Adjacent mutations in the gating loop of Kir6.2 produce neonatal diabetes and hyperinsulinism
2009; Springer Nature; Volume: 1; Issue: 3 Linguagem: Inglês
10.1002/emmm.200900018
ISSN1757-4684
AutoresKenju Shimomura, Sarah E. Flanagan, Brittany Zadek, Mark Lethby, Lejla Zubcevic, Christophe A. Girard, Oliver Petz, Roope Männikkö, Ritika R. Kapoor, Khalid Hussain, Mars Skae, Peter Clayton, Andrew T. Hattersley, Sian Ellard, Frances M. Ashcroft,
Tópico(s)Cardiac Ischemia and Reperfusion
ResumoResearch Article12 June 2009Open Access Adjacent mutations in the gating loop of Kir6.2 produce neonatal diabetes and hyperinsulinism Kenju Shimomura Kenju Shimomura Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Sarah E. Flanagan Sarah E. Flanagan Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Brittany Zadek Brittany Zadek Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Mark Lethby Mark Lethby Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Lejla Zubcevic Lejla Zubcevic Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Christophe A. J. Girard Christophe A. J. Girard Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Oliver Petz Oliver Petz St. Vincenz Hospital Coesfeld, Childrens Hospital, Germany Search for more papers by this author Roope Mannikko Roope Mannikko Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Ritika R. Kapoor Ritika R. Kapoor London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust and The Institute of Child Health, University College London, UK Search for more papers by this author Khalid Hussain Khalid Hussain London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust and The Institute of Child Health, University College London, UK Search for more papers by this author Mars Skae Mars Skae Department of Endocrinology, Royal Manchester Children's Hospital, Central Manchester & Manchester Children's University Hospitals NHS Trust, UK Search for more papers by this author Peter Clayton Peter Clayton Department of Endocrinology, Royal Manchester Children's Hospital, Central Manchester & Manchester Children's University Hospitals NHS Trust, UK Search for more papers by this author Andrew Hattersley Andrew Hattersley Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Sian Ellard Sian Ellard Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Frances M. Ashcroft Corresponding Author Frances M. Ashcroft [email protected] Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Kenju Shimomura Kenju Shimomura Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Sarah E. Flanagan Sarah E. Flanagan Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Brittany Zadek Brittany Zadek Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Mark Lethby Mark Lethby Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Lejla Zubcevic Lejla Zubcevic Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Christophe A. J. Girard Christophe A. J. Girard Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Oliver Petz Oliver Petz St. Vincenz Hospital Coesfeld, Childrens Hospital, Germany Search for more papers by this author Roope Mannikko Roope Mannikko Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Ritika R. Kapoor Ritika R. Kapoor London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust and The Institute of Child Health, University College London, UK Search for more papers by this author Khalid Hussain Khalid Hussain London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust and The Institute of Child Health, University College London, UK Search for more papers by this author Mars Skae Mars Skae Department of Endocrinology, Royal Manchester Children's Hospital, Central Manchester & Manchester Children's University Hospitals NHS Trust, UK Search for more papers by this author Peter Clayton Peter Clayton Department of Endocrinology, Royal Manchester Children's Hospital, Central Manchester & Manchester Children's University Hospitals NHS Trust, UK Search for more papers by this author Andrew Hattersley Andrew Hattersley Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Sian Ellard Sian Ellard Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK Search for more papers by this author Frances M. Ashcroft Corresponding Author Frances M. Ashcroft [email protected] Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK Search for more papers by this author Author Information Kenju Shimomura1, Sarah E. Flanagan2, Brittany Zadek1, Mark Lethby1, Lejla Zubcevic1, Christophe A. J. Girard1, Oliver Petz3, Roope Mannikko1, Ritika R. Kapoor4, Khalid Hussain4, Mars Skae5, Peter Clayton5, Andrew Hattersley2, Sian Ellard2 and Frances M. Ashcroft *,1 1Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK 2Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, UK 3St. Vincenz Hospital Coesfeld, Childrens Hospital, Germany 4London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust and The Institute of Child Health, University College London, UK 5Department of Endocrinology, Royal Manchester Children's Hospital, Central Manchester & Manchester Children's University Hospitals NHS Trust, UK *Tel: +44 (0) 1865-285810; Fax: +44 (0) 1865-285813 EMBO Mol Med (2009)1:166-177https://doi.org/10.1002/emmm.200900018 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract KATP channels regulate insulin secretion from pancreatic β-cells. Loss- and gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause hyperinsulinism of infancy and neonatal diabetes, respectively. We report two novel mutations in the gating loop of Kir6.2 which cause neonatal diabetes with developmental delay (T293N) and hyperinsulinism (T294M). These mutations increase (T293N) or decrease (T294M) whole-cell KATP currents, accounting for the different clinical phenotypes. The T293N mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly decreasing channel inhibition by ATP and increasing whole-cell currents. T294M channels exhibit a dramatically reduced Po(0) in the homozygous but not in the pseudo-heterozygous state. Unlike wild-type channels, hetT294M channels were activated by MgADP in the absence but not in the presence of MgATP; however, they are activated by MgGDP in both the absence and presence of MgGTP. These mutations demonstrate the importance of the gating loop of Kir channels in regulating Po(0) and further suggest that Mg-nucleotide interaction with SUR1 may reduce ATP inhibition at Kir6.2. The paper explained PROBLEM: The ATP-sensitive potassium (KATP) channel is a tiny, gated pore in the cell membrane of insulin-secreting β-cells that regulates insulin secretion. When this channel is shut, insulin is secreted and when it is open insulin secretion is prevented. Channel activity is regulated by changes in blood glucose levels: when blood glucose levels rise (as after a meal), ATP is generated by the β-cell, and binds to the channel and causes its closure. This stimulates insulin release and the hormone then restores the blood glucose concentration to its resting level. Mutations in the KATP channel are a common cause of neonatal diabetes (diabetes that develops within the first six months of life) and of hyperinsulinism of infancy (in which insulin secretion is unregulated leading to very low blood glucose levels). The aim of this paper is to identify the molecular mechanism underlying two novel disease-causing mutations in the pore-forming subunit of the KATP channel (Kir6.2 or KCNJ11). RESULTS: We describe here a novel Kir6.2 mutation (T293N) that causes neonatal diabetes in conjunction with developmental delay and muscle weakness. Functional analysis revealed that the mutant channel failed to close in response to ATP. This is expected to impair insulin secretion, thus causing diabetes. It might also reduce the activity of brain neurons, thus leading to the neurological problems experienced by the patients. Treatment with glibenclamide (a sulphonylurea drug known to bind to and close the KATP channel) partially blocked the KATP channel in functional studies. It also reduced the insulin requirement of the patient and improved walking, speech and fine motor skills. We also identified a loss-of-function mutation in the same gene at the adjacent residue (T294M) that was associated with hyperinsulinism of infancy. This mutation caused the channel to remain shut even when ATP or metabolism were low. This is expected to cause continuous insulin secretion accounting for the disease phenotype. IMPACT: These results provide novel insights into how the opening and closing of the KATP channel is regulated. INTRODUCTION Naturally occurring mutations can provide important insights into ion channel function. This is especially true of mutations in the ATP-sensitive potassium (KATP) channel, where mutations can cause either too much or too little insulin secretion, giving rise to hyperinsulinaemic hypoglycaemia of infancy and neonatal diabetes, respectively (for a review, see Ashcroft, 2007). KATP channels couple the metabolic state of the cell to its electrical activity and are expressed in a wide range of cell types, including neurones, muscle and endocrine tissue (reviewed by Ashcroft, 2007; Nichols, 2006). In pancreatic β-cells, KATP channels have a crucial role in insulin secretion (Fig S1 of Supporting information). Their closure in response to metabolically generated ATP produces membrane depolarization, activation of voltage-gated Ca2+ channels, Ca2+ influx and thereby insulin granule exocytosis (Ashcroft, 2007; Henquin, 2000; Nichols, 2006). Conversely, KATP channel opening leads to membrane hyperpolarization, thus reducing Ca2+ influx and switching off insulin secretion. This metabolic regulation is mediated by changes in the intracellular concentration of adenine nucleotides with ATP inducing KATP channel closure by binding to the Kir6.2 subunit of the channel (Tucker et al, 1997) and Mg-nucleotides (MgATP, MgADP) stimulating channel opening via the SUR1 subunit (Gribble et al, 1997; Nichols et al, 1996; Shyng et al, 1997). The balance between these two opposing effects of nucleotides determines the level of channel activity in the cell. Mutations in KCNJ11 and ABCC8, the genes encoding the pore-forming (Kir6.2) and regulatory (SUR1) subunits, respectively, of the KATP channel cause impaired insulin secretion. Loss-of-function mutations cause hyperinsulinism of infancy (HI), a condition in which insulin is continuously secreted, independent of the plasma glucose level (De Leon & Stanley, 2007; Dunne et al, 2004). This is a serious condition because the patient can suffer irreversible brain damage as a consequence of the resulting hypoglycaemia. Most of the mutations have been found in SUR1, but 24 have been identified in Kir6.2 (Flanagan et al, 2009). Gain-of-function mutations in KATP channel genes give rise to neonatal diabetes, a rare disorder characterized by elevated glucose levels within the first six months of life (for reviews, see Ashcroft, 2007; Flanagan et al, 2009; Hattersley & Ashcroft, 2005). All mutations result in neonatal diabetes but this can either be permanent (PNDM) or follow a remitting relapsing course. In some cases, the patient experiences developmental delay, epilepsy and muscle weakness in conjunction with neonatal diabetes, a condition called DEND syndrome (Hattersley & Ashcroft, 2005). In a milder form of this syndrome, known as intermediate DEND syndrome (iDEND), the patients are not affected by epilepsy. Although mutations in either Kir6.2 or SUR1 can cause all these different conditions, both iDEND and DEND syndromes are more commonly associated with Kir6.2 mutations than SUR1 mutations. All Kir6.2 mutations cause a reduced KATP channel sensitivity to inhibition by MgATP, but this is greater for mutations that cause the more severe disease phenotypes (Koster et al, 2008; Masia et al, 2007; Proks et al, 2005b; Shimomura et al, 2006). In β-cells, the increased KATP current impairs insulin release, thus causing diabetes (Girard et al, 2009; Koster et al, 2000). In neurones, it leads to the neurological symptoms of DEND syndrome. In this paper, we identify two novel KCNJ11 mutations in patients with the opposite phenotypes of iDEND (T293N) and HI (T294M). These mutations lie side-by-side in the gating loop of Kir6.2. Functional analysis revealed that the T293N mutation produces a marked reduction in KATP channel inhibition by MgATP, due to an increase in the intrinsic channel open probability (Po(0)). This leads to a large increase in the resting whole-cell KATP current, which can account for the iDEND phenotype of the patient. In contrast, the T294M mutation reduced Po(0) to unmeasurable levels, thereby reducing the whole-cell current and stimulating insulin secretion. RESULTS Patient characteristics and genetics A novel heterozygous KCNJ11 mutation (c.878C>A; p.Thr293Asn or T293N) was identified in a girl born to second cousins of Turkish descent (Fig 1, case 1). DNA was not available from the parents but neither of them is known to be diabetic. The proband had a birth weight of 2.6 kg and presented with diabetic ketoacidosis at 10 weeks of age. She has developmental delay with severe muscle weakness in the trunk and legs, but no epilepsy, indicating she has iDEND syndrome. She was initially treated with insulin but glibenclamide (1–1.1 mg/kg/day) was added from 15 months of age, which enabled the insulin dose to be reduced from 0.9 to 0.5 U/kg/day. Improvements in her walking, speech and fine motor skills were noted after starting glibenclamide. Figure 1. Partial pedigrees showing inheritance of KCNJ11 mutations. Circles represent females and squares indicate males. An arrow with the letter P points to the proband in each family. Filled symbols denote patients with hyperinsulinism and vertical hatching represents neonatal diabetes. Unaffected heterozygous mutation carriers are denoted by a dot. The genotype is shown below each symbol: N denotes a normal allele and N/N a normal genotype. The residue number and amino acid change are given for mutation carriers. Download figure Download PowerPoint Two patients with congenital hyperinsulinism (Fig 1, cases 2 and 3) were heterozygous for a second KCNJ11 mutation, T294M (c.881C>T; p.Thr294Met). Case 2 is a female born at 38 weeks gestation with a birth weight of 4.1 kg, who developed severe hypoglycaemia soon after birth. She required high concentrations of intravenous glucose infusions to maintain normoglycaemia and a hypoglycaemia screen at 10 days of age confirmed hyperinsulinism with a plasma insulin concentration of 110 pM at a blood glucose concentration of 1.2 mM. She did not respond to diazoxide treatment and underwent a sub-total pancreatectomy at four weeks of age. Histological analysis showed hyperplasia throughout the pancreas with enlarged nuclei. The T294M mutation was inherited from her unaffected father: her unaffected paternal grandmother also carried the mutation (Fig 1, case 2). Analysis of microsatellite markers across the chromosome 11p15.1–11p15.5 region showed loss of heterozygosity of the maternal allele consistent with a giant focal lesion. The heterozygous germline T294M mutation is therefore homozygous within the pancreas. Case 3 is a male born at 37 weeks gestation with a birth weight of 4.8 kg. Hyperinsulinaemic hypoglycaemia was diagnosed at one week of age (glucose 2.3 mM, insulin 531 pM) and euglycaemia was achieved with diazoxide therapy (starting dose 10 mg/kg/day, current dose at 19 months of age is 4 mg/kg/day). His mother is also heterozygous for the T294M mutation: she had glycosuria during pregnancy but has no history of hypoglycaemia. A second mutation was not detected within the coding region of the ABCC8 gene. Effects of T293N and T294M mutations on whole-cell KATP currents To determine the molecular mechanism of the disease, wild-type and mutant KATP channels were expressed in Xenopus oocytes. Under resting conditions, wild-type KATP channels are closed due to the high intracellular ATP concentration ([ATP]i) (Fig 2A and B). However, substantial currents were activated by the metabolic inhibitor sodium azide, which lowers [ATP]i and thus opens KATP channels. The ability of the KATP channel blockers glibenclamide (Fig 2A and B) and tolbutamide (Fig 2C, Fig S2 of Supporting information) to inhibit these currents confirmed their identity. Figure 2. Effects of the T293N and T294M mutations on the whole-cell currents. A.. Representative wild-type, hetT293M and homT293M whole-cell currents evoked by a voltage step from −10 to −30 mV. The bars indicate the application of 3 mM azide or 100 µM glibenclamide (glib). B–C.. Mean steady-state whole-cell KATP channel current (as indicated) before (white bar) and after the application of 3 mM azide (grey bar) and in the presence of 3 mM azide and either 100 µM glibenclamide (B, black bar) or 0.5 mM tolbutamide (C, black bar). Note B and C were separate sets of experiments using different oocytes. The number of oocytes is indicated below the bar. D.. Mean steady-state whole-cell evoked by a voltage step from −10 mV to −30 mV before (white bar) and after application of 3 mM azide (light grey bar), in the presence of 3 mM azide and 0.34 mM diazoxide (dark grey bar) and in the presence of 3 mM azide and 0.5 mM tolbutamide (black bar). Download figure Download PowerPoint In contrast to wild-type channels, substantial resting K+ currents were present in oocytes expressing homomeric Kir6.2-T293N/SUR1 (homT293N) channels (Fig 2A–C). These currents were further increased by azide suggesting that homT293N channels are not fully open at resting [ATP]i in the oocyte. Coinjection of wild-type and mutant Kir6.2, to simulate the heterozygous state, produced resting currents (hetT293N) that were much greater than wild-type and further increased by azide (Fig 2A–C). Mutant channels were significantly less sensitive to both glibenclamide and tolbutamide at concentrations that maximally block wild-type channels. Glibenclamide (100 µM) blocked wild-type channels by 96 ± 1% (n = 5) compared with 66 ± 9% (n = 5) for hetT293N channels and 59 ± 4% (n = 5) for homT293N channels (Fig 2B). Inhibition by 0.5 mM tobutamide was 97 ± 1% (n = 5) for wild-type channels, 36 ± 2% (n = 5) for hetT293N channels and 24 ± 3% (n = 5) for homT293N channels (Fig 2C). Thus the T293N mutation appears to induce a greater reduction in block by tolbutamide than glibenclamide. In contrast to wild-type channels, homT294M channels failed to open in response to either azide or the KATP channel opener diazoxide (Fig 2D). Resting hetT294M currents were also unmeasureable, and although some current was activated by both azide and diazoxide it was significantly less than that observed for wild-type channels (66% and 51% smaller, respectively: Fig 2D, Fig S2 of Supporting information). The currents were fully blocked by tolbutamide. These differences in the magnitude of the whole-cell currents are consistent with the clinical phenotypes associated with the mutations. T293N channels exhibit reduced ATP sensitivity The increase in resting whole-cell KATP currents suggests that the T293N mutation may reduce channel inhibition by ATP, as found for other iDEND mutations. To explore this idea, we measured ATP-concentration response curves in inside-out membrane patches. Because ATP does not interact with SUR1 to stimulate channel activity in the absence of Mg2+ (Gribble et al, 1998), experiments were first carried out in the absence of Mg2+: this enables the effect of the mutation on ATP block at Kir6.2 to be studied in isolation from Mg-nucleotide activation at SUR1. Figure 3A shows that both homT293N and hetT293N currents were less ATP sensitive: half-maximal block (IC50) was produced by 778 µM, 37 µM and 6 µM ATP for homT293N, hetT293N and wild-type channels respectively (Table 1). The concentration–response curve for homT293N currents was best fit by assuming that a small fraction (4%) of channels are never closed by ATP. These data indicate that the mutation decreases the ATP sensitivity of the channel, at least in part, via Kir6.2. Figure 3. Effects of the T293N mutation on KATP channel ATP sensitivity. ATP concentration–response relations in the absence (A) and presence (B) of Mg2+. Left: KATP currents recorded in response to voltage ramps from −110 to +110 mV in an inside-out patch excised from oocytes expressing wild-type or homT293N channels. The dashed line indicates the zero current level. The bar indicates application of 100 µM ATP (A) or 1 mM ATP (B). Right: Mean relationships between [ATP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for Kir6.2/SUR1 (○, n = 5), and heterozygous (•, n = 6) or homomeric (▪, n = 5) Kir6.2-T293N/SUR1 channels in the absence of Mg2+ (A); or for wild-type (○, n = 8), hetT293N (•, n = 5) or homT293N (▪, n = 5) currents in the presence of Mg2+ (B). The lines are drawn to equation 1, with the following parameters. A: wild-type (IC50 = 5 µM, h = 0.9), hetT293N (IC50 = 36 µM, h = 1.2), homoT293N (IC50 = 793 µM, h = 1.5, a = 0.03). B: Kir6.2/SUR1 (IC50 = 14 µM, h = 1) and het293N (IC50 = 391 µM, h = 0.7, a = 0.2). The line through the hom293N data is drawn by hand. Download figure Download PowerPoint Table 1. Characteristics of mutations IC50 (µM; 0 mM Mg2+) IC50 (µM; 2 mM Mg2+) %I (3 mM MgATP) WT 6 ± 1 (n = 5) 17 ± 2 (n = 8) 0.8 ± 0.3 (n = 8) hetT293N 37 ± 7 (n = 6) 324 ± 48 (n = 5) 34 ± 2 (n = 5) homT293N 778 ± 92 (n = 5) n.m. >90% IC50 (µM; 0 mM Mg2+) IC50 (µM; 2 mM Mg2+) %I (3 mM MgATP) WT 6 ± 1 (n = 10) 16 ± 2 (n = 10) 0.7 ± 0.3 (n = 10) hetT294M 6 ± 3 (n = 6) 7 ± 2 (n = 10) 1.0 ± 0.4 (n = 10) Values in parentheses indicate the number of oocytes. n.m., not meausureable. %I = current recorded in an inside-out patch in the presence of 3 mM MgATP expressed as a percentage of that in the absence of MgATP. The values were calculated from the fit of equation 1 to the data obtained for individual oocytes. In the intact pancreatic β-cell, the effect of the T293N mutation on ATP inhibition at Kir6.2 will be modified by the stimulatory effect of MgATP at SUR1. Thus, we next compared ATP concentration–response curves in the presence of Mg2+ (Fig 3B). HomT293N currents were significantly less ATP sensitive than in the absence of Mg2+, being blocked less than 20% even at MgATP concentrations as high as 10 mM. The IC50 for hetT293N channels (324 µM) was also less than that of wild-type channels (17 µM; Table 1). As found for other mutations causing neonatal diabetes (Proks et al, 2005a), the increase in the IC50 produced by Mg2+ was greater for hetT293N channels (nine-fold) than for wild-type channels (3-fold) (compare Fig 4A and B). In addition, a large fraction of hetT293N current was unblocked at 3 mM MgATP (34 ± 2%, n = 5). Figure 4. Effects of Mg2+ on ATP sensitivity. Comparison of ATP concentration–response curves of wild-type (A) het293N (B) and hetT294M (C) channels in the absence (○) and presence (•) of Mg2+. Same data as in Fig 2 and 3. Download figure Download PowerPoint As Mg2+ is always present inside the cell, and ATP concentrations probably lie above 1 mM in physiological conditions (Gribble et al, 2000), these results suggest that there will be a substantial increase in the resting whole-cell KATP currents for both hetT293N and homT293N channels, as observed experimentally (Fig 2A–C). T293N channels have an increased intrinsic open probability Mutations that reduce the ATP sensitivity of the KATP channel can act by impairing ATP binding or transduction, or by influencing the intrinsic (ligand-independent) gating of the channel (Ashcroft 2007; Masia et al, 2007; Proks et al, 2005b; Shimomura et al, 2006). We therefore examined the effect of the T293N mutation on the single-channel kinetics. Experiments were carried out in the absence of ATP, where intrinsic gating can be assessed. We found that the intrinsic Po(0) was markedly increased by the T293N mutation (Fig 5), being 0.87 ± 0.03 (n = 5) for homT293N compared with 0.27 ± 0.04 (n = 6) for wild-type channels. This increase in Po(0) is sufficient to account for the lower ATP sensitivity of the mutant channel (Enkvetchakul et al, 2000; Proks et al, 2005b). Figure 5. The T293N mutation increases Po(0). Representative single KATP channel currents recorded at −60 mV from inside-out patches from oocytes expressing SUR1 plus either Kir6.2 or Kir6.2-T293N. The dashed line indicates the zero current level. Download figure Download PowerPoint The increased Po(0) is produced by an increase in the duration of the bursts of openings and a decrease in the duration and frequency of the long closed states. Mean burst duration was 16 ± 3 ms (n = 5) for wild-type channels and 399 ± 19 ms (n = 5) for homT293N channels. Wild-type channels had two interburst states of 4.2 ± 1.3 and 59 ± 3 ms (n = 5), with relative frequencies of 10 and 5% respectively (the balance being made up of intraburst closed times). In contrast, homT293N channels had a single interburst state of 13 ± 1 ms (n = 5) with a frequency of 0.9%. T294M channels are expressed at the surface membrane No currents were detected in inside-out patches excised from oocytes expressing homT294M channels, even in the absence of added nucleotide. This might reflect a reduced density of mutant channels in the plasma membrane or the mutant channels could be present but permanently closed (due to a markedly decreased Po(0)). To determine which of these possibilities was correct, we measured the surface expression of T294M channels in COS-7 cells. As previously reported (Zerangue et al, 1999), Kir6.2 did not reach the surface membrane in the absence of SUR1, but was easily detected when Kir6.2 and SUR1 were co-expressed (Fig 6A). Co-expression of SUR1 with Kir6.2-T294M resulted in less surface expression, but this was still substantial. This suggests that the lack of homT294M currents observed in excised patches is, in part, due to a lower channel density, caused by impaired trafficking of channels to the plasma membrane. Nevertheless, the fact that the expression is 60% of wild-type indicates many homT294M channels are present, but that they are closed (i.e. they have a Po(0) of zero). Figure 6. Effects of the T294M mutation on channel density and ATP sensitivity. A.. Mean surface expression for wild-type and mutant KATP channels in COS-7 cells, measured using a chemiluminnescence assay. Wild-type Kir6.2 was expressed alone or in combination with SUR1. B,C.. Mean relationships between [ATP] and KATP conductance (G) expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2/SUR1 (○, n = 10), and hetKir6.2-T294M/SUR1 (•, n = 6) channels in the absence of Mg2+ (B); or for Kir6.2/SUR1 (○, n = 10), and heterozygous (•, n = 10) Kir6.2-T294M/SUR1 currents in the presence of Mg2+ (C). The lines are drawn to equation 1, with the following parameters. B: Kir6.2/SUR1 (IC50 = 5 µM, h = 1.1), hetT294M (IC50 = 4 µM, h = 0.7). C: Kir6.2/SUR1 (IC50 = 14 µM, h = 1.1), hetT294M (IC50 = 6 µM, h = 0.7). Download figure Download PowerPoint In the heterozygous state, there will be a mixed population of channels that contain a variable number of mutant subunits (from 0 to 4). The Po(0) of hetT294M channels was therefore measured in multi-channel patches (Fig S3 of Supporting information). This represents the average Po(0) of the channel population: it was not different from wild-type, being 0.34 ± 0.03 (n = 15) for hetT294M and 0.27 ± 0.03 (n = 11) for wild-type channels. ATP-sensitivity of hetT294M channels Although no homT294M currents were detected in inside-out patches, large hetT294M currents were observed. Figure 6B and C shows that although the ATP sensitivity of hetT294M currents was similar to wild-type in the absence of Mg2+ (IC50 6 µM in both cases), it was greater than wild-type in the presence of Mg2+ (IC50 = 7 and 16 µM, respectively) (Table 1). However, there was no obvious difference in ATP block
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