 M 1 m, 2 d Near total Hypoglycemia Focal * C717X/- 32 F 1 m, 22 d Near total Cure Focal * 1874del c/- 33 M 2 m, 16 d 30% Cure Focal Q954X/- 34 F 2 m, 7 d 85% Hypoglycemia Normal ND 3 m, 4 d Near total Hypoglycemia Normal (continued) the patient underwent a mere 30% pancreatectomy. Login to comment
173 Another novel mutation, R74W (Table 3), was identified in patient no. 18 which was inherited from the father. Login to comment
187 base pair deletions, a novel splice site mutation, and the R74W mutation described in patient no. 18 above (Table 3) were identified in each of five other patients (no. 31, nos. 29 and 32, no. 23, and no. 30, respectively). Login to comment
207 ABCC8 Exon 2 c220t 74 Arg to Trp -Bsp MI 18, 30 ABCC8 Exon 12 c1795t 598 Arg to stop codon -Sin I 19 ABCC8 Exon 13 1874del c 625 Frameshift None 32 ABCC8 Intron 15 2116 ϩ 1 g to t NA 5' splice site alteration None 23 ABCC8 Exon 16 c2151a 717 Cys to stop codon -Bbv I 31 ABCC8 Exon 25 c2995t 999 Arg to stop codon ϩHph I 19 ABCC8 Exon 29 3576 del g 1192 Frameshift None 29 KCNJ11 Exon 1 g401c 134 Gly to Ala None 17 KCNJ11 Exon 1 c797t 266 Pro to Leu None 17 -, base change abolishes a restriction enzyme site; ϩ, base change creates a restriction enzyme site; NA, not applicable. Login to comment
248 A novel mutation described in this study, R74W, was present in one diffuse HI patient and one focal HI patient. Login to comment

[hide] Genotype-phenotype correlations in children with c... J Clin Endocrinol Metab. 2005 Feb;90(2):789-94. Epub 2004 Nov 23. Henwood MJ, Kelly A, Macmullen C, Bhatia P, Ganguly A, Thornton PS, Stanley CA
Genotype-phenotype correlations in children with congenital hyperinsulinism due to recessive mutations of the adenosine triphosphate-sensitive potassium channel genes.
J Clin Endocrinol Metab. 2005 Feb;90(2):789-94. Epub 2004 Nov 23., [PMID:15562009]

Abstract [show]
Congenital hyperinsulinism (HI) is most commonly caused by recessive mutations of the pancreatic beta-cell ATP-sensitive potassium channel (K(ATP)), encoded by two genes on chromosome 11p, SUR1 and Kir6.2. The two mutations that have been best studied, SUR1 g3992-9a and SUR1 delF1388, are null mutations yielding nonfunctional channels and are characterized by nonresponsiveness to diazoxide, a channel agonist, and absence of acute insulin responses (AIRs) to tolbutamide, a channel antagonist, or leucine. To examine phenotypes of other K(ATP) mutations, we measured AIRs to calcium, leucine, glucose, and tolbutamide in infants with recessive SUR1 or Kir6.2 mutations expressed as diffuse HI (n = 8) or focal HI (n = 14). Of the 24 total mutations, at least seven showed evidence of residual K(ATP) channel function. This included positive AIR to both tolbutamide and leucine in diffuse HI cases or positive AIR to leucine in focal HI cases. One patient with partial K(ATP) function also responded to treatment with the channel agonist, diazoxide. Six of the seven patients with partial defects had amino acid substitutions or insertions; whereas, the other patient was compound heterozygous for two premature stop codons. These results indicate that some K(ATP) mutations can yield partially functioning channels, including cases of hyperinsulinism that are fully responsive to diazoxide therapy.

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54 Gene Haplotype Calcium (␮U/ml) Leucine (␮U/ml) Glucose (␮U/ml) Tolbutamide (␮U/ml) Diazoxide responsive Diffuse HI 1 SUR1 delF1388/D1472H 6 2 13 -2 No 2 Kir6.2 G134A/P266L 20 3 36 -2 No 3 SUR1 g3992-9a/g1630ϩ1a 11 16 -2 No 4 SUR1 N188S/D1472N 7 1 7 7 No 5 SUR1 R598X/R999X 32 1 72 27 No 6 SUR1 R495Q/R1215Q -2 15 44 30 No 7 SUR1 R74W/R1215Q 52 28 20 98 No 8 SUR1 g3992-9a/K1337N 2 18 39 33 Yes Focal HI 9 SUR1 F27S 17 -1 16 29 No 10 SUR1 F686S 12 2 27 12 No 11 SUR1 E501K 6 3 9 10 No 12 SUR1 3576delg 9 6 9 12 No 13 SUR1 g3992-9a 5 8 25 9 No 14 SUR1 g3992-9a 3 8 40 21 No 15 SUR1 c2924-10a 4 8 67 29 No 16 Kir6.2 A101D 1 8 177 88 No 17 SUR1 R1215W 7 9 15 6 No 18 Kir6.2 R136L 8 10 115 21 No 19 SUR1 g3992-9a 40 15 35 -0.3 No 20 SUR1 6aa insertion in exon 5 6 16 22 15 No 21 SUR1 R1215W 38 47 58 15 No 22 Kir6.2 R301H 16 55 75 14 No Controls (␮U/ml, mean Ϯ SD) KATP HI (n ϭ 7) 28 Ϯ 16 5 Ϯ 8 12 Ϯ 9 4 Ϯ 6 No GDH-HI (n ϭ 7) 2.3 Ϯ 5.4 42 Ϯ 27 120 Ϯ 52 94 Ϯ 56 Yes Normal (n ϭ 6) 3 Ϯ 4 1.4 Ϯ 2.8 56 Ϯ 26 48 Ϯ 32 Yes a To convert insulin (␮U/ml to pmol/liter), multiply by 6.0. identified in other patients. Login to comment
107 Degree of residual channel function in KATP mutations Null Indeterminate Partial SUR1 g3992-9a g1630ϩ1a R598X/R999X delF1388 N188S/D1472N R495Q/R1215Q F27S 3576delg R74W/R1215Q F686S K1337N E501K 6 aa insertion in exon 5 c2924-10a R1215W Kir6.2 G134A/P266L R301H A101D R136L FIG. 1. Login to comment

[hide] Mutation spectra of ABCC8 gene in Spanish patients... Hum Mutat. 2006 Feb;27(2):214. Fernandez-Marmiesse A, Salas A, Vega A, Fernandez-Lorenzo JR, Barreiro J, Carracedo A
Mutation spectra of ABCC8 gene in Spanish patients with Hyperinsulinism of Infancy (HI).
Hum Mutat. 2006 Feb;27(2):214., [PMID:16429405]

Abstract [show]
Hyperinsulinism of Infancy (HI) is a clinical disorder characterized by deregulation of insulin secretion that leads to profound hypoglycemia. Mutations in genes encoding the ATP-regulated potassium channels of the pancreatic beta-cell, namely ABCC8 (SUR1) and KCNJ11 (Kir6.2), are the major genetic known cause of the disease. To elucidate the genetic etiology of HI in the uncharacterized Spanish population, we conducted extensive sequencing analysis of the ABCC8 (83.5Kb) and KCNJ11 (1.7Kb) genes in 34 Spanish HI patients. Mutations in ABCC8 were detected for both alleles in 13 patients, while ten patients carried only one mutation in one of the ABCC8 alleles. We have detected 22 novel and seven previously described mutations in ABCC8, approximately 60% of them lead to a premature termination signal, which would result in truncated SUR1 proteins. No mutations were found in the KCNJ11 gene. In addition, we report for the first time a 3914bp macrodeletion associated with the HI disorder. The potential pathogenicity of several additional variants is discussed. The spatial pattern of three pathological mutations suggests possible geographical founder effects. This work reveals for first time the involvement of KATP channels in the pathogenesis of an important proportion (approximately 68%) of Spanish HI patients. The spectrum of mutations in Spanish HI patients provides an important tool for diagnosis and prognosis of HI patients in the Spanish population, as well as for genetic counseling of HI families.

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106 Mutations c.220C>T (p.R74W), c.331G>C (p.G111R), and c.563A>G (p.N188S) are situated in the TMD0 domain of SUR1 which is implicated in the strong association between SUR1 and Kir6.2 and modulate trafficking and gating of the channel (Chan et al. 2003). Login to comment
107 Although R74W was found for first time in our population, Nestorowicz et al. (1998) described a different amino acid change at the same codon (R74Q). Login to comment
137 Clinical characteristics of HI Spanish patients that carry at least one mutation in ABCC8 Mutationsg Pa Ob Sex Mc Td PCe PTf Pchrh Mchrh 1a Gal M >p90 DZ 5 (>90%) OT, NF, GC p.R248X c.3576delG 1b Gal F >p50 DZ, OT, NF - OT, NF, NGT p.R248X c.3576delG 3 Gal F >p90 DZ 2 (95%) - c.584 585insA c.584 585insA 4 Gal M >p75 DZ 4 (95%) DZ, NGT c.584 585insA c.584 585insA 5 Gal M >p50 OT, DZ 16 (90%) - c.1347 1348delGA - 8 Cast M >p75 DZ, OT, GC - - p.M233R - 9 Cast F >p75 DZ 0.5 (85%) DZ, OT, PC (99%) p.G111R - 12 And M - - - - c.4612 -2 A>T p.D310N 14 Cat M >p75 DZ - - p.R934X c.3992-9 G>A 17 Cat F >p90 DZ, OT - - c.3133 3152del c.4619 4620insT 18 Cat M <p50 DZ, CNF 0.5 (95%) DZ c.1732 1746dup - 19 Can M <p50 DZ, NF, OT 2 (99%) - c.1332+4438 1631-9207del c.1332+4438 1631-9207del 20 Cat M - DZ, NF, GC - - c.2142delG p.T1131P 21 Cat F >p50 DZ, NF - - - i - i 23 Bal M >p90 CNF - - c.4310 G>A c.1732 1746dup 25 Mor M - DZ, OT yes (EXITUS) p.N188S, c.4123-19 C>T p.N188S, c.4123-19 C>T 27 Cast F >p75 DZ, CNF 24 (75%) PC (99%) p.R598X p.L1451P 28 Cat M >p90 DZ, CNF - - p.R1251X p.L1148R 30 Cast M >p90 DZ, OT 5 (95%) DZ, OT (EXITUS) p.R74W - 31 Gal F >p90 DZ 0.5 (95%) DZ - p.K719T 32 Cat F >p90 DZ - - - p.N1296K 33 Cast F >p75 DZ, OT 1 (95%) DZ, OT c.3291 3292delGC - 34 Val F >p90 DZ, NF - (EXITUS) p.P551R - a P = patient. Login to comment
147 Genetic variants found in ABCC8 gene from HI Spanish cohort Mutations considered pathogenic nt change a aa change a Type E/Ic Domaind Patient Refe PSIC f Polypheng C h c.220C>T p.R74W MIS E2 CL1 P30 NR 2.257 PrD Highly c.331G>C p.G111R MIS E3 TM3 P9 [1] 1.672 PsD Moderately c.563A>G p.N188S MIS E4 TM5 P25 [2] 1.494 Benign Highly c.698T>G p.M233R MIS E5 CL3 P8 NR 2.428 PrD Highly c.584_585insA p.Y195X FS E5 ─ P3, P4 NR ─ ─ ─ c.742C>T p.R248X NON E5 ─ P1a, P1b [3] ─ ─ ─ c.928G>A p.D310N MIS E6 CL3 P12 NR 1.614 PsD Highly c.1347_1348delGA p.V449VfsX493 FS E9 ─ P5 NR ─ ─ ─ c.1332+4438_1631-9207del p.I445FfsX447 FS ─ ─ P19 NR ─ ─ ─ c.1652C>G p.P551R MIS E11 TM10 P34 NR 2.1 PsD Highly c.1732_1746dup p.A578_L582dup IFins E12 ─ P18, P23 NR ─ ─ ─ c.1792C>T p.R598X NON E12 ─ P27 NR ─ ─ ─ c.2156 A>C p.K719T MIS E16 CL6 P31 NR 1.927 PsD Highly c.2142delG p.Q714QfsX724 FS E16 ─ P20 NR ─ ─ ─ c.2394-2A>G ─ AS I19 ─ P21 NR ─ ─ ─ c.2800C>T p.R934X NON E23 ─ P14 NR ─ ─ ─ c.3133_3152del p.L1045LfsX1107 FS E25 ─ P17 [6] ─ ─ ─ c.3291_3292delGC p.L1097LfsX1113 FS E26 ─ P33 NR c.3391A>C p.T1131P MIS E27 CL7 P20 NR 1.777 PsD Moderately c.3443T>G p.L1148R MIS E28 TM14 P28 NR 1.722 PsD Highly c.3576delG p.L1191LfsX1207 FS E29 ─ P1a, P1b, NR ─ ─ ─ c.3751C>T p.R1251X NON E30 ─ P28 NR ─ ─ ─ c.3888C>G p.N1296K MIS E32 TM17 P32 NR 1.924 PsD Highly c.3992-9G>A ─ AS I 32 ─ P14 [4] ─ ─ ─ c.4123-19C>T ─ AS I33 ─ P25 [5] ─ ─ ─ c.4310G>A ─ AS E35 ─ P23 [4] ─ ─ ─ c.4352T>C p.L1451P MIS E36 CL9 P27 NR 1.797 PsD Highly c.4612-2 A>T ─ AS I38 ─ P12 NR ─ ─ ─ c.4619_4620insT p.H1540AfsX1559 FS E39 ─ P17 NR ─ ─ ─ Polimorphisms and unclassified variants nt change a aa change a Type E/Ic SNPid Patientsi Controls NCBI j Exclusion c. 207T>C p.P69P SYN E2 rs1048099 28/46 ─ 0.50 S c. 330C>T p.A110A SYN E3 rs8192695 2/48 ─ 0.04 S c. Login to comment

[hide] Preoperative evaluation of infants with focal or d... J Clin Endocrinol Metab. 2004 Jan;89(1):288-96. Stanley CA, Thornton PS, Ganguly A, MacMullen C, Underwood P, Bhatia P, Steinkrauss L, Wanner L, Kaye R, Ruchelli E, Suchi M, Adzick NS
Preoperative evaluation of infants with focal or diffuse congenital hyperinsulinism by intravenous acute insulin response tests and selective pancreatic arterial calcium stimulation.
J Clin Endocrinol Metab. 2004 Jan;89(1):288-96., [PMID:14715863]

Abstract [show]
Infants with congenital hyperinsulinism often require pancreatectomy. Recessive mutations of the ATP-dependent plasma membrane potassium channel (K(ATP)) genes, SUR1 and K(ir)6.2, cause diffuse hyperinsulinism. K(ATP) channel mutations can also cause focal disease through loss of heterozygosity for maternal 11p, resulting in expression of a paternal mutation. This study evaluated whether focal vs. diffuse hyperinsulinism could be diagnosed by acute insulin response (AIR) tests and whether arterial calcium stimulation/venous sampling (ASVS) could localize focal lesions. Fifty infants with diazoxide-unresponsive hyperinsulinism were studied. Focal lesions occurred in 70% of the cases. Positive AIR calcium occurred in 17 of 30 focal and 10 of 13 diffuse cases (P < 0.04). Positive AIR tolbutamide occurred in 27 of 30 focal vs. seven of 13 diffuse cases (P < 0.02); K(ATP) channel mutations were identified in four of the latter. ASVS localized the lesion in 24 of 33 focal cases (73%) but correctly diagnosed diffuse disease in only four of 13 cases. These results indicate that preoperative AIR tests do not distinguish focal vs. diffuse disease because some K(ATP) channel mutations retain responsiveness to tolbutamide. The ASVS test can be used to localize focal lesions in infants. The combination of ASVS, careful intraoperative histologic analysis, and surgical expertise succeeded in correcting hypoglycemia in 86% of the infants with focal hyperinsulinism.

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205 of alleles SUR1 region Exon 1 F27Sa 1 Exon 2 R74W 2 Exon 4 536-9 del atgga 1 Exon 4 N188S 1 Exon 5 ins 6a.a. 1 Exon 10 R495Qa 1 Exon 10 E501Ka 1 Intron 10 g 1630ϩ1 aa,b 1 Exon 12 R598X 1 Exon 13 1874 del c 1 Exon 15 F686Sa 1 Exon 16 C717X 1 Intron 24 c 2924-9 aa 1 Exon 24 2835-8 del agaga 2 Exon 24 Q954X 1 Exon 25 R999X 2 Exon 29 3576 del g 1 Exon 29 R1215Q 2 Exon 29 R1215Wa,b 2 Intron 32 g 3992-9 a 4 Exon 33 L1350Qa 1 Exon 33 G1401Ra 1 Exon 34 S1387F 1 Exon 34 delF1388 1 Exon 36 D1472Ha 1 Exon 36 D1472Na 1 Kir6.2 region A102Na 1 G134A 1 R136La 1 P266L 1 R301Ha 1 a Novel mutations. Login to comment

[hide] Molecular and immunohistochemical analyses of the ... Mod Pathol. 2006 Jan;19(1):122-9. Suchi M, MacMullen CM, Thornton PS, Adzick NS, Ganguly A, Ruchelli ED, Stanley CA
Molecular and immunohistochemical analyses of the focal form of congenital hyperinsulinism.
Mod Pathol. 2006 Jan;19(1):122-9., [PMID:16357843]

Abstract [show]
Congenital hyperinsulinism is a rare pancreatic endocrine cell disorder that has been categorized histologically into diffuse and focal forms. In focal hyperinsulinism, the pancreas contains a focus of endocrine cell adenomatous hyperplasia, and the patients have been reported to possess paternally inherited mutations of the ABCC8 and KCNJ11 genes, which encode subunits of an ATP-sensitive potassium channel (K(ATP)). In addition, the hyperplastic endocrine cells show loss of maternal 11p15, where imprinted genes such as p57(kip2) reside. In order to evaluate whether all cases of focal hyperinsulinism are caused by this mechanism, 56 pancreatectomy specimens with focal hyperinsulinism were tested for the loss of maternal allele by two methods: immunohistochemistry for p57(kip2) (n=56) and microsatellite marker analysis (n=27). Additionally, 49 patients were analyzed for K(ATP) mutations. Out of 56 focal lesions, 48 demonstrated clear loss of p57(kip2) expression by immunohistochemistry. The other eight lesions similarly showed no nuclear labeling, but the available tissue was not ideal for definitive interpretation. Five of these eight patients had paternal K(ATP) mutations, of which four demonstrated loss of maternal 11p15 within the lesion by microsatellite marker analysis. All of the other three without a paternal K(ATP) mutation showed loss of maternal 11p15. K(ATP) mutation analysis identified 32/49 cases with paternal mutations. There were seven patients with nonmaternal mutations whose paternal DNA material was not available, and one patient with a mutation that was not present in either parent's DNA. These eight patients showed either loss of p57(kip2) expression or loss of maternal 11p15 region by microsatellite marker analysis, as did the remaining nine patients with no identifiable K(ATP) coding region mutations. The combined results from the immunohistochemical and molecular methods indicate that maternal 11p15 loss together with paternal K(ATP) mutation is the predominant causative mechanism of focal hyperinsulinism.

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93 KATP mutationsa Nuclear labeling of p57kip2 Microsatellite marker analysis at 11p15 Remarks on histology Lesion Islets in normal area 1 g3992-9a/ + ND 2 R1494Q/ + ND 3 V21D/ + ND 4 g3992-9a/ + ND 5 3576 del g/ Small lesion + ND 6 R74W/ Small normal area and weak Loss of maternal allele 7 C717X/ + Loss of maternal allele 8 1874 del c/ + ND 9 Q954X/ + ND 10 g3992-9g/ + Loss of maternal allele 11 E501K/ + Loss of maternal allele 12 R136Lb / Weak Loss of maternal allele 13 c2924-9a/ + Loss of maternal allele Focal lesion occupies large area of pancreas 14 g3992-9a/ + ND 15 3084 del g/ + ND 16 R302Hb / + Loss of maternal allele 17 g3992-9a/ + ND 18 536-539 del atgg/ + ND 19 R1215W/ + Loss of maternal allele 20 R999X/ + ND 21 L1350Q/ + ND 22 G1401R/ Weak Loss of maternal allele 23 g2041-21a/ + Loss of maternal allele 24 G7R/ Weak Loss of maternal allele 25 g3992-9a/ + Loss of maternal allele Rare nonadjacent large islet cell nuclei 26 g3992-9a/ + ND 27 Q954X/ + ND 28 delF1388/ + ND 29 Q472X/ + ND 30 G40Db / + Loss of maternal allele 31 S116Pb / + ND 32 g3992-9a/ + ND 33 g2116+1t, nonmaternal + ND 34 A101Db , nonmaternal Small normal area Loss of maternal allele Focal lesion occupies large area of pancreas 35 F27S, nonmaternal Weak Loss of maternal allele 36 G1379R, nonmaternal + ND 37 1631 del t, nonmaternal + ND 38 R1215W, nonmaternal + Loss of maternal allele 39 L503P, nonmaternal + Loss of maternal allele 40 F686S, de novo + Loss of maternal allele 41 1332+4 del c, maternalc + Loss of maternal allele 42 / + Loss of maternal allele 43 / + ND 44 / Small lesion + Loss of maternal allele 45 / + Loss of maternal allele 46 / + Loss of maternal allele 47 / + ND 48 / + Loss of maternal allele 49 / + ND 50 ND + ND 51 ND + ND 52 ND + Loss of maternal allele Rare nonadjacent large islet cell nuclei 53 ND + Loss of maternal allele Focal lesion occupies large area of pancreas All 10 pancreatic specimens studied from patients with diffuse hyperinsulinism did not show loss of p57kip2 labeling of the islet cell nuclei (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
76 p.?/N (c.1333-1013AOG/N) Yes No Focal Hypoglycaemia resolved after removal of focal lesion 42 37, 4480 Female !1 1.7 5.6 A1263T/N (c.3787GOA/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 45 39, 3960 Male !1 2.4 23.6 p.?/N (c.1-?_c.1176C1?/N) No Focal Hypoglycaemia resolved after removal of focal lesion 46 34, 3800 Male !1 0.8 201 D855E/N (c.2565COA/N) No Focal Hypoglycaemia resolved after removal of focal lesion 47 41, 4780 Male !1 2.4 15.9 M429X/N (c.1254_1284dup/N) No Focal Hypoglycaemia resolved after removal of focal lesion 48 40, 3800 Female !1 2.4 5.9 M429X/N (c.1254_1284dup/N) No Diffuse On octreotide at 7 months 49 40, 4100 Male !1 0.8 9.1 R74W/N (c.220COT/N) No Focal Hypoglycaemia resolved after removal of focal lesion 51 38, 3730 Female !1 1.9 21.6 E995X (c.2983GOT) No Focal Hypoglycaemia resolved after removal of focal lesion KCNJ11 7 38, 3780 Female !1 1.7 29.6 M209I/N (c.627GOA/N) Yes Diffuse On Dzx at 2.8 years 12 40, 3450 Male !1 2 6.7 R54H/N (c.161GOA/N) Yes - Off Dzx after 4 months 26 36, 3880 Male !1 !1.0 6.1 E292K/N (c.874GOA/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 28 40, 4600 Male !1 1.3 38.87 R136fs/N (c.405dupG/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 35 38, 4120 Female !1 2.2 12.0 T294M/N (c.881COT/N) No Diffuse (PVS) Near-total pancreatectomy (95%) 36 37, 3960 Male !1 1.9 7.0 G312C/N (c.934GOT/N) No Focal Hypoglycaemia resolved after removal of focal lesion 43 40, 4100 Female !1 1.0 35 I284del/N (c.850_852delATC/N) Yes Diffuse Off Dzx at 4 years of age 44 40, 4580 Female 32 2.2 7 p.*391Rext*94/N (c.1171TO C/N) No Focal Partial pancreatectomy, currently on Dzx 50 37, 3980 Male !1 0.5 2.9 T62M/N (c.185COT/N) Yes - Off Dzx at 1 month 52 40, 5190 Male !1 2.5 8.4 R177W/N (c.529AOT/N) Yes Diffuse On Dzx at 3 months of age 53 40, 3920 Male !1 2.1 9.2 E292K/N (c.874GOA/N) Yes Diffuse Off Dzx at 5 months of age Dzx, diazoxide; PET, positron emission tomography; PVS, pancreatic venous sampling; GA, gestational age; Wt, weight; LOH, loss of heterozygosity; Resp, responsiveness. Login to comment