ABCMdb

PMID: 17584766

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., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Login to comment 33 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 46 ABCC8 p.Leu225Pro Furthermore, changes in gating were not observed for the L225P mutation that also lies within TMD0 (19). Login to comment 47 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu We first compared the effect of the F132L mutation in SUR1 on the kinetics of single KATP channel currents. Login to comment 55 ABCC8 p.Phe132Leu The F132L mutation dramatically increased the burst duration and reduced the time spent in the interburst intervals. Login to comment 58 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu The ATP sensitivity of SUR1-F132L mutant channels was further decreased in the presence of 2 mM Mg2+ (Fig. 2B). Login to comment 84 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu (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 ABCC8 p.Phe132Leu Thus, these data suggest that MgGDP activation may be enhanced by the F132L mutation in SUR1. Login to comment 109 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu This was not only true for wild-type TMD0, but also for hetTMD0-F132L and homTMD0-F132L (Table 1). Login to comment 118 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu Thus, we next examined whether the F132L mutation altered surface expression of the KATP channel complex. Login to comment 141 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu Our results further show that the F132L mutation impairs this physical association. Login to comment 152 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu (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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 172 ABCC8 p.Leu225Pro 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 175 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu (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 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu Mg2+ produced a dramatic reduction in the ATP sensitivity of homKir6.2/SUR1-F132L and hetKir6.2/SUR1-F132L channels. Login to comment 195 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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 ABCC8 p.Phe132Leu 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