ABCMdb

PMID: 17317760

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., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro RESULTS-L225P-containing KATP channels were significantly more active in the intact cell than in wild-type channels. Login to comment 4 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. Login to comment 6 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro L225P causes increased stimulation of recombinant KATP channels by Mg nucleotides and increased activity in the intact cell. Login to comment 17 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro RESULTS AND DISCUSSION A neonatal diabetes patient with a L225P mutation in SUR1. Login to comment 55 ABCC8 p.Leu225Pro RESULTS AND DISCUSSION A neonatal diabetes patient with a L225P mutation in SUR1. Login to comment 61 ABCC8 p.Leu225Pro Reconstituted KATP channels containing L225P exhibit increased Mg-nucleotide stimulation. Login to comment 62 ABCC8 p.Leu225Pro Reconstituted KATP channels containing L225P exhibit increased Mg-nucleotide stimulation. Login to comment 64 ABCC8 p.Leu225Pro Heterozygous t674c mutation in the proband leads to heterozygous L225P point mutation. Login to comment 65 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 66 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro L225P channels are more stimulated by Mg nucleotides. Login to comment 84 ABCC8 p.Leu225Pro L225P and D880N were engineered into SUR1 to examine their functional consequences. Login to comment 85 ABCC8 p.Leu225Pro L225P and D880N were engineered into SUR1 to examine their functional consequences. Login to comment 86 ABCC8 p.Leu225Pro Channel activity in the on-cell configuration (before excision) was significantly increased by L225P but not altered by D880N (Fig. 1B). Login to comment 87 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro L225P channels are more active in the intact cell. Login to comment 92 ABCC8 p.Leu225Pro L225P channels are more active in the intact cell. Login to comment 93 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro L225P-containing channels are more active in the intact cell. Login to comment 97 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 98 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 103 ABCC8 p.Leu225Pro *P < 0.05 compared with L225P by Student`s t test. Login to comment 104 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro increased Rb fluxes measured for L225P channels must reflect differences in activity rather than in expression. Login to comment 105 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro L225P does not alter sulfonylurea sensitivity of the channel. Login to comment 109 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 110 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro L225P effects are dependent on intact nucleotide hydrolysis at the NBFs. Login to comment 115 ABCC8 p.Leu225Pro L225P effects are dependent on intact nucleotide hydrolysis at the NBFs. Login to comment 117 ABCC8 p.Lys719Met ABCC8 p.Leu225Pro 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 118 ABCC8 p.Lys719Met ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 121 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro L225P does not alter sulfonylurea sensitivity of KATP channels. Login to comment 124 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro A: Representative currents recorded from inside-out membrane patches from COS cells expressing wild-type (top) or L225P (bottom) channels. Login to comment 125 ABCC8 p.Leu225Pro A: Representative currents recorded from inside-out membrane patches from COS cells expressing wild-type (top) or L225P (bottom) channels. Login to comment 131 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro The actual result was (50%) L225P activity (Fig. 4C), consistent with the "same subunit" model. Login to comment 134 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 135 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro L225P alters transduction of Mg-nucleotide stimulation from the core of the same SUR1 subunit. Login to comment 137 ABCC8 p.Leu225Pro L225P alters transduction of Mg-nucleotide stimulation from the core of the same SUR1 subunit. Login to comment 138 ABCC8 p.Leu225Pro L225P and D1506A are indicated by green and red circles, respectively. Login to comment 139 ABCC8 p.Leu225Pro L225P and D1506A are indicated by green and red circles, respectively. Login to comment 140 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 141 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 145 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro 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 147 ABCC8 p.Leu225Pro 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 148 ABCC8 p.Leu225Pro 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 149 ABCC8 p.Phe132Leu ABCC8 p.Leu213Arg ABCC8 p.Ile1424Val 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Leu213Arg ABCC8 p.Ile1424Val ABCC8 p.Ile1424Val 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 ABCC8 p.Phe132Leu ABCC8 p.Leu225Pro ABCC8 p.Ile1424Val 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 ABCC8 p.Phe132Leu ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Ile1424Val 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 ABCC8 p.Phe132Leu ABCC8 p.Leu225Pro ABCC8 p.Ile1424Val 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 ABCC8 p.Leu225Pro In contrast with L225P, D880N did not affect Mg-nucleotide stimulation or on-cell channel activity. Login to comment 155 ABCC8 p.Leu225Pro In contrast with L225P, D880N did not affect Mg-nucleotide stimulation or on-cell channel activity. Login to comment 156 ABCC8 p.Leu225Pro 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 ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Ile1424Val The carrier of the I1424V mutation (which is mechanistically similar to L225P) has also been successfully treated with sulfonyureas (12). Login to comment 158 ABCC8 p.Leu225Pro ABCC8 p.Ile1424Val The carrier of the I1424V mutation (which is mechanistically similar to L225P) has also been successfully treated with sulfonyureas (12). Login to comment 159 ABCC8 p.Leu225Pro 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 160 ABCC8 p.Leu225Pro 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