ABCMdb

ABCC8 p.Phe132Leu

ClinVar:	c.394T>G , p.Phe132Val D , Pathogenic/Likely pathogenic
Predicted by SNAP2:	A: D (53%), C: N (72%), D: D (80%), E: D (75%), G: D (63%), H: D (59%), I: N (57%), K: D (75%), L: N (66%), M: N (61%), N: D (71%), P: D (75%), Q: D (66%), R: D (66%), S: D (53%), T: N (57%), V: N (57%), W: N (57%), Y: N (78%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: N, G: D, H: N, I: N, K: N, L: N, M: N, N: D, P: D, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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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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129 The recently identified PNDM-causing SUR1 F132L mutation is an example, so are many Kir6.2 mutations reported earlier (8, 29). Login to comment
155 As a control, we also analyzed the Po of the F132L mutant. Login to comment
156 Consistent with that reported previously (30), the Po of F132L (0.71 Ϯ 0.05) tends to be higher than that of WT (Fig. 4), although the difference did not reach statistical significance. 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
273 Several SUR1-TMD0 mutations have been reported to reduce physical association between TMD0 and Kir6.2 in co-immunoprecipitation experiments, including CHI-causing A116P and V187D mutations and PNDM-causing F132L mutation (10, 30). Login to comment
274 The former two do not affect the gating properties of the channel (13), whereas the F132L mutation reduces ATP sensitivity by increasing channel intrinsic Po (30). Login to comment
287 In this regard, it is important to note that we have found the neonatal diabetes-causing mutation F132L also significantly reduces channel expression at the cell surface (57.05 Ϯ 1.75% of WT; n ϭ 3), and that sulfonylureas restore mutant channel surface expression to the same level as WT (109.2 Ϯ 7.05; n ϭ 3). Login to comment
154 As a control, we also analyzed the Po of the F132L mutant. Login to comment
286 In this regard, it is important to note that we have found the neonatal diabetes-causing mutation F132L also significantly reduces channel expression at the cell surface (57.05 afe; 1.75% of WT; n afd; 3), and that sulfonylureas restore mutant channel surface expression to the same level as WT (109.2 afe; 7.05; n afd; 3). 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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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
172 Again, the F132L mini-channels were included as a control because they have increased Po and decreased ATP sensitivity compared with WT mini-KATP channels (Proks et al., 2007). 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
181 Mini-KATP channels with the F132L mutation were used as a positive control; F132L was identified in patients with severe neonatal diabetes and has been shown to increase intrinsic Po in full-length and mini-KATP channels (Proks et al., 2006, 2007). Login to comment
186 WT, E128K, or F132L Figure 5. E128K full-length KATP channels have decreased PIP2 response. Login to comment
391 Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. Login to comment

[hide] Molecular basis of neonatal diabetes in Japanese p... J Clin Endocrinol Metab. 2007 Oct;92(10):3979-85. Epub 2007 Jul 17. Suzuki S, Makita Y, Mukai T, Matsuo K, Ueda O, Fujieda K
Molecular basis of neonatal diabetes in Japanese patients.
J Clin Endocrinol Metab. 2007 Oct;92(10):3979-85. Epub 2007 Jul 17., [PMID:17635943]

Abstract [show]
CONTEXT: Neonatal diabetes mellitus (NDM) is classified clinically into a transient form (TNDM), in which insulin secretion recovers within several months, and a permanent form (PNDM), requiring lifelong medication. However, these conditions are genetically heterogeneous. OBJECTIVE: Our objective was to evaluate the contribution of the responsible gene and delineate their clinical characteristics. PATIENTS AND METHODS: The chromosome 6q24 abnormality and KCNJ11 and ABCC8 mutations were analyzed in 31 Japanese patients (16 with TNDM and 15 with PNDM). Moreover, FOXP3 and IPF1 mutations were analyzed in a patient with immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome and with pancreatic agenesis, respectively. RESULTS: A molecular basis for NDM was found in 23 patients: 6q24 in eleven, KCNJ11 in nine, ABCC8 in two, and FOXP3 in one. All the patients with the 6q24 abnormality and two patients with the KCNJ11 mutation proved to be TNDM. Five mutations were novel: two (p.A174G and p.R50G) [corrected] in KCNJ11, two (p.A90V and p.N1122D) in ABCC8, and one (p.P367L) in FOXP3. Comparing the 6q24 abnormality and KCNJ11 mutation, there were some significant clinical differences: the earlier onset of diabetes, the lower frequency of diabetic ketoacidosis at onset, and the higher proportion of the patients with macroglossia at initial presentation in the patients with 6q24 abnormality. In contrast, two patients with the KCNJ11 mutations manifested epilepsy and developmental delay. CONCLUSIONS: Both the 6q24 abnormality and KCNJ11 mutation are major causes of NDM in Japanese patients. Clinical differences between them could provide important insight into the decision of which gene to analyze in affected patients first.

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161 The reported mutations F132L (4) and H1023Y (5) are located in TMD0 and TMD2, respectively, and functional studies confirmed that these mutations reduced ATP sensitivity. Login to comment

[hide] Mutations in the ABCC8 gene encoding the SUR1 subu... Diabetes Obes Metab. 2007 Nov;9 Suppl 2:28-39. Patch AM, Flanagan SE, Boustred C, Hattersley AT, Ellard S
Mutations in the ABCC8 gene encoding the SUR1 subunit of the KATP channel cause transient neonatal diabetes, permanent neonatal diabetes or permanent diabetes diagnosed outside the neonatal period.
Diabetes Obes Metab. 2007 Nov;9 Suppl 2:28-39., [PMID:17919176]

Abstract [show]
AIM: Mutations in the ABCC8 gene encoding the SUR1 subunit of the pancreatic ATP-sensitive potassium channel cause permanent neonatal diabetes mellitus (PNDM) and transient neonatal diabetes mellitus (TNDM). We reviewed the existing literature, extended the number of cases and explored genotype-phenotype correlations. METHODS: Mutations were identified by sequencing in patients diagnosed with diabetes before 6 months without a KCNJ11 mutation. RESULTS: We identified ABCC8 mutations in an additional nine probands (including five novel mutations L135P, R306H, R1314H, L438F and M1290V), bringing the total of reported families to 48. Both dominant and recessive mutations were observed with recessive inheritance more common in PNDM than TNDM (9 vs. 1; p < 0.01). The remainder of the PNDM probands (n = 12) had de novo mutations. Seventeen of twenty-five children with TNDM inherited their heterozygous mutation from a parent. Nine of these parents had permanent diabetes (median age at diagnosis: 27.5 years, range: 13-35 years). Recurrent mutations of residues R1183 and R1380 were found only in TNDM probands and dominant mutations causing PNDM clustered within exons 2-5. CONCLUSIONS: ABCC8 mutations cause PNDM, TNDM or permanent diabetes diagnosed outside the neonatal period. There is some evidence that the location of the mutation is correlated with the clinical phenotype.

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No. Sentence Comment
85 Five residues are sites for different amino acid substitutions: V86A/G, F132L/V, D212I/N, R1183Q/W and R1380C/H/L. Login to comment
86 Nine mutations were observed in more than one proband; R1183W (c.3547C>T) was identified in five probands, R1380C (c.4138C>T) in three probands and the remainder; F132L (c.394T>C), D209E (c.627C>A), T229I (c.686C>T), L582V (c.1744C>G), R826W (c.2476C>T), R1183Q (c.3548G>A) and R1380L (c.4139G>T) were each observed in two probands. Login to comment
161 Affected probands and family members can be separated into three distinct groups based T229I/T229I ABCC8 mutations Transient Neonatal Diabetes Mellitus Recessive homozygous mutations R826W (2) H1024Y R1183Q (2) R1183W (5) R1314H R1380C (3) R1380H R1380L (2) D209E D212I D212N R306H V324M C435R L451P L582V (2) Dominant heterozygous mutations Permanent Neonatal Diabetes Mellitus E382K/E382K A1185E/A1185E Mosaic N72S Recessive homozygous or mosaic mutations P45L/G1401R E208K/Y263D T229I/V1523L L438F/M1290V P207S/c.536del4 E1327K+V1523A/ c.1327ins10 Recessive compound heterozygous mutations 1K Dominant heterozygous mutations D209E Q21 L213R L225P(2) I1425V V86A V86G F132L (2) F132V L135P Fig. 2 A diagram illustrating the inheritance of ABCC8 mutations in probands with permanent and transient forms of neonatal diabetes. Login to comment
163 Permanent Neonatal Diabetes Mellitus Transient Neonatal Diabetes Mellitus 1 5 10 15 20 25 30 35 39 N72S V86A V86G F132L F132V L135PP45L P207S E208K D209E Q211K L213R L225P T229I Y263D D209E D212I D212N T229I R306H V324M L438F L451P E382K R826W R1183W R1183Q A1185E E1327K R1314H M1290V R1380C R1380H R1380L G1401R V1523A V1523L H1024YC435R L582V I1425V Fig. 3 The location of missense mutations causing neonatal diabetes within the coding sequence of ABCC8. Login to comment
171 The patient with developmental delay, epilepsy and neonatal diabetes (DEND) syndrome and the F132L mutation was not able to discontinue insulin [17]. Login to comment
175 A second patient with the same mutation (F132L) had developmental delay but no epilepsy. Login to comment
176 No neurological features were reported in R1183W/Q A1185E E1327K G1401R V1523A/L NBD1 NBD2 outside membrane inside P45L N72S F132L/V L135P P207S E208K D209E Q211K D212I/N L213R L225P T229I Y263D E382K V86A/G L438F C435R R1380C/H/L L451P R826W TMD0 TMD1 TMD2 R306H V324M L582V H1024Y I1425V R1314H M1290V Fig. 4 A schematic of the membrane topologies of SUR1 showing the location of the ABCC8 missense mutations causing neonatal diabetes. Login to comment
197 Genotype-phenotype Correlation Most of the dominantly acting mutations located in exons 2-5 of the ABCC8 gene (V86A/G, F132L/V, L135P, D209E, Q211K, L213R and L225P) cause PNDM. Login to comment
224 Furthermore, different mutations at the same residue (V86A/G, F132L/V, D212I/N, R1183Q/W and R1380C/H/L) cause either PNDM (V86 and F132) or biphasic TNDM (D212, R1183 and R1380), suggesting a different pathological mechanism. Login to comment
227 The cluster of neonatal diabetes causing mutations in the first five exons of the ABCC8 gene that encode these regions might cause diabetes by increasing the open stability of the channel through interaction with the Kir6.2 subunit as demonstrated for the F132L mutation [17]. Login to comment
247 Functional data have only been published for 8/39 ABCC8 missense mutations to date (F132L [16]; I1425V and H1024Y [13]; mutations (P207S, T229I, A1185E and V1523L [14]; L225P [16]). 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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4 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. Login to comment
98 These observations suggest that activation by distinct pathways converges toward the same Rho phenotype, which we have shown to reflect a particular conformation of the SUR/Kir6.2 interface. The Mutation F132L in SUR1 Induces a Rho-resistant Phenotype- The mutation F132L in TMD0 of SUR1 is responsible for neonatal diabetes (15). Login to comment
109 In the same study, the F132L mutation was shown by co-immunoprecipitation to diminish FIGURE 1. Login to comment
118 It remains that, when probed with the Rho protection assay, the F132L mutation had distinct effects on full-length SUR1 and on the isolated TMD0 domain. Login to comment
121 In conclusion, the mutation F132L in SUR1 produces a Rho-resistant phenotype like the activators MgADP, zinc, or SR47063. Login to comment
123 This suggests that the mutation F132L mimics the presence of openers and causes channel hyperactivity by inducing a conformational change that alters the TMD0/Kir6.2 interface. The Mutation E126A in SUR2A Induces a Rho-sensitive Phenotype-Residue Phe132 is located in the predicted second intracellular loop of SUR1 (24). 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
133 As for the F132L mutation, we attempted to test the effect of the mutation E126A on TMD0 of SUR2A alone but were unable to record sufficient channel activity from oocytes co-expressing TMD0E126AϩKir6.2⌬C36, thus precluding any valid characterization. Login to comment
173 The neonatal diabetes F132L mutation in SUR1 leads to a constitutive Rho-insensitive conformation, similar to that of the TMD0 ؉ Kir6.2 channel. Login to comment
174 A and B, currents recorded in inside-out patches from Xenopus oocytes co-expressing Kir6.2 and either wild-type SUR1 (A) or the F132L mutant (B). 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
214 This suggests that although the F132L and E126A mutations lock the TMD0-Kir6.2 interface in distinct conformations where the Rho-binding site is either always masked or always accessible, other regions of SUR are unaffected and can interact with Kir6.2 to modulate its gating. 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
225 This suggests that the conformation of TMD0 in all these cases: F132L, TMD0 alone, and activators, could be similar. Login to comment
226 Thus, the channel hyperactivity caused by the F132L mutation would reflect the stabilization of TMD0 in the same activated conformation that is triggered by activators. Login to comment
100 These observations suggest that activation by distinct pathways converges toward the same Rho phenotype, which we have shown to reflect a particular conformation of the SUR/Kir6.2 interface. The Mutation F132L in SUR1 Induces a Rho-resistant Phenotype- The mutation F132L in TMD0 of SUR1 is responsible for neonatal diabetes (15). Login to comment
111 In the same study, the F132L mutation was shown by co-immunoprecipitation to diminish FIGURE 1. Login to comment
120 It remains that, when probed with the Rho protection assay, the F132L mutation had distinct effects on full-length SUR1 and on the isolated TMD0 domain. 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
135 As for the F132L mutation, we attempted to test the effect of the mutation E126A on TMD0 of SUR2A alone but were unable to record sufficient channel activity from oocytes co-expressing TMD0E126Aaf9;Kir6.2èc;C36, thus precluding any valid characterization. Login to comment
175 The neonatal diabetes F132L mutation in SUR1 leads to a constitutive Rho-insensitive conformation, similar to that of the TMD0 &#2d19; Kir6.2 channel. Login to comment
176 A and B, currents recorded in inside-out patches from Xenopus oocytes co-expressing Kir6.2 and either wild-type SUR1 (A) or the F132L mutant (B). 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
192 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
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
227 This suggests that the conformation of TMD0 in all these cases: F132L, TMD0 alone, and activators, could be similar. Login to comment
228 Thus, the channel hyperactivity caused by the F132L mutation would reflect the stabilization of TMD0 in the same activated conformation that is triggered by activators. Login to comment
99 These observations suggest that activation by distinct pathways converges toward the same Rho phenotype, which we have shown to reflect a particular conformation of the SUR/Kir6.2 interface. The Mutation F132L in SUR1 Induces a Rho-resistant Phenotype- The mutation F132L in TMD0 of SUR1 is responsible for neonatal diabetes (15). Login to comment
110 In the same study, the F132L mutation was shown by co-immunoprecipitation to diminish FIGURE 1. Login to comment
119 It remains that, when probed with the Rho protection assay, the F132L mutation had distinct effects on full-length SUR1 and on the isolated TMD0 domain. Login to comment
122 In conclusion, the mutation F132L in SUR1 produces a Rho-resistant phenotype like the activators MgADP, zinc, or SR47063. Login to comment
134 As for the F132L mutation, we attempted to test the effect of the mutation E126A on TMD0 of SUR2A alone but were unable to record sufficient channel activity from oocytes co-expressing TMD0E126Aaf9;Kir6.2èc;C36, thus precluding any valid characterization. Login to comment
215 This suggests that although the F132L and E126A mutations lock the TMD0-Kir6.2 interface in distinct conformations where the Rho-binding site is either always masked or always accessible, other regions of SUR are unaffected and can interact with Kir6.2 to modulate its gating. Login to comment

[hide] Permanent neonatal diabetes due to activating muta... Rev Endocr Metab Disord. 2010 Sep;11(3):193-8. Edghill EL, Flanagan SE, Ellard S
Permanent neonatal diabetes due to activating mutations in ABCC8 and KCNJ11.
Rev Endocr Metab Disord. 2010 Sep;11(3):193-8., [PMID:20922570]

Abstract [show]
The ATP-sensitive potassium (K(ATP)) channel is composed of two subunits SUR1 and Kir6.2. The channel is key for glucose stimulated insulin release from the pancreatic beta cell. Activating mutations have been identified in the genes encoding these subunits, ABCC8 and KCNJ11, and account for approximately 40% of permanent neonatal diabetes cases. The majority of patients with a K(ATP) mutation present with isolated diabetes however some have presented with the Developmental delay, Epilepsy and Neonatal Diabetes syndrome. This review focuses on mutations in the K(ATP) channel which result in permanent neonatal diabetes, we review the clinical and functional effects as well as the implications for treatment.

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57 For example the R50Q KCNJ11 mutation and the F132V ABCC8 mutation cause isolated diabetes whilst the R50P and F132L mutations cause DEND syndrome [22, 27, 40]. Login to comment
85 One of the most notable R1183W/Q A1185E E1327K G1401R V1523A/L V1524M R1531A NBD1 NBD2 outside membrane inside P45L N72S F132L/V L135P P207S E208K D209E Q211K D212I/N L213R L225P T229I Y263D A269D/N E382K V86A/G R1380C/H/L C435R L438F M1290V L451P R826W R1314H TMD0 TMD1 TMD2 R306H V324M L582V H1024Y I1425V A90V Y356C R521Q N1123D R1153G T1043TfsX74 Fig. 3 Schematic representation of 50 ABCC8 mutations which cause neonatal diabetes. Login to comment

[hide] Review. SUR1: a unique ATP-binding cassette protei... Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):257-67. Aittoniemi J, Fotinou C, Craig TJ, de Wet H, Proks P, Ashcroft FM
Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator.
Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):257-67., [PMID:18990670]

Abstract [show]
SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

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98 Co-immunoprecipitation (but not necessarily surface trafficking) is abolished by mutation of F132L in CL2 (Proks et al. 2006a) and of Y195E at the start of CL3 (figure 2). Login to comment
102 For example, the F132L mutation in SUR1 disrupts the physical binding of Kir6.2 and TMD0 (Proks et al. 2007). Login to comment
104 This indicates that the F132L mutation must disrupt an inhibitory interaction between Kir6.2 and TMD0, leaving a stimulatory interaction intact. Login to comment
169 Only one mutation (F132L) has been shown to act this way to date (Proks et al. 2006a, 2007). Login to comment
171 The F132L mutation increases the duration of the bursts of the KATP channel openings and reduces the frequency and duration of the interburst closed states. Login to comment
185 Such naturally occurring mutations TNDM PNDM DEND TNDM PNDM DEND iDEND WT P206L D212N P45L N72S P207S E208K+Y263D D212I T229I A1185E V1522L+Y229I F132L 0 0.05 0.10 0.15 fractionofcurrentremaining in3mMMgATP(a) (b) (i) (ii) Figure 4. Login to comment
204 (a) (b) P45L N72S F132L NH2 A90V V86G COOHL135P exoplasmic cytoplasmic Walker A Walker A linker Walker B linker Walker B V324M E382K C435R L438F L582V R826W H1023Y N1122D R1183Q A1185E R1314H E1327K R1380 L I1425V V1524 L P207S E208K Q211K D212I/N L225P T229I Y263D A269D R306H D209E L213R TMD0 TMD1 TMD2 NBD1 NBD2 CL3 linker site 1 site 2 NBD1 NBD2 R826W R1380 L E1327K I1425V V1524 L Figure 5. Login to comment
97 Co-immunoprecipitation (but not necessarily surface trafficking) is abolished by mutation of F132L in CL2 (Proks et al. 2006a) and of Y195E at the start of CL3 (figure 2). Login to comment
101 For example, the F132L mutation in SUR1 disrupts the physical binding of Kir6.2 and TMD0 (Proks et al. 2007). Login to comment
103 This indicates that the F132L mutation must disrupt an inhibitory interaction between Kir6.2 and TMD0, leaving a stimulatory interaction intact. Login to comment
172 Only one mutation (F132L) has been shown to act this way to date (Proks et al. 2006a, 2007). Login to comment
174 The F132L mutation increases the duration of the bursts of the KATP channel openings and reduces the frequency and duration of the interburst closed states. Login to comment
188 Such naturally occurring mutations TNDM PNDM DEND TNDM PNDM DEND iDEND WT P206L D212N P45L N72S P207S E208K+Y263D D212I T229I A1185E V1522L+Y229I F132L 0 0.05 0.10 0.15 fraction of current remaining in 3 mM MgATP (a) (b) (i) (ii) Figure 4. Login to comment
207 (a) (b) P45L N72S F132L NH2 A90V V86G COOH L135P exoplasmic cytoplasmic Walker A Walker A linker Walker B linker Walker B V324M E382K C435R L438F L582V R826W H1023Y N1122D R1183Q A1185E R1314H E1327K R1380 L I1425V V1524 L P207S E208K Q211K D212I/N L225P T229I Y263D A269D R306H D209E L213R TMD0 TMD1 TMD2 NBD1 NBD2 CL3 linker site 1 site 2 NBD1 NBD2 R826W R1380 L E1327K I1425V V1524 L Figure 5. Login to comment

[hide] A heterozygous activating mutation in the sulphony... Hum Mol Genet. 2006 Jun 1;15(11):1793-800. Epub 2006 Apr 13. Proks P, Arnold AL, Bruining J, Girard C, Flanagan SE, Larkin B, Colclough K, Hattersley AT, Ashcroft FM, Ellard S
A heterozygous activating mutation in the sulphonylurea receptor SUR1 (ABCC8) causes neonatal diabetes.
Hum Mol Genet. 2006 Jun 1;15(11):1793-800. Epub 2006 Apr 13., [PMID:16613899]

Abstract [show]
Neonatal diabetes is a genetically heterogeneous disorder with nine different genetic aetiologies reported to date. Heterozygous activating mutations in the KCNJ11 gene encoding Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, are the most common cause of permanent neonatal diabetes. The sulphonylurea receptor (SUR) SUR1 serves as the regulatory subunit of the K(ATP) channel in pancreatic beta cells. We therefore hypothesized that activating mutations in the ABCC8 gene, which encodes SUR1, might cause neonatal diabetes. We identified a novel heterozygous mutation, F132L, in the ABCC8 gene of a patient with severe developmental delay, epilepsy and neonatal diabetes (DEND syndrome). This mutation had arisen de novo and was not present in 150 control chromosomes. Residue F132 shows evolutionary conservation across species and is located in the first set of transmembrane helices (TMD0) of SUR1, which is proposed to interact with Kir6.2. Functional studies of recombinant K(ATP) channels demonstrated that F132L markedly reduces the sensitivity of the K(ATP) channel to inhibition by MgATP and this increases the whole-cell K(ATP) current. The functional consequence of this ABCC8 mutation mirrors that of KCNJ11 mutations causing neonatal diabetes and provides new insights into the interaction of Kir6.2 and SUR1. As SUR1 is expressed in neurones as well as in beta cells, this mutation can account for both neonatal diabetes and the neurological phenotype. Our results demonstrate that SUR1 mutations constitute a new genetic aetiology for neonatal diabetes and that they act by reducing the K(ATP) channel's ATP sensitivity.

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4 We identified a novel heterozygous mutation, F132L, in the ABCC8 gene of a patient with severe developmental delay, epilepsy and neonatal diabetes (DEND syndrome). Login to comment
7 Functional studies of recombinant KATP channels demonstrated that F132L markedly reduces the sensitivity of the KATP channel to inhibition by MgATP and this increases the whole-cell KATP current. Login to comment
55 One patient (ISPAD68) was heterozygous for a novel mutation (F132L) in the ABCC8 gene encoding SUR1. Login to comment
58 The novel F132L mutation results in the substitution of leucine for phenylalanine at residue 132 (p.Phe132Leu) in exon 3 of the ABCC8 gene (c.394T.C; Fig. 1A). Login to comment
60 The F132L mutation was not found in 150 normal chromosomes. Login to comment
66 The region on either side of F132L is also highly conserved (Fig. 1B). Login to comment
68 Clinical characteristics of patient with F132L SUR1 mutation ISPAD68 was a boy born in 1978 with a birth weight of 2200 g to a 29-year-old mother who had previously given birth to two healthy older children. Login to comment
74 F132L mutation, conservation of F132 across species and location of F132L in SUR1. Login to comment
75 (A) Sequencing of ISPAD68 showing a heterozygous mutation (c.394T.C) resulting in the substitution of phenylalanine (TTC) by leucine (CTC) at residue 132 (F132L) of the ABCC8 gene (arrow). Login to comment
81 (C) Schematic of the proposed membrane topologies of SUR1 and Kir6.2 showing the location of F132L (arrowed) in SUR1. Login to comment
104 SUR1 mutant KATP channels are not closed by resting ATP levels To analyse the functional effects of the SUR1-F132L mutation, we studied recombinant KATP channels expressed in Xenopus oocytes. Login to comment
107 In contrast, significant whole-cell Kþ currents were present in the absence of metabolic inhibition (resting currents) in oocytes expressing either homomeric or heterozygous SUR1-F132L mutant channels (Fig. 2). Login to comment
112 SUR1-F132L KATP channels have reduced ATP sensitivity To explore the molecular basis of the reduced metabolic sensitivity of mutant KATP channels, we tested the ability of ATP to block wild-type and mutant channels in inside-out patches. Login to comment
115 (A) Whole-cell currents recorded from Xenopus oocytes coexpressing Kir6.2 and either SUR1 (WT) or SUR1-F132L (homF132L) or both SUR1 and SUR1-F132L (hetF132L) in response to voltage steps of +20 mV from a holding potential of 210 mV. Login to comment
126 These results indicate that the F132L mutation in SUR1 markedly reduces the ability of ATP to inhibit the KATP channel. Login to comment
127 DISCUSSION We report a novel heterozygous mutation, F132L, in the ABCC8 gene encoding SUR1 in a patient with DEND syndrome. Login to comment
130 Genetic evidence for the pathogenicity of the F132L mutation is strong; it is a spontaneous mutation, absent from 150 normal chromosomes and affects a residue that shows evolutionary conservation across species. Login to comment
131 Functional studies also support the pathogenicity of the F132L mutation by revealing that the mutation dramatically reduces the inhibitory potency of MgATP. Login to comment
139 Our results suggest that this physical interaction may be mediated, in part, via the second cytosolic loop of SUR1 and that the F132L mutation influences the interaction of SUR1 with Kir6.2. Login to comment
143 (A) KATP currents recorded in inside-out patches excised from oocytes coexpressing Kir6.2 and either SUR1 or SUR1-F132L (Kir6.2/SUR1-homF132L) or both SUR1 and SUR1-F132L (Kir6.2/SUR1-hetF132L). Login to comment
147 (B) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2/SUR1 (open circles, n ¼ 6), and heterozygous (solid circles, n ¼ 6) or homomeric (solid squares, n ¼ 7) Kir6.2/SUR1-F132L channels. Login to comment
154 The F132L mutation causes a dramatic decrease in the ability of ATP to inhibit the activity of the KATP channel in both the homozygous and simulated heterozygous states. Login to comment
170 Predicted response to sulphonylurea treatment In the presence of the sulphonylurea tolbutamide (0.5 mmol/l), heterozygous F132L SUR1 channels were blocked by 72%. Login to comment
178 Given the location of residue F132 in TMD0, a region known to regulate KATP channel gating (26,27), it seems reasonable to speculate that the F132L mutation acts by stabilizing the channel open state, thus indirectly reducing its ATP sensitivity. Login to comment
186 Functional analysis demonstrates that the F132L SUR1 mutation acts in a similar way to Kir6.2 mutations causing DEND syndrome by producing a marked reduction in the ability of ATP to block the KATP channel. Login to comment

[hide] Effective treatment with oral sulfonylureas in pat... Diabetes Care. 2008 Feb;31(2):204-9. Epub 2007 Nov 19. Rafiq M, Flanagan SE, Patch AM, Shields BM, Ellard S, Hattersley AT
Effective treatment with oral sulfonylureas in patients with diabetes due to sulfonylurea receptor 1 (SUR1) mutations.
Diabetes Care. 2008 Feb;31(2):204-9. Epub 2007 Nov 19., [PMID:18025408]

Abstract [show]
OBJECTIVE: Neonatal diabetes can result from mutations in the Kir6.2 or sulfonylurea receptor 1 (SUR1) subunits of the ATP-sensitive K(+) channel. Transfer from insulin to oral sulfonylureas in patients with neonatal diabetes due to Kir6.2 mutations is well described, but less is known about changing therapy in patients with SUR1 mutations. We aimed to describe the response to sulfonylurea therapy in patients with SUR1 mutations and to compare it with Kir6.2 mutations. RESEARCH DESIGN AND METHODS: We followed 27 patients with SUR1 mutations for at least 2 months after attempted transfer to sulfonylureas. Information was collected on clinical features, treatment before and after transfer, and the transfer protocol used. We compared successful and unsuccessful transfer patients, glycemic control before and after transfer, and treatment requirements in patients with SUR1 and Kir6.2 mutations. RESULTS: Twenty-three patients (85%) successfully transferred onto sulfonylureas without significant side effects or increased hypoglycemia and did not need insulin injections. In these patients, median A1C fell from 7.2% (interquartile range 6.6-8.2%) on insulin to 5.5% (5.3-6.2%) on sulfonylureas (P = 0.01). When compared with Kir6.2 patients, SUR1 patients needed lower doses of both insulin before transfer (0.4 vs. 0.7 units x kg(-1) x day(-1); P = 0.002) and sulfonylureas after transfer (0.26 vs. 0.45 mg x kg(-1) x day(-1); P = 0.005). CONCLUSIONS: Oral sulfonylurea therapy is safe and effective in the short term in most patients with diabetes due to SUR1 mutations and may successfully replace treatment with insulin injections. A different treatment protocol needs to be developed for this group because they require lower doses of sulfonylureas than required by Kir6.2 patients.

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54 Doses Table 1-Clinical characteristics of patients with SUR1 mutations according to success of treatment with sulfonylureas Characteristic All patients Patients with successful sulfonylurea treatment Patients with unsuccessful sulfonylurea treatment P* n 27 23 4 Mutation (number of patients) NA V86G†, P45L/G1401R- (2)†, D209E (3)†, T229I/V1523L†, Q211K†, V86A (2)†, E1507G, V215I/V607M, E208K/Y263D†, R1380L (2)‡, D212I (3)§, T229I/T229I‡, R1183W§, L225P†, R826W, and D209N F132L (2)†, F132V†, and N72S† (mosaic). Login to comment
75 Two of these patients with F132V and F132L mutations had increased C-peptide levels following the transfer, but it was decided that the response was insufficient to discontinue insulin. Login to comment
56 Doses Table 1-Clinical characteristics of patients with SUR1 mutations according to success of treatment with sulfonylureas Characteristic All patients Patients with successful sulfonylurea treatment Patients with unsuccessful sulfonylurea treatment P* n 27 23 4 Mutation (number of patients) NA V86Gߤ, P45L/G1401R- (2)ߤ, D209E (3)ߤ, T229I/V1523Lߤ, Q211Kߤ, V86A (2)ߤ, E1507G, V215I/V607M, E208K/Y263Dߤ, R1380L (2)ߥ, D212I (3)&#a7;, T229I/T229Iߥ, R1183W&#a7;, L225Pߤ, R826W, and D209N F132L (2)ߤ, F132Vߤ, and N72Sߤ (mosaic). Login to comment
77 Two of these patients with F132V and F132L mutations had increased C-peptide levels following the transfer, but it was decided that the response was insufficient to discontinue insulin. Login to comment

[hide] Mechanism of KATP hyperactivity and sulfonylurea t... FEBS Lett. 2011 Nov 16;585(22):3555-9. Epub 2011 Oct 19. Babenko AP, Vaxillaire M
Mechanism of KATP hyperactivity and sulfonylurea tolerance due to a diabetogenic mutation in L0 helix of sulfonylurea receptor 1 (ABCC8).
FEBS Lett. 2011 Nov 16;585(22):3555-9. Epub 2011 Oct 19., [PMID:22020219]

Abstract [show]
Activating mutations in different domains of the ABCC8 gene-coded sulfonylurea receptor 1 (SUR1) cause neonatal diabetes. Here we show that a diabetogenic mutation in an unexplored helix preceding the ABC core of SUR1 dramatically increases open probability of (SUR1/Kir6.2)(4) channel (KATP) by reciprocally changing rates of its transitions to and from the long-lived, inhibitory ligand-stabilized closed state. This kinetic mechanism attenuates ATP and sulfonylurea inhibition, but not Mg-nucleotide stimulation, of SUR1/Kir6.2. The results suggest a key role for L0 helix in KATP gating and together with previous findings from mutant KATP clarify why many patients with neonatal diabetes require high doses of sulfonylureas.

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14 Consistent with our hypothesis, the diabetogenic F132L in TMD0 of SUR1 increased KATP activity in the absence of nucleotides [15]. Login to comment
93 Consistent with the proposal, F132L in TMD0 and activating mutations in M0 alter intrinsic gating [15,39,40]. Login to comment

[hide] Mechanism of action of a sulphonylurea receptor SU... Hum Mol Genet. 2007 Aug 15;16(16):2011-9. Epub 2007 Jun 21. Proks P, Shimomura K, Craig TJ, Girard CA, Ashcroft FM
Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome.
Hum Mol Genet. 2007 Aug 15;16(16):2011-9. Epub 2007 Jun 21., [PMID:17584766]

Abstract [show]
Activating mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. Here, we analyse the molecular mechanism of action of the heterozygous mutation F132L, which lies in the first set of transmembrane helices (TMD0) of SUR1. This mutation causes severe developmental delay, epilepsy and permanent neonatal diabetes (DEND syndrome). We show that the F132L mutation reduces the ATP sensitivity of K(ATP) channels indirectly, by altering the intrinsic gating of the channel. Thus, the open probability is markedly increased when Kir6.2 is co-expressed with mutant TMD0 alone or with mutant SUR1. The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Our results explain how a mutation in an accessory subunit can produce enhanced activity of the K(ATP) channel pore (formed by Kir6.2). They also provide further evidence that interactions between TMD0 of SUR1 and Kir6.2 are critical for K(ATP) channel gating and identify a residue crucial for this interaction at both physical and functional levels.

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0 Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome Peter Proks{ , Kenju Shimomura{ , Tim J. Craig, Heidi de Wet, Christophe A.J. Girard and Frances M. Ashcroft* University Laboratory of Physiology, Oxford University, Oxford OX1 3PT, UK Received April 23, 2007; Revised and Accepted June 10, 2007 Activating mutations in the genes encoding the ATP-sensitive potassium (KATP) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. Login to comment
1 Here, we analyse the molecular mechanism of action of the heterozygous mutation F132L, which lies in the first set of transmembrane helices (TMD0) of SUR1. Login to comment
3 We show that the F132L mutation reduces the ATP sensitivity of KATP channels indirectly, by altering the intrinsic gating of the channel. Login to comment
5 The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Login to comment
33 Recently, we identified the first gain-of-function mutation in SUR1 (F132L) that causes DEND syndrome and showed that it exhibits reduced inhibition by MgATP (17). Login to comment
44 This suggests that mutations in F132L associated with ND may influence the sensitivity of the channel to MgATP indirectly, by altering the single-channel kinetics. Login to comment
47 In this paper, we examine the molecular mechanism by which the F132L mutation in SUR1 influences KATP channel inhibition by ATP. Login to comment
50 RESULTS F132L alters the intrinsic gating of KATP channels The aim of this paper is to determine the molecular mechanism by which the F132L mutation reduces the ATP sensitivity of the KATP channel. Login to comment
52 We first compared the effect of the F132L mutation in SUR1 on the kinetics of single KATP channel currents. Login to comment
55 The F132L mutation dramatically increased the burst duration and reduced the time spent in the interburst intervals. Login to comment
58 The intrinsic open probability (PO) of SUR1-F132L channels was significantly greater (P , 0.05) than that of wild-type channels, being 0.72 + 0.03 (n ¼ 8) for SUR1-F132L, compared with 0.26 + 0.03 (n ¼ 6) for SUR1 channels. Login to comment
59 These differences in channel kinetics suggest that the SUR1-F132L mutation influences KATP channel ATP sensitivity indirectly, via changes in channel gating, as is found for some Kir6.2 mutations. Login to comment
60 Because all patients carrying the F132L mutation are heterozygotes, their pancreatic beta-cells will contain a mixture of wild-type and mutant SUR1. Login to comment
64 Because the channels in the heterozygous population will have different PO, we compared the mean PO of the heterozygous channel population with that of homomeric F132L channels (see Materials and Methods for details). Login to comment
69 Single-channel currents recorded at 260 mV from inside-out membrane patches excised from oocytes expressing Kir6.2/SUR1, Kir6.2/ SUR1-F132L, Kir6.2DC, Kir6.2DC/TMD0 and Kir6.2DC/TMD0-F132L. Login to comment
72 There was a further increase in PO when the F132L mutation was introduced into TMD0 (to 0.82 + 0.02; n ¼ 6). Login to comment
73 These results confirm that the first five transmembrane domains of SUR1 modulate the gating of Kir6.2 (22-24) and show that the F132L mutation enhances this effect. Login to comment
74 Effects of the F132L mutation on the ATP sensitivity of SUR1 channels To explore the effects of the F132L mutation further, we compared the ATP sensitivity of KATP channels composed of Kir6.2 and either wild-type or mutant SUR1. Login to comment
78 The ATP sensitivity of SUR1-F132L mutant channels was further decreased in the presence of 2 mM Mg2+ (Fig. 2B). Login to comment
84 Effects of F132L mutation on the nucleotide activation Because MgATP and MgADP interact with both the NBDs of SUR1, as well as with Kir6.2, it is not easy to separate the stimulatory (via SUR1) and inhibitory (via Kir6.2) effects of these nucleotides. Login to comment
87 A potential problem, however, is that F132L channels have a high intrinsic open probability, which makes it difficult to detect whether Mg-nucleotides cause channel activation. Login to comment
92 Top: KATP currents recorded in response to successive voltage ramps from 2110 to +100 mV in an inside-out patch excised from an oocyte expressing Kir6.2/SUR1 or homKir6.2/ SUR1-F132L channels, as indicated. Login to comment
94 Bottom: (A) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2/ SUR1 (open circle, n = 6), and heterozygous (filled circle, n = 6) or homomeric (filled square, n = 12) Kir6.2/SUR1-F132L channels. Login to comment
98 (B) Mean relationship between [MgATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2/SUR1 (open circle, n = 7), and heterozygous (filled circle, n = 7) or homomeric (filled square, n = 10) Kir6.2/SUR1-F132L channels. Login to comment
107 Thus, these data suggest that MgGDP activation may be enhanced by the F132L mutation in SUR1. Login to comment
109 Effects of F132L mutation on the ATP sensitivity of TMD0 channels We next compared the ATP sensitivity of Kir6.2DC/TMD0 channels with heterozygous and homomeric TMD0-F132L channels (Fig. 4). Login to comment
113 The F132L mutation further reduced the ATP sensitivity of Kir6.2DC/TMD0 channels (Fig. 4A): the IC50 for ATP inhibition was 2.1 and 4.2 mM for hetTMD0-F132L and homTMD0-F132L channels, respectively (Table 1). Login to comment
115 This was not only true for wild-type TMD0, but also for hetTMD0-F132L and homTMD0-F132L (Table 1). Login to comment
118 The pedestal was of similar magnitude in both the absence and presence of Mg2+ , being 10% for wild-type channel, 25% for hetTMD0-F132L and 40% for homTMD0-F132L channels at 10 mM ATP. Login to comment
119 The F132L mutation disrupts the interaction between TMD0 and Kir6.2 Our results suggest that the F132L mutation may influence KATP channel gating by altering the interaction between SUR1 and Kir6.2. Login to comment
122 Xenopus oocytes were co-injected with Kir6.2DC-HA and either wild-type TMD0, TMD0-F132L or a mixture of both (to simulate heterozygosity). Login to comment
124 The F132L mutation reduced the binding of TMD0 to Kir6.2 by 90% (P , 0.001), indicating that F132 is crucial for this interaction. Login to comment
125 Heterozygous expression of TMD0-F132L led to an 75% reduction in binding (Fig. 5A), but this was not significantly different from that of homTMD0-F132L (P ¼ 0.28). Login to comment
128 Mean hetKir6.2/SUR1-F132L and homKir6.2/SUR1-F132L currents recorded in the presence of 200 mM ATPgAA and 200 mM ATPgAA+100 mM MgGDP, as indicated. Login to comment
132 Values of IC50 for ATP inhibition of various KATP channels in both Mg2þ -containing and Mg2þ -free solutions Channel IC50 Mg2þ -free 2 mM Mg2þ Kir6.2/SUR1 7 + 1 mM (n ¼ 7) 14 + 1 mM (n ¼ 7) hetKir6.2/SUR1-F132L 30 + 1 mM (n ¼ 7) 122 + 23 mM (n ¼ 8) homKir6.2/SUR1-F132L 51 + 7 mM (n ¼ 8) 910 + 180 mM (n ¼ 10) Kir6.2DC/TMD0 700 + 65 mM (n ¼ 6) 603 + 32 mM (n ¼ 6) hetKir6.2DC/TMD0-F132L 2.10 + 0.16 mM (n ¼ 6) 2.57 + 0.13 mM (n ¼ 6) homKir6.2DC/TMD0-F132L 4.20 + 1.05 mM (n ¼ 6) 6.05 + 0.94 mM (n ¼ 6) A possible explanation for the reduced physical interaction between TMD0 and SUR1 produced by the F132L mutation is that the interaction is state-dependent, occurring only when the channel is in the closed state. Login to comment
139 Thus, we next examined whether the F132L mutation altered surface expression of the KATP channel complex. Login to comment
141 Co-expression with SUR1/TMD0 enhanced surface expression of both Kir6.2 and Kir6.2DC: however, this was unaffected by the F132L mutation (Fig. 6). Login to comment
142 DISCUSSION Molecular mechanism of action Single-channel analysis revealed that the F132L mutation dramatically enhances the burst duration and the intrinsic open probability (PO) of both homomeric Kir6.2/ SUR1-F132L and Kir6.2/TMD0-F132L channels. Login to comment
143 Thus, F132L is a gating mutation that alters the ATP-sensitivity of the KATP channel indirectly, via stabilization of the channel open state (30,34). Login to comment
149 Our results further show that the F132L mutation impairs this physical association. Login to comment
152 Top: KATP currents recorded at 260 mV in an inside-out patch excised from an oocyte expressing Kir6.2DC/TMD0 or homozygous Kir6.2DC/TMD0-F132L channels. Login to comment
154 Bottom: (A) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2DC /TMD0 (open circle, n = 6) and heterozygous (filled circle, n = 6) or homomeric (filled square, n = 6) Kir6.2DC /TMD0-F132L channels. Login to comment
158 (B) Mean relationship between [MgATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2DC /TMD0 (open circle, n = 5) and heterozygous (filled circle, n = 7) or homomeric (filled square, n = 10) Kir6.2DC /TMD0-F132L channels. Login to comment
165 Because the F132L mutation markedly affected channel gating, whether in TMD0 or full-length SUR1, the physical association of these two subunits may be required for the modulation of Kir6.2 kinetics by SUR1. Login to comment
169 Because the F132L mutation increases the PO of Kir6.2/TMD0 channels even further, it appears that there may be an additional inhibitory effect of TMD0 that is abolished by mutation of F132. Login to comment
175 Although we cannot rule out an allosteric effect, the fact the F132L mutation greatly reduces the physical interaction between Kir6.2 and TMD0 suggests that the intracellular loop within which F132 lies must be in close proximity to the cytoplasmic domains of Kir6.2. Login to comment
176 Heterozygosity and nucleotide sensitivity Our results demonstrate that both the ATP and MgATP concentration-inhibition curves for heterozygous Kir6.2/ SUR1-F132L channels are intermediate between those of wild-type and homKir6.2/SUR1-F132L channels. Login to comment
178 (A) Co-immunoprecipitation of FLAG-tagged TMD0 and Kir6.2DC, using wild-type or F132L TMD0 and wild-type Kir6.2DC. Login to comment
192 Mg2+ produced a dramatic reduction in the ATP sensitivity of homKir6.2/SUR1-F132L and hetKir6.2/SUR1-F132L channels. Login to comment
195 This suggests that the mechanism by which nucleotide binding/hydrolysis at the NBDs of SUR1 is translated in opening of the Kir6.2 pore is also enhanced by the F132L mutation. Login to comment
204 It is not yet clear if there is an overlap between the ATP sensitivity of the two phenotypes (DEND and I-DEND) and whether additional factors present in patients may further enhance the severity of the syndrome caused by the F132L mutation. Login to comment
248 Co-immunoprecipitation of TMD0 and Kir6.2 Oocytes were injected with 5 ng of Kir6.2DC mRNA and 5 ng of either TMD0, mutant TMD0 (F132L or C166S) or a 50:50 mix of wild-type and mutant TMD0 mRNAs. Login to comment

[hide] Update of mutations in the genes encoding the panc... Hum Mutat. 2009 Feb;30(2):170-80. Flanagan SE, Clauin S, Bellanne-Chantelot C, de Lonlay P, Harries LW, Gloyn AL, Ellard S
Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism.
Hum Mutat. 2009 Feb;30(2):170-80., [PMID:18767144]

Abstract [show]
The beta-cell ATP-sensitive potassium (K(ATP)) channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis it is therefore not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1 (SUR1). It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinism of infancy, while activating mutations in KCNJ11 and ABCC8 have recently been described that result in the opposite phenotype of diabetes. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment on diagnosing patients with mutations in these genes.

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121 Syndromic PNDM A total of 10 activating KCNJ11 mutations (R50P, R50G, Q52R, G53D, V59G, C166F, C166Y, I167L, I296L, and G334D) and one ABCC8 mutation (F132L) have been reported that result in a severe phenotype of development delay, epilepsy, and neonatal diabetes, termed DEND syndrome [Gloyn et al., 2004b, 2006; Flanagan et al., 2006; Proks et al., 2006a; Masia et al., 2007b; Shimomura et al., 2007; Suzuki et al., 2007]. Login to comment

[hide] Mutations in the ABCC8 (SUR1 subunit of the K(ATP)... Clin Endocrinol (Oxf). 2009 Sep;71(3):358-62. Epub 2008 Nov 18. Klupa T, Kowalska I, Wyka K, Skupien J, Patch AM, Flanagan SE, Noczynska A, Arciszewska M, Ellard S, Hattersley AT, Sieradzki J, Mlynarski W, Malecki MT
Mutations in the ABCC8 (SUR1 subunit of the K(ATP) channel) gene are associated with a variable clinical phenotype.
Clin Endocrinol (Oxf). 2009 Sep;71(3):358-62. Epub 2008 Nov 18., [PMID:19021632]

Abstract [show]
OBJECTIVE: Mutations in the ABCC8 gene encoding the SUR1 subunits of the beta-cell K-ATP channel cause neonatal diabetes (ND) mellitus. We aimed to determine the contribution of ABCC8 gene to ND in Poland, to describe the clinical phenotype associated with its mutations and to examine potential modifying factors. PATIENTS: The Nationwide Registry of ND in Poland includes patients diagnosed before 6 months of age. In total 16 Kir6.2 negative patients with ND, 14 permanent and 2 relapsed transient, were examined. MEASUREMENTS: ABCC8 gene mutations were detected by direct sequencing. Mutation carriers' characteristics included clinical data and biochemical parameters. In addition, we performed the hyperinsulinaemic euglycaemic clamp and tested for islet-specific antibodies in diabetic subjects. RESULTS: We identified two probands with permanent ND (one heterozygous F132V mutation carrier and one compound heterozygote with N23H and R826W mutations) and two others with relapsed transient ND (heterozygotes for R826W and V86A substitutions, respectively). One subject, a heterozygous relative with the R826W mutation, had adult onset diabetes. There were striking differences in the clinical picture of the mutation carriers as the carrier of two mutations, N23H and R826W, was controlled on diet alone with HbA(1c) of 7.3%, whereas the F132V mutation carrier was on 0.66 IU/kg/day of insulin with HbA(1c) of 11.7%. The C-peptide level varied from 0.1 ng/ml (F132V) to 0.75 ng/ml (V86A). We also observed a variable insulin resistance, from moderate (M = 5.5 and 5.6 mg/kg/min, respectively, in the two R826W mutation carriers) to severe (M = 2.6 mg/kg/min in the F132V mutation carrier). We were able to transfer two patients off insulin to sulphonylurea (SU) and to reduce insulin dose in one other patient. Interestingly, there was no response to SU in the most insulin resistant F132V mutation carrier despite high dose of glibenclamide. All examined auto-antibodies were present in one of the subjects, the V86A mutation carrier, although this did not seem to influence the clinical picture, as we were able to transfer this girl off insulin. CONCLUSION: Mutations in SUR1 are the cause of about 15% of Kir6.2 negative permanent ND in Poland. The clinical phenotype of SUR1 diabetic mutation carriers is heterogeneous and it appears to be modified by variable sensitivity to insulin.

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66 The causative relationships between both de novo variants, V86A and F132V, and diabetic phenotype are evident, particularly as different mutations at these residues (F132L and V86G) were previously described in other cases of neonatal diabetes4,18 in addition to another case of PNDM with the V86A mutation.20 The R826W mutation was found in two families in this study but they are not known to share a common ancestor. Login to comment
80 Interestingly, both previously reported carriers of the mutation at this residue, F132L, showed some degree of developmental delay.18 A genotype/phenotype correlation, that includes the clinical picture and the response to the SU, has been reported for both KCNJ11 and ABCC8 mutations,22,23 although the correlation for Kir6Æ2 is strongest. Login to comment

[hide] A mutation in the TMD0-L0 region of sulfonylurea r... Diabetes. 2007 May;56(5):1357-62. Epub 2007 Feb 22. Masia R, De Leon DD, MacMullen C, McKnight H, Stanley CA, Nichols CG
A mutation in the TMD0-L0 region of sulfonylurea receptor-1 (L225P) causes permanent neonatal diabetes mellitus (PNDM).
Diabetes. 2007 May;56(5):1357-62. Epub 2007 Feb 22., [PMID:17317760]

Abstract [show]
OBJECTIVE: We sought to examine the molecular mechanisms underlying permanenent neonatal diabetes mellitus (PNDM) in a patient with a heterozygous de novo L225P mutation in the L0 region of the sulfonylurea receptor (SUR)1, the regulatory subunit of the pancreatic ATP-sensitive K(+) channel (K(ATP) channel). RESEARCH DESIGN AND METHODS: The effects of L225P on the properties of recombinant K(ATP) channels in transfected COS cells were assessed by patch-clamp experiments on excised membrane patches and by macroscopic Rb-flux experiments in intact cells. RESULTS: L225P-containing K(ATP) channels were significantly more active in the intact cell than in wild-type channels. In excised membrane patches, L225P increased channel sensitivity to stimulatory Mg nucleotides without altering intrinsic gating or channel inhibition by ATP in the absence of Mg(2+). The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. L225P did not alter channel inhibition by sulfonylurea drugs, and, consistent with this, the patient responded to treatment with oral sulfonylureas. CONCLUSIONS: L225P underlies K(ATP) channel overactivity and PNDM by specifically increasing Mg-nucleotide stimulation of the channel, consistent with recent reports of mechanistically similar PNDM-causing mutations in SUR1. The mutation does not affect sulfonylurea sensitivity, and the patient is successfully treated with sulfonylureas.

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149 Two reports have identified three SUR1 mutations associated with PNDM (F132L, L213R, and I1424V) and five associated with transient neonatal diabetes (11,12). Login to comment
150 Common to F132L and I1424V is an increased sensitivity of the channel to Mg nucleotides, such that channel overactivity results at physiological nucleotide concentrations. Login to comment
151 Our results are consistent with these previous studies, as L225P increases channel sensitivity to Mg nucleotides without altering intrinsic gating or inhibition by ATP. Login to comment
152 The effects of L225P are similar to those of the PNDM-associated I1424V mutation (12) but not as severe as those of the DEND (Developmental Delay, Epilepsy, and Neonatal Diabetes Syndrome)-associated F132L mutation (11). Login to comment
153 The effects of L225P are similar to those of the PNDM-associated I1424V mutation (12) but not as severe as those of the DEND (Developmental Delay, Epilepsy, and Neonatal Diabetes Syndrome)-associated F132L mutation (11). Login to comment

[hide] Permanent neonatal diabetes caused by dominant, re... Am J Hum Genet. 2007 Aug;81(2):375-82. Epub 2007 Jun 29. Ellard S, Flanagan SE, Girard CA, Patch AM, Harries LW, Parrish A, Edghill EL, Mackay DJ, Proks P, Shimomura K, Haberland H, Carson DJ, Shield JP, Hattersley AT, Ashcroft FM
Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects.
Am J Hum Genet. 2007 Aug;81(2):375-82. Epub 2007 Jun 29., [PMID:17668386]

Abstract [show]
Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.

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27 Apparent spontaneous mutations were confirmed by testing parental and proband DNA samples with use of a panel of six microsatellite markers on chromosome 11p15.11 Heterozygous de novo mutations V86A, V86G, F132L, F132V, D209E, Q211K, and L225P were present in eight patients (table 2). Login to comment
73 Details of ABCC8 Mutations and Clinical Information ISPAD Number Mutation (Protein Effect) Nucleotide Change Zygosity Age at Diagnosis (wk) Birth Weighta (Percentile) Neurological Feature Developmental Delay Muscle Weakness Epilepsy 123 V86Ab c.257TrC Heterozygous 8 2,900 (9) No No No 124 V86G c.257TrG Heterozygous 5 2,900 (13) No No No 68 F132Lb c.394TrC Heterozygous 13 2,200 (!1) Yes Yes Yes 125 F132L c.394TrC Heterozygous 26 2,440 (9) Yes Yes No 82 F132V c.394TrG Heterozygous 20 NA No No No 46 D209E c.627CrA Heterozygous 5 2,720 (13) No No No 134 Q211Kb c.631CrA Heterozygous 16 2,400 (3) No No No 122 L225Pc c.674TrC Heterozygous 4 2,500 (11) No No No 117 E382K c.1144GrA Homozygous 8 2,700 (4) No No No 118 A1185E c.3554CrA Homozygous 0 4,200 (95) No Yes Yes 116 N72S c.215ArG Mosaic 5 3,870 (74) No No No 47 P45L ϩ G1401R [c.134CrT] ϩ [c.4201GrA] Compound heterozygous 6 2,520 (18) Yes Yes No 119 E208K ϩ Y263D [c.622GrA] ϩ [c.787TrG] Compound heterozygous 13 2,950 (28) Yes No No 120 T229I ϩ V1523L [c.686CrT] ϩ [c.4567GrT] Compound heterozygous 4 NA No No No 78 P207S ϩ Y179X [c.619CrT] ϩ [c.536_539delATGG] Compound heterozygous 8 3,290 (29) No No No 121 [E1327K; V1523A] ϩ T1043QfsX74 [c.3979GrA; 4568CrT] ϩ [c.3127_3129delACCinsCAGCCAGGACCTG] Compound heterozygous 1 2,380 (!1) No No No a NA p not available. Login to comment
94 To simulate the patient`s genotype, we coinjected Kir6.2 mRNA with hetF132L (1:1 mix of WT and F132L SUR1 mRNAs); homA1185E or P207S (mutant SUR1 only); or V1523LϩT229I (1:1 mix of V1523L and T229I SUR1). Login to comment
102 ForIC p 15 h p 1.11 n p 650 hetSUR1-F132L (a), mM, , and . Login to comment
103 For homSUR1-P207S (b), mM, , and . Login to comment
133 To simulate the heterozygous state (e.g., F132L), Kir6.2 was coexpressed with a 1:1 mixture of wild-type and mutant SUR1 mRNA. Login to comment
95 To simulate the patient`s genotype, we coinjected Kir6.2 mRNA with hetF132L (1:1 mix of WT and F132L SUR1 mRNAs); homA1185E or P207S (mutant SUR1 only); or V1523Laf9;T229I (1:1 mix of V1523L and T229I SUR1). Login to comment
136 To simulate the heterozygous state (e.g., F132L), Kir6.2 was coexpressed with a 1:1 mixture of wild-type and mutant SUR1 mRNA. Login to comment

[hide] New ABCC8 mutations in relapsing neonatal diabetes... Diabetes. 2007 Jun;56(6):1737-41. Epub 2007 Mar 27. Vaxillaire M, Dechaume A, Busiah K, Cave H, Pereira S, Scharfmann R, de Nanclares GP, Castano L, Froguel P, Polak M
New ABCC8 mutations in relapsing neonatal diabetes and clinical features.
Diabetes. 2007 Jun;56(6):1737-41. Epub 2007 Mar 27., [PMID:17389331]

Abstract [show]
Activating mutations in the ABCC8 gene that encodes the sulfonylurea receptor 1 (SUR1) regulatory subunit of the pancreatic islet ATP-sensitive K(+) channel (K(ATP) channel) cause both permanent and transient neonatal diabetes. Recently, we have described the novel mechanism where basal Mg-nucleotide-dependent stimulatory action of SUR1 on the Kir6.2 pore is increased. In our present study, we identified six new heterozygous ABCC8 mutations, mainly in patients presenting the transient form of neonatal diabetes (six of eight), with a median duration of initial insulin therapy of 17 months (range 0.5-38.0). Most of these mutations map to key functional domains of SUR1. Whereas Kir6.2 mutations are a common cause of permanent neonatal diabetes and in a few cases associate with the DEND (developmental delay, epilepsy, and neonatal diabetes) syndrome, SUR1 mutations are more frequent in transient (52%) compared with permanent (14%) neonatal diabetes cases screened for ABCC8 in our series. Although ketoacidosis is frequent at presentation, SUR1 mutations associate mainly with transient hyperglycemia, with possible recurrence later in life. One-half of the SUR1 neonatal diabetic patients presented with de novo mutations. In some familial cases, diabetes is not always present in the adult carriers of SUR1 mutations, supporting variability in their clinical expressivity that remains to be fully explained.

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78 and in one patient previously reported with a SUR1 mutation (F132L) and severe DEND (developmental delay, epilepsy, and neonatal diabetes) syndrome (21). Login to comment
82 and in one patient previously reported with a SUR1 mutation (F132L) and severe DEND (developmental delay, epilepsy, and neonatal diabetes) syndrome (21). Login to comment

[hide] Activation of the K(ATP) channel by Mg-nucleotide ... J Gen Physiol. 2010 Oct;136(4):389-405. Proks P, de Wet H, Ashcroft FM
Activation of the K(ATP) channel by Mg-nucleotide interaction with SUR1.
J Gen Physiol. 2010 Oct;136(4):389-405., [PMID:20876358]

Abstract [show]
The mechanism of adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel activation by Mg-nucleotides was studied using a mutation (G334D) in the Kir6.2 subunit of the channel that renders K(ATP) channels insensitive to nucleotide inhibition and has no apparent effect on their gating. K(ATP) channels carrying this mutation (Kir6.2-G334D/SUR1 channels) were activated by MgATP and MgADP with an EC(50) of 112 and 8 microM, respectively. This activation was largely suppressed by mutation of the Walker A lysines in the nucleotide-binding domains of SUR1: the remaining small ( approximately 10%), slowly developing component of MgATP activation was fully inhibited by the lipid kinase inhibitor LY294002. The EC(50) for activation of Kir6.2-G334D/SUR1 currents by MgADP was lower than that for MgATP, and the time course of activation was faster. The poorly hydrolyzable analogue MgATPgammaS also activated Kir6.2-G334D/SUR1. AMPPCP both failed to activate Kir6.2-G334D/SUR1 and to prevent its activation by MgATP. Maximal stimulatory concentrations of MgATP (10 mM) and MgADP (1 mM) exerted identical effects on the single-channel kinetics: they dramatically elevated the open probability (P(O) > 0.8), increased the mean open time and the mean burst duration, reduced the frequency and number of interburst closed states, and eliminated the short burst states. By comparing our results with those obtained for wild-type K(ATP) channels, we conclude that the MgADP sensitivity of the wild-type K(ATP) channel can be described quantitatively by a combination of inhibition at Kir6.2 (measured for wild-type channels in the absence of Mg(2+)) and activation via SUR1 (determined for Kir6.2-G334D/SUR1 channels). However, this is not the case for the effects of MgATP.

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420 Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. Login to comment
424 Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. Login to comment

[hide] Permanent neonatal diabetes mellitus--the importan... Eur J Clin Invest. 2011 Mar;41(3):323-33. doi: 10.1111/j.1365-2362.2010.02409.x. Epub 2010 Nov 4. Rubio-Cabezas O, Klupa T, Malecki MT
Permanent neonatal diabetes mellitus--the importance of diabetes differential diagnosis in neonates and infants.
Eur J Clin Invest. 2011 Mar;41(3):323-33. doi: 10.1111/j.1365-2362.2010.02409.x. Epub 2010 Nov 4., [PMID:21054355]

Abstract [show]
BACKGROUND: The differential diagnosis of various types and forms of diabetes is of great practical importance. This is particularly true for monogenic disease forms, where some spectacular applications of pharmacogenetics have recently been described. DESIGN: For many years the distinct character of diabetes diagnosed in the first weeks and months of life remained unnoticed. The results of the search for type 1 diabetes-related autoantibodies, description of the HLA haplotypes distribution and analysis of clinical features in patients diagnosed in the first 6 months of life provided the initial evidence that the etiology of their disease might be different from that of autoimmune diabetes. RESULTS: Over the last decade, mutations in about a dozen of genes have been linked to the development of Permanent Neonatal Diabetes Mellitus (PNDM). The most frequent causes of PNDM are heterozygous mutations in the KCNJ11, INS and ABCC8 genes. Although PNDM is a rare phenomenon (one case in about 200,000 live births), this discovery has had a large impact on clinical practice as most carriers of KCNJ11 and ABCC8 gene mutations have been switched from insulin to oral sulphonylureas with an improvement in glycemic control. In this review we summarize the practical aspects of diabetes differential diagnosis in neonates and infants. CONCLUSIONS: Genetic testing should be advised in all subjects with PNDM as it may influence medical care in subjects with these monogenic forms of early onset diabetes.

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307 Clin Endocrinol (Oxf) 2009;71:358-62. 50 Proks P, Shimomura K, Craig TJ, Girard CA, Ashcroft FM. Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. Login to comment

[hide] Three C-terminal residues from the sulphonylurea r... J Physiol. 2008 Jul 1;586(13):3075-85. Epub 2008 May 1. Dupuis JP, Revilloud J, Moreau CJ, Vivaudou M
Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the K(ATP) channel subunits SUR2A and Kir6.2.
J Physiol. 2008 Jul 1;586(13):3075-85. Epub 2008 May 1., [PMID:18450778]

Abstract [show]
Cardiac ATP-sensitive potassium (K(ATP)) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory--but not inhibitory--transduction processes. These results indicate that, within the K(ATP) channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.

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37 However, most of these works concluded that there was a major role of TMD0 in the control of the Kir6.2 open probability (Po) but none of them defined it as essential in pharmacological responsiveness of the channel, except for the report of a heterozygous mutation in cytoplasmic loop 2, F132L, responsible for changes in ATP and tolbutamide sensitivity (Proks et al. 2006). Login to comment

[hide] Differential mechanisms of Cantu syndrome-associat... J Gen Physiol. 2015 Dec;146(6):527-40. doi: 10.1085/jgp.201511495. Cooper PE, Sala-Rabanal M, Lee SJ, Nichols CG
Differential mechanisms of Cantu syndrome-associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel.
J Gen Physiol. 2015 Dec;146(6):527-40. doi: 10.1085/jgp.201511495., [PMID:26621776]

Abstract [show]
Cantu syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium ((86)Rb(+)) efflux assays, we show that KATP channels formed with P429L, A475V, or C1039Y mutants enhance KATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive KATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.

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232 Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. 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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348 However, it has been shown that many mutations identified in PNDM (Gloyn et al., 2004; Proks et al., 2004, 2005, 2006; Koster et al., 2005) also reduce channel biogenesis efficiency, including Q52R, V59G/M, R201C/H and I296L in Kir6.2 (Lin et al., 2006a) as well as F132L in SUR1 (Pratt et al., 2009) when expressed heterologously as homomeric mutant channels (Table 2). Login to comment
356 Mutation Surface expression Gating References increased by SU property SUR1 F132L Yes Increased Po Pratt et al., 2009 V324M N.D. Increased MgADP sensitivity Zhou et al., 2010 Kir6.2 C42R N.D. Increased Po Yorifuji et al., 2005 Q52R Yes Increased Po Proks et al., 2004; Lin et al., 2006a V59G Yes Increased Po Proks et al., 2004; Lin et al., 2006a V59M Yes Increased Po Koster et al., 2005; Lin et al., 2006a R201C Yes Decreased ATP inhibition Proks et al., 2004; Lin et al., 2006a R201H Yes Decreased ATP inhibition Proks et al., 2004; Lin et al., 2006a Pro226_ Pro232del N.D. Increased Po Lin et al., 2013 I296L Yes Increased Po Proks et al., 2005; Lin et al., 2006a CONCLUSIONS AND PERSPECTIVES Pharmacological chaperones have emerged as promising therapeutic tools for treating diseases resulting from defective protein folding and/or trafficking. Login to comment