ABCMdb

PMID: 12627323

Reimann F, Huopio H, Dabrowski M, Proks P, Gribble FM, Laakso M, Otonkoski T, Ashcroft FM
Characterisation of new KATP-channel mutations associated with congenital hyperinsulinism in the Finnish population.
Diabetologia. 2003 Feb;46(2):241-9. Epub 2003 Jan 9., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population. Login to comment 8 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. Login to comment 9 ABCC8 p.Leu1551Val The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of KATP current activation observed in response to metabolic inhibition in intact oocytes. Login to comment 10 ABCC8 p.Leu1551Val The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 µmol/l to 120 µmol/l). Login to comment 36 ABCC8 p.Glu1506Lys The Finnish SUR1 mutation E1506K is one exception, showing a dominant mode of inheritance [5]. Login to comment 40 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Three of these mutations are found in the C-terminal domain of SUR1 (L1551V, A1457T and V1550D), and one is found in the N-terminus of Kir6.2 (K67N). Login to comment 64 ABCC8 p.Glu1506Lys ABCC8 p.Val187Asp The mutations V187D and E1506K have been described previously [3, 5]. Login to comment 94 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Metabolic poisoning with 3 mmol/l azide induced a large increase in Kir6.2/SUR1 currents, a smaller increase in Kir6.2-K67N/SUR1 and Kir6.2/SUR1-L1551V currents, and no significant change in Kir6.2/SUR1-A1457T or Kir6.2/SUR1-V1550D currents. Login to comment 95 ABCC8 p.Leu1551Val The increase in Kir6.2/ SUR1-L1551V current (3.1±0.7 fold, n=8) was comparable to that observed for Kir6.2∆C36 expressed in the absence of SUR (5.9±1.1 fold, n=5), but much smaller than that found for Kir6.2/SUR1 currents (89±15 fold, n=19). Login to comment 98 ABCC8 p.Leu1551Val Kir6.2/SUR1 currents started to increase about 200 s after azide addition, and reached a steady state at about 700 s. Kir6.2/SUR1-L1551V currents showed a similar latency, but a very much slower rate of current increase. Login to comment 101 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Addition of 340 µmol/l diazoxide, in the continued presence of azide, increased Kir6.2/SUR1 and Kir6.2-K67N/SUR1 currents but did not activate Kir6.2/ SUR1-L1551V, Kir6.2/SUR1-A1457T or Kir6.2/SUR1-V1550D currents. Login to comment 105 ABCC8 p.Leu1551Val Latencies and times to steady state of azide-activated currents Latency (s) Time to steady n state (s) Kir6.2/SUR1 209±10 712±26 33 Kir6.2-K67N/SUR1 259±20 a 916±54 c 11 Kir6.2/SUR1-L1551V 203±40 NS 1430±30 c, d 6 Kir6.2∆C36 30±3 c 1890±90 c, d 6 Statistical significance is shown for comparison with wild type Kir6.2/SUR1: NS, non significant; a p<0.05, b p<0.01, c p<0.001. Login to comment 110 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr In contrast, no measurable currents were observed in patches excised from oocytes coexpressing Kir6.2 and either SUR1-A1457T or SUR1-V1550D. Login to comment 111 ABCC8 p.Leu1551Val The L1551V mutation caused a tenfold reduction in current. Login to comment 115 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr Kir6.2-HA was detectable in the plasma membrane when coexpressed with wild-type SUR1, but not when coexpressed with either SUR1-A1457T or SUR1-V1550D (Fig. 3B). Login to comment 116 ABCC8 p.Leu1551Val Although coexpression of SUR1-L1551V resulted in detectable surface expression of Kir6.2-HA, the signal was about 40-fold lower than that found for wild-type SUR1. Login to comment 117 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Thus, these mutations prevent (A1457T, V1550D) or reduce (L1551V) plasma membrane targeting of KATP channels expressed in oocytes. Login to comment 121 ABCC8 p.Leu1551Val Pharmacological and nucleotide sensitivity of mutant channels. We next investigated the properties of Kir6.2-K67N/SUR1 and Kir6.2/SUR1-L1551V channels in detail. Login to comment 123 ABCC8 p.Leu1551Val Kir6.2/SUR1-L1551V currents were inhibited by ATP with an IC50 of 119±12 µmol/l, and a Hill coeffi- 244 F. Reimann et al.: Characterisation of new KATP-channel mutations associated with congenital hyperinsulinism Fig. 2. Login to comment 125 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Oocytes were co-injected (as indicated) with mRNAs encoding Kir6.2 plus either SUR1, SUR1-A1457T, SUR1-V1550D or SUR1-L1551V, or with Kir6.2-K67N plus SUR1, or with Kir6.2∆36 alone. Login to comment 127 ABCC8 p.Leu1551Val Inset: mean whole-cell current amplitudes for Kir6.2-L1551V measured at -100 mV shown on an expanded scale cient of 0.99±0.10 (n=8; Fig. 4A). Login to comment 130 ABCC8 p.Leu1551Val The block of Kir6.2/ SUR1-L1551V produced by MgADP reflects the inhibitory action of ADP on Kir6.2, which becomes evident when the stimulatory action of the nucleotide (mediated via SUR1) is abolished. Login to comment 131 ABCC8 p.Leu1551Val Kir6.2/SUR1-L1551V channels were also unaffected by diazoxide and inhibited by tolbutamide to a lesser extent than wild type Kir6.2/SUR1 currents (Fig. 4B). Login to comment 132 ABCC8 p.Leu1551Val The altered properties of Kir6.2/SUR1-L1551V channels might be attributable either to an increase in the channel open probability [25], or to a functional uncoupling of Kir6.2 from mutant SUR1. Login to comment 133 ABCC8 p.Leu1551Val To distinguish between these possibilities, we measured the single-channel properties of Kir6.2/SUR1-L1551V expressed in oocytes. Login to comment 134 ABCC8 p.Leu1551Val The L1551V mutation did not significantly affect the single-channel conductance, but increased the channel open probability (Po) from 0.22 [25] to 0.65. Login to comment 136 ABCC8 p.Leu1551Val As discussed below, these effects probably explain the observed reduction in the inhibitory effects of both ATP and tolbutamide on Kir6.2/SUR1-L1551V currents. Login to comment 148 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val The number of oocytes is given above the bars. Statistical significance (t test) was tested against uninjected oocytes for Kir6.2HA coinjected with SUR1, SUR1-A1457T, SUR1-V1550D or SUR1-L1551V. Login to comment 151 ABCC8 p.Leu1551Val ABCC8 p.Leu1551Val ABCC8 p.Leu1551Val Single-channel kinetics of Kir6.2/SUR1 and Kir6.2/ SUR1-L1551V channels Kir6.2/SUR1a Kir6.2/SUR1-L1551V Open probability (Po) 0.22 0.65±0.05 Mean open time (τo) (ms) 1.69 2.05±0.09 Mean short closed time (τf) (ms) 0.33 0.41±0.02 Mean long closed time (τs) (ms) 16.1 15.6±2.8 Percentage <s 18 2.2±0.4 Mean burst duration (ms) 13.5 109±22 a Kinetic parameters of single Kir6.2/SUR1 [24] and Kir6.2/ SUR1-L1551V channels (n=6) 246 F. Reimann et al.: Characterisation of new KATP-channel mutations associated with congenital hyperinsulinism Fig. 4A-C. Login to comment 152 ABCC8 p.Leu1551Val ABCC8 p.Leu1551Val (A) ATP-sensitivity of Kir6.2/SUR1-L1551V and Kir6.2-K67N/SUR1 currents. Mean ATP concentration-response relations for Kir6.2/SUR1 (n=5), Kir6.2/SUR1-L1551V (n=8), Kir6.2-K67N/SUR1 (n=4) and Kir6.2∆C36 (n=5) currents. Login to comment 154 ABCC8 p.Leu1551Val The line is the best fit of the data to the Hill equation for Kir6.2/SUR1-L1551V (IC50= 119±12 µmol/l, h=1.0±0.1) and Kir6.2-K67N/SUR1 (IC50= 6.9±0.8 µmol/l, h=0.9±0.1). Login to comment 156 ABCC8 p.Leu1551Val ABCC8 p.Leu1551Val (B) Effects of MgADP, tolbutamide and diazoxide on Kir6.2/SUR1, Kir6.2/ SUR1-L1551V and Kir6.2-K67N/SUR1 currents. Mean macroscopic slope conductance recorded in the presence of ADP (100 µmol/l), ATP (100 µmol/l), ATP plus diazoxide (100 and 340 µmol/l, respectively), or ATP plus ADP (100 µmol/l each), or tolbutamide (100 µmol/l), expressed as percentage of the slope conductance in control solution, for Kir6.2/SUR1, Kir6.2/ SUR1-L1551V and Kir6.2-K67N/SUR1 currents. Login to comment 169 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr Two of the mutations in SUR1 (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. Login to comment 171 ABCC8 p.Leu1551Val KATP channels containing the L1551V mutation in SUR1 had impaired surface expression and altered channel properties, including loss of MgADP activation. Both these factors are likely to reduce KATP channel activity in pancreatic beta cells. Login to comment 174 ABCC8 p.Leu1551Val Kir6.2/SUR1-L1551V currents showed reduced sensitivity to ATP-inhibition, loss of activation by MgADP and diazoxide and impaired block by tolbutamide. Login to comment 176 ABCC8 p.Leu1551Val The inhibitory effect of MgADP on Kir6.2/SUR1-L1551V currents was less than that observed when MgADP activation was abolished by mutation of the Walker A lysines in SUR1 [32, 33]. Login to comment 179 ABCC8 p.Leu1551Val The lower ATP sensitivity, loss of MgADP activation, and reduced surface expression probably all contribute to the smaller magnitude and slower activation of Kir6.2/SUR1-L1551V channels on metabolic poisoning in intact oocytes. Login to comment 185 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr Two mutations in SUR1 (A1457T and V1550D), that did not result in functional channels in Xenopus oocytes, were each found only once in the Finnish population [23]. Login to comment 186 ABCC8 p.Val187Asp In both cases they occurred as part of a complex heterozygous genotype, with the SUR1 mutation V187D on the second allele [3]. Login to comment 187 ABCC8 p.Val187Asp As SUR1-V187D also abolished KATP channel activity [3], these two subjects would be predicted to have no functional KATP channels, consistent with the observed severe, drug-resistant CHI phenotype [23]. Login to comment 188 ABCC8 p.Leu1551Val The third mutation in SUR1 (L1551V) was found in two siblings, who had a milder form of CHI, which was responsive to diazoxide [23]. Login to comment 189 ABCC8 p.Leu1551Val The mild phenotype and diazoxide-sensitivity of these subjects is consistent with the finding that both siblings were heterozygous for the L1551V mutation and had an apparently normal second SUR1 allele. Login to comment 190 ABCC8 p.Glu1506Lys Although CHI is usually a recessive condition, dominant SUR1 mutations (e.g. SUR1-E1506K) have been reported [5]. Login to comment 191 ABCC8 p.Leu1551Val The subjects in this study inherited the L1551V mutation from their father, who is not known to have had CHI [23]. Login to comment 193 ABCC8 p.Leu1551Val ABCC8 p.Leu1551Val However, if this were the case for the L1551V family, the hyperinsulinaemia should not have been sensitive to diazoxide, as the hyperplastic tissue would express only diazoxide-insensitive SUR1-L1551V subunits. Login to comment 195 ABCC8 p.Leu1551Val An alternative idea is that the phenotypic effect of the L1551V mutation depends on the genetic background of the individual, and that it produces CHI only in those people who carry polymorphisms in other genes that predispose towards enhanced insulin secretion. Login to comment