A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications
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- Tammaro, P., Flanagan, S.E., Zadek, B. et al. Diabetologia (2008) 51: 802. doi:10.1007/s00125-008-0923-1
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Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects.
The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg−1 day−1). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes.
Heterozygous (het) and homozygous L164P KATP channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients.
Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.
KeywordsKATP channelKCNJ11Kir6.2Neonatal diabetesType 2 diabetes
intracellular ATP concentration
neonatal diabetes with severe neurological complications
ATP-sensitive K+ channel
permanent neonatal diabetes mellitus
channel open probability
ATP-sensitive potassium (KATP) channels link cellular metabolism to membrane electrical activity by regulating K+ fluxes across the plasma membrane . They are found in multiple tissues but are of particular importance in regulating insulin secretion from pancreatic beta cells [1, 2]. At substimulatory glucose concentrations, KATP channels are open so that the membrane potential is hyperpolarised and Ca2+ influx and insulin secretion are prevented . Glucose metabolism enhances ATP production, resulting in closure of KATP channels, stimulation of electrical activity, opening of voltage-gated Ca2+ channels and exocytosis of insulin granules.
KATP channels are hetero-octamers of Kir6.x and sulfonylurea receptor (SUR) subunits [4–6]. Four inwardly rectifying subunits (Kir6.2 in pancreatic beta cells) form the pore of the channel and four auxiliary SUR subunits (SUR1 in pancreatic beta cells) associate with the tetrameric pore and regulate its gating [7–9]. Binding and/or hydrolysis of Mg-nucleotides by the intracellular nucleotide-binding domains (NBDs) of SUR produces channel opening [10–13]. It is believed that reciprocal changes in the intracellular concentrations of ATP and MgADP are involved in the metabolic regulation of KATP channels.
Over the last 4 years, many different missense mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) have been shown to cause permanent neonatal diabetes mellitus (PNDM). This is a rare disorder characterised by high blood glucose levels that manifests within the first 6 months of life. A subgroup of mutations were associated with a more severe clinical profile characterised by Delayed development of motor, intellectual and social skills, muscle weakness, Epilepsy, facial dysmorphism and Neonatal Diabetes (DEND syndrome) [14–16]. Mutations in Kir6.2 have also been found to cause a remitting relapsing form of neonatal diabetes that resembles transient neonatal diabetes mellitus [17, 18].
All Kir6.2 mutations analysed to date were heterozygous and most were de novo mutations [14, 19–21]. In most cases, the diabetes they caused could be successfully treated with sulfonylureas [14, 20, 22], which directly close KATP channels by binding to the SUR1 subunit of the channel . All PNDM mutations result in a reduced KATP channel sensitivity to inhibition by MgATP in vitro [15, 24]. This is expected to cause an increased KATP current amplitude and reduced insulin secretion. Studies to date suggest that the severity of the clinical phenotype reflects the extent of the reduction of the channel ATP sensitivity. Thus, mutations that produce a small increase in KATP current in the presence of physiological concentrations of MgATP (1–5 mmol/l) lead to PNDM, whereas mutations that cause a larger increase in KATP current give rise to DEND syndrome .
In this paper, we identify a KCNJ11 mutation (L164P) that causes neonatal diabetes without obvious neurological complications. In functional studies, we show that the L164P mutation produces a large increase in the resting whole-cell current and a marked reduction in KATP channel sensitivity to inhibition by ATP. These effects are a secondary consequence of an increase in the channel open probability (Po) produced by the mutation. Surprisingly, other mutations that produce a similar increase in Po cause DEND syndrome. The L164P mutant channel was also far less blocked by the sulfonylurea tolbutamide, which explains why the patients were unable to transfer to glibenclamide therapy.
Informed consent was obtained from all individuals investigated (or from their parents if they were children).
Molecular genetic analysis
Genomic DNA was extracted from peripheral lymphocytes using standard procedures. The KCNJ11 gene was amplified and sequenced as described . Other family members were also tested for the novel mutation. Family relationships were confirmed using a combination of six microsatellites on chromosome 11: D11S902, D11S419, D11S1397, D11S1901, D11S921 and D11S1888.
Female Xenopus laevis were anaesthetised with ethyl 3-aminobenzoate methanesulfonate salt (MS222; 2 g/l added to the water). One ovary was removed via a mini-laparotomy, the incision sutured and the animal allowed to recover. Subsequently, animals were operated on for a second time, but under terminal anaesthesia. Immature stage V–VI oocytes were incubated for 60 min with 1 mg/ml collagenase (Type V; Sigma, Poole, UK) and manually defolliculated. All procedures were carried out in accordance with UK Home Office Legislations and the University of Oxford ethical guidelines. Oocytes were coinjected with ∼0.8 ng wild-type or mutant Kir6.2 mRNA and ∼4 ng mRNA encoding SUR. The final injection volume was 50 nl per oocyte. Isolated oocytes were maintained in Barth’s solution and studied 1–4 days after injection.
Wild-type or mutant Kir6.2 (GenBank D50581) were coexpressed with SUR1 in Xenopus oocytes (GenBank L40624) as described . Because all the patients were heterozygous for the L164P mutation, their pancreatic beta cells will contain a mixture of wild-type and mutant Kir6.2 subunits. To simulate this heterozygosity, we coinjected Xenopus oocytes with a 1:1 mixture of mutant and wild-type Kir6.2 together with SUR1 mRNA. This is expected to give rise to a mixed population of channels composed of homomeric wild-type channels, homomeric mutant channels and heteromeric channels containing between one and three mutant subunits . We refer to this global channel population as heterozygous channels.
Whole-cell currents were recorded using a two-electrode voltage clamp in response to voltage steps of ±20 mV from a holding potential of −10 mV, filtered at 1 kHz and digitised at 4 kHz. Oocytes were perfused with a solution containing (in mmol/l): 90 KCl, 1 MgCl2, 1.8 CaCl2 and 5 HEPES (pH 7.4 with KOH). Metabolic inhibition was produced by 3 mmol/l sodium azide.
Macroscopic currents were recorded from giant inside-out patches using an EPC10 amplifier (List Medical Electronics, Darmstadt, Germany) controlled with Pulse v8.74 software (Heka Electronik, Lambrecht, Germany). Macroscopic currents were elicited by 3 s voltage ramps from −110 to +100 mV (holding potential 0 mV), or recorded at a constant potential of −60 mV. They were filtered at 0.5 kHz and digitised at 1 kHz. The pipette solution contained (mmol/l): 140 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with KOH). The internal (bath) solution contained (mmol/l): 107 KCl, 1 K2SO4, 2 MgCl2, 10 EGTA, 10 HEPES (pH 7.2 with KOH) and Mg-nucleotides as indicated. Experiments were conducted at 20–22°C. Solutions were changed using a local perfusion system consisting of tubes of 200 μm diameter into which the tip of the patch pipette was inserted.
Single-channel currents were measured at −60 mV, filtered at 5 kHz and digitised at 20 kHz. Unitary amplitude and Po were measured from the Gaussian fit to all-points amplitude histograms of tracts of current of 30–90 s duration.
Data were analysed with in-house routines developed in the IgorPro platform (Wavematrics, Portland, OR, USA). Data are given as means ± SEM in the text and in the figures. Statistical significance was evaluated using a two-tailed Student t test and p < 0.05 taken to indicate a significant difference.
Patient characteristics and genetics
Three unrelated probands with permanent neonatal diabetes were shown to be heterozygous for the KCNJ11 gene mutation L164P (c.491T>C, p.Leu164Pro). All were female and had unaffected parents of different ethnic origin. Two patients have been reported previously [27, 28]. Mutation testing and microsatellite analysis of DNA from both parents and the child for two of the cases confirmed that the mutation had arisen de novo (the parents of the third case were not available for testing). None of the patients had any neurological complications or obvious developmental delay.
The first patient is currently 8 years old. She was born in Singapore from Sri Lankan parents, at 38 weeks of gestation with a birthweight of 2.6 kg. She developed diabetes at 30 weeks of age. When she came to Australia at the age of 6.5 years, her HbA1c level was elevated (7.4%) and her diabetes required insulin treatment (0.45 U kg−1 day−1). Glibenclamide treatment (1.0 mg kg−1 day−1) was trialled for 2 months (at 7 years of age) but did not produce a decrease in her insulin requirement. She continues to require insulin (up to 0.7 U kg−1 day−1) with an HbA1c ranging from 7.2 to 8.2%. She has normal developmental milestones, her neurological examination is normal and she has no evidence of diabetic complications.
The second patient, born in Australia of Afghan origin, is currently 2.7 years old. She was born at term following an uneventful pregnancy, with a birthweight of 2.7 kg. She presented at 8 weeks in diabetic ketoacidosis and was subsequently treated (at 2.5 months for 4 weeks, then at 5 months for 3 months) with glibenclamide (up to 1.1 mg kg−1 day−1, regimens of two times per day or three times per day), but had no reduction in insulin requirement (0.4 U kg−1 day−1) . Her mean HbA1c since diagnosis has been 7.6% (range 7.2–8.7%). Assessment at 5 months by a neurologist was entirely normal, with a normal EEG. Subsequently, she has had normal developmental milestones and her neurological examinations have been normal at all follow up visits every 3 months. Diabetic complications have not been assessed due to the young age of the patient.
The third patient is from Slovakia . She was born at term with a birthweight of 2.6 kg, and was diagnosed with diabetes at 5 weeks of age. She is currently 20 years old and is treated with insulin (1.24 U kg−1 day−1). Glibenclamide transfer was not attempted because the patient also suffers from hepatitis C. She has poor glycaemic control, with an HbA1c level of 15.2%, and various diabetic complications including retinopathy and nephropathy. Psychomotoric development in childhood was normal. A neurological examination suggested the presence of diabetic motoric polyneuropathy in the lower extremities but there were no further neurological findings. Although her IQ was not tested, she successfully completed a specialised business school training suggesting normal mental development.
Functional analysis: effects on whole-cell KATP currents
The sulfonylurea tolbutamide (0.5 mmol/l) blocked whole-cell KATP currents by 98% but had no effect on homL164P currents (4% block). HetL164P channels were blocked by only 54% (Fig. 1).
Functional analysis: effects on KATP channel ATP sensitivity
ATP sensitivity of wild-type and mutant channels
IC50 (2 mmol/l Mg2+)
h (2 mmol/l Mg2+)
A (2 mmol/l Mg2+)
%Imax (3 mmol/l MgATP)
9.6 ± 1.6
1.30 ± 0.08
15.8 ± 3.0
0.99 ± 0.05
0.01 ± 0.01
100 ± 8*
1.10 ± 0.05
118 ± 29*
1.16 ± 0.34
0.29 ± 0.07*
33.6 ± 3.4*
98 ± 1*
Molecular mechanism of the reduced ATP sensitivity
Mutations that reduce the ATP sensitivity of the KATP channel can act in several ways. They may prevent ATP binding directly. They may impair the mechanism by which nucleotide binding is coupled to channel gating. They may also stabilise the intrinsic open state of the channel (i.e. that in the absence of ATP), which shifts the gating equilibrium in the presence of ATP towards channel opening and thus indirectly reduces the channel ATP sensitivity (e.g. [15, 30, 31]).
Single-channel parameters for wild-type and homL164P channels
0.39 ± 0.05
4.0 ± 0.1
0.86 ± 0.01*
4.1 ± 0.1
Effects on KATP channel ATP sensitivity in the presence of Mg2+
Previous studies have shown that Kir6.2 mutations associated with neonatal diabetes may not only decrease the sensitivity of Kir6.2 to ATP, but can also enhance channel activation by Mg-nucleotides . We therefore explored the effect of the L164P mutation on the ATP sensitivity in the presence of 2 mmol/l Mg2+.
Surprisingly, there was no difference in the effect of ATP in the presence and absence of Mg2+ on homL164P channels (Fig. 2c,d). This contrasts with what has been observed for mutations in the ATP-binding site that completely abolished ATP inhibition in the absence of Mg2+, such as R50P and G334D: channels containing these mutations were activated by MgATP [33, 34]. The IC50 for ATP inhibition of hetL164P channels was also not significantly affected by Mg2+, although the pedestal of unblocked current at high ATP concentrations was increased by about 50% (Table 1). At 3 mmol/l MgATP, a concentration within the physiological range, the unblocked current was 34%, substantially greater than that found for wild-type channels (<1%; Table 1).
Functional analysis: effects on MgADP sensitivity
Despite the fact that 100 μmol/l MgATP blocked mutant channels less, the lower extent of activation of hetL164P channels by 30 μmol/l MgADP is not due to the fact that channel activity is already maximal, as the amplitude is still significantly less than that in control solution (Fig. 5).
We describe the clinical and functional effects of a KCNJ11 mutation, L164P, associated with permanent neonatal diabetes. This mutation causes a marked reduction in KATP channel inhibition by ATP primarily by stabilising the open state of the channel. This leads to an increase in the whole-cell KATP current, and in beta cells is expected to result in a reduction in insulin secretion. Surprisingly, unlike other mutations that reduced the channel ATP sensitivity by a similar amount, no motor or mental developmental delay was associated with the L164P mutation.
Structural considerations and molecular basis for reduced ATP sensitivity
In a homology model of Kir6.2 , L164 lies partway along the permeation pathway, 35 Å away from the ATP-binding site (Fig. 3). It is therefore unlikely that it acts by reducing ATP binding directly. The side-chains of L164 point into the pore, forming a hydrophobic girdle that is narrow enough to prevent the passage of water and hydrated K+ ions , which suggests that L164 may form a hydrophobic gate within the pore. Functional studies support this idea. Following mutation of L164 to cysteine, cadmium ions were able to block the KATP channel with high affinity [36, 37], suggesting that the four cysteines (one on each subunit) come together close enough to form a high-affinity binding site for Cd2+. These data indicate that the side-chain of L164 must face into the pore, and that the pore is very narrow at this position. Substitution of a proline for L164 is expected to produce a kink in the α-helix  and disrupt the hydrophobic gate. It is therefore not surprising that the L164P mutation caused a dramatic effect on the Po. Mutation of L164 to cysteine, alanine, valine, threonine or glycine also produces a very large increase in Po [30, 36, 37, 39], consistent with the importance of this residue in channel gating.
The fact that the L164P mutation enhanced Po can explain, in part, the reduced ATP sensitivity of the channel. It is also possible that the mutation may have effects additional to stabilisation of the channel open state.
Effect of Mg2+ on Kir6.2-L164P/SUR1 channel ATP sensitivity
In contrast to all PNDM mutations studied to date , MgATP was unable to enhance the activity of either homL164P or hetL164P channels. The lack of MgATP activation of homL164P channels may be due to the fact that Po is already very high and thus there is no scope for further activation. However, little MgATP activation was also observed for hetL164P channels. This is in marked contrast to other mutations that caused enhanced Po (I296L, , V59G ) where MgATP activated both homomeric and heterozygous channels. It is therefore possible that the L164P mutation reduces the efficacy of coupling between SUR1 and Kir6.2, and thereby decreases the ability of MgATP to stimulate channel activity. In support of this idea, MgADP activation of hetL164P channels was also reduced. This may explain the inability of Mg2+ to reduce ATP inhibition of hetL164P channels, since it is well established that MgATP must be hydrolysed to MgADP to stimulate channel activity.
The reason for the lack of Mg-nucleotide activation of hetL164P channels is unclear. Because L164 lies within the pore, far away from the NBDs of SUR1, the effect must be mediated allosterically. It is possible that this is mediated by an interaction between the backbone of L164 itself, or transmembrane domain 2 (within which L164 lies), and the transmembrane domains of SUR1. In the absence of an atomic resolution structure of the KATP channel complex, however, this cannot be definitely determined.
HomL164P channels were not blocked by tolbutamide, as expected because of their high Po . The lower efficacy of tolbutamide on hetL164P channels (about 50% block) may reflect the enhanced Po of channels within the heterozygous population containing mutant subunits. It could also reflect impaired coupling between SUR1 and Kir6.2, such as that found for MgADP.
The reason for this anomaly is not clear, particularly as the resting whole-cell current was also very large. Previous studies have also indicated that such large whole-cell currents are invariably associated with more severe clinical symptoms. The lack of responsiveness of the patient to sulfonylureas is consistent with the very large increase in Po and the resting whole-cell current. It is therefore likely that hetL164P channels expressed in Xenopus oocytes are a reasonable model for the pancreatic beta cell KATP channels of the patients. We are therefore forced to conclude that compensatory mechanisms must ameliorate the extra-pancreatic effects of this mutation. The fact that the patients had very different ethnic origins suggests the compensatory mechanism is unlikely to reflect a shared genetic background: however, it does appear to be specific to the L164P mutation, which is unique (to date) in having a marked functional effect in vitro despite causing neonatal diabetes without neurological complications.
Importantly, a maximally effective concentration of tolbutamide only blocked hetL164P channels by 50%. All patients to date whose channels are blocked by <65% have not been able to transfer from insulin treatment to sulfonylurea therapy. This suggested that our patients would be unable to transfer to sulfonylurea therapy, as indeed was found to be the case. This is likely to be due to the fact that L164P destabilises the long closed state of the channel, to which sulfonylureas preferentially bind, and which is rarely entered in channels with enhanced Po. Our results further suggest that not all patients with permanent neonatal diabetes will necessarily respond to sulfonylurea treatment, and indicates that knowledge of the functional effect of the mutation is helpful for predicting the drug response in patients.
We thank the patients and their referring clinicians. Financial support was provided by the Wellcome Trust (F. M. Ashcroft, A. T. Hattersley), the Royal Society (F. M. Ashcroft), the European Union (Integrated Project EuroDia LSHM-CT-2006–518153 in the Framework Programme 6 [FP6]) of the European-Community (F. M. Ashcroft, A. T. Hattersley), the Sir Graham Wilkins studentship (S. E. Flanagan) and research grants from the Slovak Research and Development Agency (51–014205; I. Klimes) and Slovak Ministry of Health (MZ.2005/15-NEDU-01; I. Klimes). P. Tammaro holds a Junior Research Fellowship at the Wolfson College, B. Zadek holds an OXION scholarship, A. T. Hattersley is a Wellcome Trust Research Leave Fellow and F. M. Ashcroft is a Royal Society Research Professor.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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