ABCMdb

ABCC8 p.Leu225Pro

ClinVar:	c.674T>C , p.Leu225Pro D , Pathogenic
Predicted by SNAP2:	A: D (59%), C: N (61%), D: D (80%), E: D (80%), F: N (82%), G: D (80%), H: D (75%), I: N (82%), K: D (80%), M: N (72%), N: D (80%), P: D (53%), Q: D (75%), R: D (80%), S: D (75%), T: D (71%), V: N (78%), W: D (59%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: N, Q: D, R: D, S: D, T: N, V: N, W: D, Y: D,

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[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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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
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
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] 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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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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184 Finally, some mutations (e.g. H1023Y in TM12, Babenko et al. 2006; L225P in CL3, Masia et al. 2007) enhance Mg-nucleotide activation by unknown mechanisms. 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
187 Finally, some mutations (e.g. H1023Y in TM12, Babenko et al. 2006; L225P in CL3, Masia et al. 2007) enhance Mg-nucleotide activation by unknown mechanisms. 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] 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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51 The latter effect is consistent with the small negative effect of L213R on the amount of mature receptor, which is in line with observations that a comparable amphipathic L0 helix of ABCC1 attaches to the membrane [23] and L225P in a less conserved portion of the cytoplasmic linker of SUR1 does not affect N [24] or surface expression of SUR1 in the same cell line [25]. Login to comment

[hide] Neonatal diabetes caused by mutations in sulfonylu... J Clin Endocrinol Metab. 2010 Dec;95(12):E473-8. Epub 2010 Sep 1. Zhou Q, Garin I, Castano L, Argente J, Munoz-Calvo MT, Perez de Nanclares G, Shyng SL
Neonatal diabetes caused by mutations in sulfonylurea receptor 1: interplay between expression and Mg-nucleotide gating defects of ATP-sensitive potassium channels.
J Clin Endocrinol Metab. 2010 Dec;95(12):E473-8. Epub 2010 Sep 1., [PMID:20810569]

Abstract [show]
CONTEXT: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to beta-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. OBJECTIVE: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. DESIGN: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. RESULTS: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4- or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on beta-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. CONCLUSION: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.

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52 The mature form of SUR1 is clearly reduced in V324M compared with WT, E208K, or L225P. Login to comment
57 Representative recordings of WT, V324M, and L225P channels are shown. Login to comment
77 These experiments showed that V324M markedly increases the Mg-nucleotide response (most evident at the 0.5/0.1 mM ATP/ADP ratio; Fig. 1C), even more so than L225P, another SUR1 mutation reported to cause permanent ND by a similar gating abnormality (15). Login to comment
96 Each bar is the mean Ϯ SEM of 30 (WT), eight (E208K), six (L225P), five (V324M), nine (E208K/L225P), six (E208K/V324M), and six (L225P/V324M) patches. Login to comment
100 To determine the relationship between the activating effect caused by V324M in TMD1 and that caused by E208K or L225P in TMD0-L0, we compared Mg-nucleotide responsivity in channels harboring a combination of these mutations. Login to comment
101 Currents measured in 0.1 mM ATP (free [Mg2ϩ ] ϳ1 mM) showed that channels harboring double mutations were more active than those with only one mutation such that the effects of E208K, L225P, or V324M are additive. Login to comment
106 Although V324M renders a marked increase in Mg-nucleotide response, even more so than L225P previously reported to cause permanent ND (15), the effect of E208K is subtle, only statistically significant at 0.1 mM ATP/0.1 mM ADP; yet both E208K and V324M are associated with transient ND. Login to comment
108 We previously proposed that in ND surface expression efficiency of a mutant modifies the extent of manifestation of the gating defect to determine diseaseoutcome(18,19).Supportingthis,whereasE208K (and also L225P; Fig. 1B) exhibited surface expression similar to WT, V324M was expressed at only approximately 50% that of WT (Fig. 1B). Login to comment
114 This suggests that similar to L225P (15), the two mutations affect transduction of MgATP binding/hydrolysis signal from the SUR1-NBFs to Kir6.2. Login to comment
117 That the effect of E208K or L225P and of V324M are additive suggests that there may be multiple transduction pathways to regulate Kir6.2 gating. Login to comment
121 Alternatively, transduction of the glibenclamide blocking effect may employ a pathway separate from that affected by V324M, as suggested for the L225P mutation (15). 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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46 Furthermore, changes in gating were not observed for the L225P mutation that also lies within TMD0 (19). Login to comment
172 This idea is also in agreement with the fact that there are many disease-causing mutations in this region and that they do not all act in the same way: for example, the L225P mutation does not alter single-channel gating (19). 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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1 De Leon,2 Courtney MacMullen,2 Heather McKnight,2 Charles A. Stanley,2 and Colin G. Nichols1 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 Kϩ channel (KATP channel). Login to comment
2 RESEARCH DESIGN AND METHODS-The effects of L225P on the properties of recombinant KATP channels in transfected COS cells were assessed by patch-clamp experiments on excised membrane patches and by macroscopic Rb-flux experiments in intact cells. Login to comment
3 RESULTS-L225P-containing KATP channels were significantly more active in the intact cell than in wild-type channels. Login to comment
4 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 Mg2ϩ . Login to comment
5 The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. Login to comment
6 L225P did not alter channel inhibition by sulfonylurea drugs, and, consistent with this, the patient responded to treatment with oral sulfonylureas. Login to comment
7 CONCLUSIONS-L225P underlies KATP 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. Login to comment
15 Here we describe a patient with permanent neonatal diabetes mellitus (PNDM) with a novel heterozygous SUR1 mutation (L225P) located in L0. Login to comment
16 L225P causes increased stimulation of recombinant KATP channels by Mg nucleotides and increased activity in the intact cell. Login to comment
17 The effects of L225P are abolished by mutations in the NBFs of SUR1 that prevent nucleotide hydrolysis, indicating that L225P likely acts on the transduction mechanism that links nucleotide hydrolysis at the NBFs to channel gating. Login to comment
21 Two mutations were found in ABCC8: t674c, predicted to cause an L225P amino acid change, was de novo, and g2638a, predicted to cause a D880N amino acid change, was maternally transmitted. Login to comment
54 RESULTS AND DISCUSSION A neonatal diabetes patient with a L225P mutation in SUR1. Login to comment
61 Reconstituted KATP channels containing L225P exhibit increased Mg-nucleotide stimulation. Login to comment
64 Heterozygous t674c mutation in the proband leads to heterozygous L225P point mutation. Login to comment
65 B: On-cell current relative to maximal postexcision current (Irel) for wild-type (WT), L225P-containing, and D880N-containing channels expressed in COS cells. Login to comment
82 However, two heterozygous point mutations were found in ABCC8 (SUR1): L225P, which was de novo (Fig. 1A), and D880N, which was maternally transmitted. Login to comment
83 L225P channels are more stimulated by Mg nucleotides. Login to comment
84 L225P and D880N were engineered into SUR1 to examine their functional consequences. Login to comment
86 Channel activity in the on-cell configuration (before excision) was significantly increased by L225P but not altered by D880N (Fig. 1B). Login to comment
88 Compared with wild-type channels, L225P did not alter (Po,zero) (0.55 Ϯ 0.02 vs. 0.58 Ϯ 0.03) nor did it alter sensitivity to inhibitory ATP in the absence of Mg2ϩ (half-maximal inhibitory [ATP], K1/2, was 8.2 Ϯ 1.2 vs. 8.9 Ϯ 1.5 ␮mol/l, Fig. 1D). Login to comment
89 However, L225P-containing channels were significantly more active in MgATP compared with wild type (K1/2 ϭ 19.0 Ϯ 2.6 vs. 67.6 Ϯ 15.8 ␮mol/l, P Ͻ 0.05 by Student`s t test) and were significantly more stimulated by MgADP than wild type (Fig. 1E and F), while D880N-containing channels were stimulated comparably with wild type (data not shown). Login to comment
90 These results indicate that L225P causes KATP channel overactivity by specifically increasing channel stimulation by Mg nucleotides and is therefore the likely cause of PNDM in the patient. Login to comment
91 L225P channels are more active in the intact cell. Login to comment
93 Consistent with expectations, L225P flux was significantly higher than wild-type flux under both basal conditions and metabolic inhibition (Fig. 2A and B). Login to comment
94 The fractional activity of L225P channels under basal conditions (relative to metabolic inhibition) was significantly increased compared with WT (Fig. 2C), indicating that L225P channels are already close to maximal activity under basal conditions. Login to comment
95 Since L225P did not significantly alter current density in excised membrane patches compared with wild type (1.3 Ϯ 0.2 vs. 1.2 Ϯ 0.1 pA/patch), the FIG. 2. Login to comment
96 L225P-containing channels are more active in the intact cell. Login to comment
97 A and B: Relative efflux of 86 Rb؉ as a function of time in basal conditions (A) or in metabolic inhibition (B) for wild-type (WT), mixed wild-type ؉ L225P, L225P, and [L225P, D1506A] channels, as well as untransfected controls. Login to comment
103 *P < 0.05 compared with L225P by Student`s t test. Login to comment
104 increased Rb fluxes measured for L225P channels must reflect differences in activity rather than in expression. Login to comment
105 Since the patient is heterozygous for L225P, we measured Rb fluxes from cells transfected with a 1:1 mixture of wild-type and L225P cDNA. Login to comment
106 The resulting mixed WT ϩ L225P channels exhibited fluxes that were intermediate between wild-type and homomeric L225P channels (Fig. 2A and B). Login to comment
107 The fractional activity of mixed wild-type ϩ L225P channels was also significantly increased compared with wild type (Fig. 2C), consistent with KATP channel overactivity leading to suppression of insulin secretion and diabetes in the heterozygous patient. Login to comment
108 L225P does not alter sulfonylurea sensitivity of the channel. Login to comment
109 Sulfonylurea sensitivity of recombinant KATP channels was unaffected by L225P, both in excised membrane patches and in intact cells (Fig. 3B-D). Login to comment
114 L225P effects are dependent on intact nucleotide hydrolysis at the NBFs. Login to comment
117 Consistent with this, the effects of L225P on channel activity in the intact cell were abolished by combination with either of two NBF mutations (K719M or D1506A) that abolish Mg-nucleotide stimulation of the channel (19,20) (Fig. 2D). Login to comment
120 To address this question, we measured Rb fluxes for all permutations of wild-type, L225P, D1506A, and (L225P, D1506A) combinations (at a 1:1 cDNA transfection ratio) (Fig. 4C). Login to comment
123 L225P does not alter sulfonylurea sensitivity of KATP channels. Login to comment
124 A: Representative currents recorded from inside-out membrane patches from COS cells expressing wild-type (top) or L225P (bottom) channels. Login to comment
131 Similarly, the activity of L225P:D1506A corresponded with (50%) L225P activity, the activity of wild type:L225P yielded an arithmetic sum of wild-type and L225P activities ([50%] wild type ϩ [50%] L225P), and the activity of L225P:[L225P, D1506A] yielded (50%) L225P activity (Fig. 4C). Login to comment
132 When L225P is mixed with D1506A, however, the resulting activity should then be (50%) L225P, if stimulation occurs through the TMD0-L0 of the same subunit, or (50%) wild type if it occurs through the TMD0-L0 of the adjacent subunit (Fig. 4B). Login to comment
133 The actual result was (50%) L225P activity (Fig. 4C), consistent with the "same subunit" model. Login to comment
134 Likewise, when wild type and [L225P, D1506A] are mixed, the activity should be (50%) wild type if stimulation occurs through the same subunit or (50%) L225P if it occurs through the adjacent subunit (Fig. 4B). Login to comment
136 L225P alters transduction of Mg-nucleotide stimulation from the core of the same SUR1 subunit. Login to comment
138 L225P and D1506A are indicated by green and red circles, respectively. Login to comment
140 B: Predicted activity of two SUR1 mixtures (wild type [WT]:[L225P, D1506A] and L225P:D1506A) based on the "same subunit" model (left, in orange) or the "adjacent subunit" model (right, in blue). Login to comment
144 Horizontal dashed lines indicate predicted (50%) wild-type activity, predicted (50%) L225P activity, and predicted (50%) wild type ؉ (50%) L225P activity, calculated from actual wild-type and L225P data. Login to comment
147 The lack of an effect of L225P on sulfonylurea sensitivity indicates that the mutation does not affect transduction of high-affinity sulfonylurea block to the Kir6.2 pore, suggesting that there may be structurally segregated transduction pathways in TMD0-L0 for each of these regulatory inputs from the SUR1 core. 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
154 In contrast with L225P, D880N did not affect Mg-nucleotide stimulation or on-cell channel activity. Login to comment
156 Consistent with lack of effect of L225P on sulfonylurea sensitivity of recombinant channels, the patient was successfully treated with oral sulfonylureas. Login to comment
157 The carrier of the I1424V mutation (which is mechanistically similar to L225P) has also been successfully treated with sulfonyureas (12). Login to comment
159 NOTE ADDED IN PROOF We have now sequenced SUR1 exon 5 from 50 healthy control subjects and have not found the L225P mutation in 98 alleles. Login to comment
55 RESULTS AND DISCUSSION A neonatal diabetes patient with a L225P mutation in SUR1. Login to comment
62 Reconstituted KATP channels containing L225P exhibit increased Mg-nucleotide stimulation. Login to comment
66 B: On-cell current relative to maximal postexcision current (Irel) for wild-type (WT), L225P-containing, and D880N-containing channels expressed in COS cells. Login to comment
85 L225P and D880N were engineered into SUR1 to examine their functional consequences. Login to comment
87 Channel activity in the on-cell configuration (before excision) was significantly increased by L225P but not altered by D880N (Fig. 1B). Login to comment
92 L225P channels are more active in the intact cell. Login to comment
98 A and B: Relative efflux of 86 Rbd19; as a function of time in basal conditions (A) or in metabolic inhibition (B) for wild-type (WT), mixed wild-type d19; L225P, L225P, and [L225P, D1506A] channels, as well as untransfected controls. Login to comment
110 Sulfonylurea sensitivity of recombinant KATP channels was unaffected by L225P, both in excised membrane patches and in intact cells (Fig. 3B-D). Login to comment
115 L225P effects are dependent on intact nucleotide hydrolysis at the NBFs. Login to comment
118 Consistent with this, the effects of L225P on channel activity in the intact cell were abolished by combination with either of two NBF mutations (K719M or D1506A) that abolish Mg-nucleotide stimulation of the channel (19,20) (Fig. 2D). Login to comment
121 To address this question, we measured Rb fluxes for all permutations of wild-type, L225P, D1506A, and (L225P, D1506A) combinations (at a 1:1 cDNA transfection ratio) (Fig. 4C). Login to comment
125 A: Representative currents recorded from inside-out membrane patches from COS cells expressing wild-type (top) or L225P (bottom) channels. Login to comment
135 Likewise, when wild type and [L225P, D1506A] are mixed, the activity should be (50%) wild type if stimulation occurs through the same subunit or (50%) L225P if it occurs through the adjacent subunit (Fig. 4B). Login to comment
137 L225P alters transduction of Mg-nucleotide stimulation from the core of the same SUR1 subunit. Login to comment
139 L225P and D1506A are indicated by green and red circles, respectively. Login to comment
141 B: Predicted activity of two SUR1 mixtures (wild type [WT]:[L225P, D1506A] and L225P:D1506A) based on the "same subunit" model (left, in orange) or the "adjacent subunit" model (right, in blue). Login to comment
145 Horizontal dashed lines indicate predicted (50%) wild-type activity, predicted (50%) L225P activity, and predicted (50%) wild type d19; (50%) L225P activity, calculated from actual wild-type and L225P data. Login to comment
148 The lack of an effect of L225P on sulfonylurea sensitivity indicates that the mutation does not affect transduction of high-affinity sulfonylurea block to the Kir6.2 pore, suggesting that there may be structurally segregated transduction pathways in TMD0-L0 for each of these regulatory inputs from the SUR1 core. 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
155 In contrast with L225P, D880N did not affect Mg-nucleotide stimulation or on-cell channel activity. Login to comment
158 The carrier of the I1424V mutation (which is mechanistically similar to L225P) has also been successfully treated with sulfonyureas (12). Login to comment
160 NOTE ADDED IN PROOF We have now sequenced SUR1 exon 5 from 50 healthy control subjects and have not found the L225P mutation in 98 alleles. 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
75 Birth weight percentiles were calculated using population-based data,15 and, when information on gestational age was not available ( ), a gestational age of 40 wk was assumed.n p 3 b Patients ISPAD 68, 123, and 134 have been reported by us elsewhere.9,10,14 c The L225P mutation has been reported in another patient by Masia et al.8 Figure 3. Schematic representation of KATP channels in family ISPAD 78. Login to comment
76 The unaffected parents are heterozygous for the c.536del4 frameshift or P207S missense mutation. Login to comment
154 Masia R, Deleon DD, Macmullen C, McKnight H, Stanley CA, Nichols CG (2007) A mutation in the TMD0-L0 region of SUR1 (L225P) causes permanent neonatal diabetes mellitus (PNDM). Login to comment
157 Masia R, Deleon DD, Macmullen C, McKnight H, Stanley CA, Nichols CG (2007) A mutation in the TMD0-L0 region of SUR1 (L225P) causes permanent neonatal diabetes mellitus (PNDM). 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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218 A mutation in the TMD0-L0 region of sulfonylurea receptor-1 (L225P) causes permanent neonatal diabetes mellitus (PNDM). Login to comment

[hide] Compound heterozygous mutations in the SUR1 (ABCC ... Channels (Austin). 2012 Mar-Apr;6(2):133-8. doi: 10.4161/chan.19980. Epub 2012 Mar 1. Lin YW, Akrouh A, Hsu Y, Hughes N, Nichols CG, De Leon DD
Compound heterozygous mutations in the SUR1 (ABCC 8) subunit of pancreatic K(ATP) channels cause neonatal diabetes by perturbing the coupling between Kir6.2 and SUR1 subunits.
Channels (Austin). 2012 Mar-Apr;6(2):133-8. doi: 10.4161/chan.19980. Epub 2012 Mar 1., [PMID:22562119]

Abstract [show]
KATP channels regulate insulin secretion by coupling beta-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg(2+)) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of KATP channel function, which will attenuate the beta-cell response to increased metabolism and will thereby decrease insulin secretion. (86)Rb(+) flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of (86)Rb(+) efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC 50 for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals.

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170 A mutation in the TMD0-L0 region of sulfonylurea receptor-1 (L225P) causes permanent neonatal diabetes mellitus (PNDM). Login to comment