Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 8, 2005
Human Molecular Genetics 2005 14(18):2717-2726; doi:10.1093/hmg/ddi305

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Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP

Peter Proks, Christophe Girard and Frances M. Ashcroft^*

University Laboratory of Physiology, Oxford University, Parks Road, Oxford, OX1 3PT, UK

^* To whom correspondence should be addressed. Tel: +44 1865285810; Fax: +44 1865285812; Email: frances.ashcroft{at}physiol.ox.ac.uk

Received June 10, 2005; Accepted August 3, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K_ATP) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg²⁺, to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic ß-cells, K_ATP channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K_ATP channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg²⁺ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K_ATP channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K_ATP current at physiological MgATP concentrations correlates with the severity of the clinical phenotype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
Neonatal diabetes diagnosed within the first 3 months of life is usually a single gene disorder associated with impaired beta-cell function. Recently, heterozygous gain-of-function mutations in KCNJ11 were discovered to be a common cause of permanent neonatal diabetes (PNDM) (1–5). Mutations in the same gene also give rise to a spectrum of other diabetic conditions including transient neonatal diabetes (6,7), childhood diabetes (7) and adult-onset diabetes (7). In some patients, PNDM is accompanied by developmental delay (1,2,4) or by a severe neurological phenotype consisting of marked developmental delay, motor weakness and epilepsy (DEND syndrome) (1,8). KCNJ11 encodes Kir6.2, which serves as the pore-forming subunit of the ATP-sensitive K⁺ (K_ATP) channel in multiple tissues (9,10). This channel is a hetero-octameric structure comprising four Kir6.2 subunits and four regulatory sulphonylurea receptor (SUR) subunits (11). There are two different SUR genes: SUR1 is expressed in ß-cells and many brain neurones (12), and SUR2 is expressed in the heart, smooth muscle, skeletal muscle and the brain (13,14).

K_ATP channels mediate glucose-induced insulin secretion from pancreatic ß-cells by coupling cell metabolism to electrical activity of the plasma membrane (15–17). In the absence of glucose, K_ATP channels are open and keep the membrane potential at a negative level at which voltage-gated calcium channels are shut and electrical activity and insulin secretion are prevented. Conversely, the increase in glucose metabolism that results from elevation of plasma glucose closes ß-cell K_ATP channels, and leads to a membrane depolarization that opens Ca²⁺ channels, stimulates electrical activity and Ca²⁺ influx and triggers insulin release. Thus, gain-of-function mutations in Kir6.2 are predicted to result in impaired insulin secretion and diabetes, as is indeed found both in humans (1–8) and in transgenic mice (18). K_ATP channels are also closed by sulphonylurea and glinide drugs, which stimulate insulin secretion by binding to the SUR subunit of the K_ATP channel. They are widely used to treat Type 2 diabetes (19) and are also effective in PNDM (2,5,20).

A large number of cytosolic substances modulate the opening and closing of K_ATP channels, but among the most important are the adenine nucleotides, which are believed to mediate the metabolic regulation of K_ATP channel activity (16,17). These have both stimulatory and inhibitory effects on channel activity. Binding of ATP (or ADP) to Kir6.2 results in channel closure (21): this process does not require Mg²⁺ as when a truncated version of Kir6.2 (Kir6.2C) is expressed in the absence of SUR the ATP sensitivity of the channel is the same whether or not Mg²⁺ is present (22). Conversely, interaction of Mg-nucleotides (MgATP, MgADP) with the nucleotide-binding domains of SUR1 increases channel activity (21–25), and evidence suggests that MgATP must be hydrolyzed to MgADP in order to stimulate channel activity (22,26). Thus, in the presence of Mg²⁺, K_ATP channel activity is governed by the balance between the stimulatory and inhibitory effects of nucleotides. Functional analysis of cloned K_ATP channels has revealed that all KCNJ11 mutations examined to date produce a marked decrease in the ability of ATP to block the K_ATP channel, either by directly impairing ATP binding to Kir6.2 or as a secondary consequence of altering the intrinsic gating of the channel (1,6,27,28). Most of these studies were carried out in the absence of Mg²⁺, in order to study the inhibitory effects of ATP on Kir6.2 in isolation from the stimulatory effects of MgATP mediated by SUR. However, it is possible that KCNJ11 mutations may also influence the interaction of Kir6.2 with SUR1, and thereby modulate the stimulatory action of Mg-nucleotides. Thus, in this article, we studied the effect of MgATP on K_ATP channels carrying KCNJ11 mutations.

We focused on the two Kir6.2 residues that are most commonly mutated: R201 and V59. Mutations at R201 have been found in 24 patients (1,3,4,29,30). All but three have PNDM without neurological features. Mutations at V59 cause a more severe phenotype. In 10/13 patients with the V59M mutation, neonatal diabetes was accompanied by developmental delay (1,2,4); one patient with the V59G mutation had DEND syndrome (1). To simulate the heterozygous state found in all patients, we coexpressed wild-type and mutant Kir6.2 with SUR1 and we refer to the mixed population of homomeric and heteromeric channels that results as heterozygous channels.

The results reported here show that Mg²⁺ produces a much greater shift in the ATP sensitivity of heterozygous K_ATP channels carrying KCNJ11 mutations than in that of the wild-type channel. This suggests that, in addition to reducing ATP block at Kir6.2, the mutations markedly enhance the stimulatory effect of Mg-nucleotides mediated by SUR1. As a consequence, K_ATP currents at ATP concentrations within the physiological range are strikingly increased. This is expected to hyperpolarize the pancreatic ß-cell and contribute to the reduced insulin secretion found in patients with KCNJ11 mutations. There was a good correlation between the current magnitude through heterozygous channels at physiological [ATP]_i and disease severity.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
Comparison of MgATP and ATP sensitivity
We first compared ATP concentration-inhibition curves for wild-type, heteterozygous (het) R201C and homomeric (hom) R201C channels in the absence (Fig. 1A) and presence (Fig. 1B) of 2 mM Mg²⁺. As previously reported, the cation caused a small shift in the IC₅₀ for ATP block of wild-type channels (from 7 µM to 13 µM, Table 1) (22). It is evident that both homR201C and hetR201C channels are very much less sensitive to ATP in the presence of Mg²⁺ than in its absence (Table 1). In addition, this effect is greater than for wild-type channels: on addition of Mg²⁺, the IC₅₀ increased 24-fold for homR201C and 30-fold for hetR201C, compared with 2-fold for wild-type channels. Importantly, there was a much greater difference in the ATP sensitivity of hetR201C channels in the presence (IC₅₀=307 µM) than in the absence (11 µM) of Mg²⁺. The physiological implications of this finding are discussed in detail subsequently.

View larger version (23K): [in this window] [in a new window]   Figure 1. (A and B) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for KATP channels comprising SUR1 and either wild-type (WT, , n=6), hetR201C (, n=6) or homR201C (, n=5) Kir6.2, in the absence (A) and presence (B) of Mg2+. The smooth curves are the best fit of Eq. 1 to the data. For wild-type, IC50=6.6 µM (A), 13 µM (B); h=1.07 (A), 1.02 (B). For hetR201C, IC50=10.4 µM (A), 307 µM (B); h=1.0 (A), 0.76 (B). For homR201C, IC50=102 µM (A), 2.4 mM (B); h=1.3 (A), 1.0 (B). The grey bars illustrate the physiological range of [ATP] in ß-cells. (C) Mean relationship between [MgATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for Kir6.2-R201C/SUR1 channels in the presence (, n=6) and absence (, n=5) of 10 µM LY292004 (LY). The smooth curves are the best fit of Eq. 1 to the data. IC50=2.4 mM (–LY); 2.0 mM (+LY); h=1.0 (–LY), 1.08 (+LY). (D) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2-R201C/SUR1 channels measured in the absence (, n=5) and presence (, n=5) of Mg2+, and for Kir6.2-R201C/SUR1-KA/KM channels in the absence (, n=6) and presence of Mg2+ (, n=6) channels. The smooth curves are the best fit of Eq. 1 to the data. For Kir6.2-R201C/SUR1, IC50=2.4 mM, h=1.0 (2 mM Mg2+) and IC50=102 µM, h=1.3 (0 mM Mg2+). For Kir6.2-R201C/SUR1-KA/KM, IC50=208 µM, h=1.96 (0 mM Mg2+) and IC50=147 µM, h=1.08 (2 mM Mg2+).

 

View this table: [in this window] [in a new window]   Table 1. ATP concentrations causing half-maximal inhibition (IC50) of wild-type, heterozygous and homomeric Kir6.2/SUR1 mutant channels measured in the absence and presence of Mg2+. n=4–11

 
Role of phosphoinositides
One mechanism by which MgATP could enhance K_ATP channel activity is via synthesis of membrane phosphoinositides such as PIP₃ and PIP₂, which enhance the activity and decrease the ATP sensitivity, of the K_ATP channel by interacting with Kir6.2 (31–33). In this case, the greater effect of MgATP on R201C channels could reflect an enhanced sensitivity to phosphoinositides. We tested this possibility using the lipid kinase inhibitor LY294002. Figure 1C shows that the MgATP sensitivity of homR201C channels was not affected by the presence of 10 µM LY294002, which blocks PIP₃ production. In the presence of the lipid kinase inhibitor, the IC₅₀ for ATP inhibition was 2.2±0.4 mM (n=6), not significantly different from that found in its absence (2.4 mM). Furthermore, 100 µM LY294002, which blocks both PIP₂ and PIP₃ production, had no effect on K_ATP channel activity in the presence of either 1 or 10 mM MgATP (n=5).

Role of SUR1
An alternative explanation for the difference in ATP-sensitivity in the presence and absence of Mg²⁺ is that MgATP hydrolysis by the nucleotide-binding domains (NBDs) of SUR1 leads to the generation of MgADP, which stimulates K_ATP channel activity and shifts the ATP dose-inhibition curve to the right (21–23,25). To address this possibility, we used a mutated form of SUR1 that is not modulated by Mg-nucleotides (24). In this construct (SUR1-KA/KM), two lysine residues critical for ATP binding/hydrolysis, K719 and K1384, were mutated (to alanine and methionine, respectively). There was no significant difference in the IC₅₀ for ATP inhibition of Kir6.2-R201C/SUR1-KAKM channels in the presence and absence of Mg²⁺ (Fig. 1D), as expected if these mutations abolish K_ATP channel activation by MgATP: the IC₅₀ were 231±50 µM (n=6) and 150±27 µM (n=6), respectively. There was also no significant difference in the IC₅₀ for ATP inhibition of Kir6.2-R201C/SUR1-KAKM channels in the presence of Mg²⁺ (150±27 µM, n=6) from that of Kir6.2-R201C/SUR1 channels in the absence of the cation (106±12 µM, n=6). However, in all cases the IC₅₀ was substantially less than that of Kir6.2-R201C/SUR1 channels in the presence of Mg²⁺ (2.4 mM). This is consistent with the idea that the large reduction in the ATP sensitivity of R201C channels produced by Mg²⁺ is mainly conferred by MgATP binding/hydrolysis at the NBDs of SUR1. In other words, the mutation enhances the stimulatory effect of MgATP that is mediated via SUR1, thereby producing an apparent reduction in the inhibitory effect of ATP when Mg²⁺ is present.

MgATP activation is altered by other gain-of-function mutations in Kir6.2
We next explored the effect of Mg²⁺ on the ATP-sensitivity of K_ATP channels containing other Kir6.2 mutations. Figure 2 compares ATP concentration-inhibition curves for channels homomeric for the R201C and R201H mutations, which cause neonatal diabetes alone, for V59M, which causes neonatal diabetes with developmental delay, and for V59G, which causes DEND syndrome. Equivalent data for wild-type, hetR201H, hetV59M and hetV59G channels are shown in Figure 3 (Fig. 1A and B for R201C data). In all cases, the cation causes a large reduction in the ATP sensitivity of both heterozygous and homomeric mutant channels, and (with the exception of V59M), this effect is much greater than for the wild-type channel (Table 1). This is consistent with the idea that MgATP activation is enhanced by the R201H, V59M and V59G mutations, as it is for R201C. However, the extent of the shift in the IC₅₀ varies, from 2-fold for wild-type and hetV59M channels to 13-fold for het R201H, and almost 40-fold for hetV59G channels. In some cases, a clear pedestal, corresponding to current that is unblocked even at saturating [ATP], is observed. This is particularly evident for hetV59M and hetV59G channels (Fig. 3), perhaps because the homomeric channels are so much less sensitive to MgATP.

View larger version (25K): [in this window] [in a new window]   Figure 2. Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for the indicated homomeric mutant channels in the presence (•) or absence of Mg2+ (). The smooth curves are the best fit of Eq. 1 to the data. For homR201C, IC50=102 µM, h=1.3 (–Mg2+) and IC50=2.4 mM, h=1.0 (+Mg2+). For homR201H, IC50=296 µM (–Mg2+), 1.95 mM (+Mg2+); h=1.8 (–Mg2+), 0.8 (+Mg2+). For homV59M, IC50=60 µM (–Mg2+), 440 µM (+Mg2+); h=0.97 (–Mg2+), 0.77 (+Mg2+). For homV59G, IC50=8.1 mM and h=0.75 (–Mg2+); in the presence of Mg2+ the line was drawn by hand. Data in the absence of Mg2+ are the same as those given by Proks et al. (27) (R201C, V59G, Q52R). The number of patches is indicated by each graph.

 

View larger version (26K): [in this window] [in a new window]   Figure 3. Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for the indicated heterozygous mutant channels in the presence () or absence of Mg2+ (). For hetV59M, IC50=17 µM (–Mg2+), 62 µM (+Mg2+); h=0.94 (–Mg2+), 0.9 (+Mg2+). The value of a for hetV59M in the presence of Mg2+ was 0.1. For hetV59G, IC50=26 µM (–Mg2+), 319 µM (+Mg2+); h=1.18 (–Mg2+), 0.8 (+Mg2+); in the absence of Mg2+ the curve was fitted assuming that 1/16 of the heterozygous population are homomeric mutant channels (IC50=8.1 mM and h=0.75); in the presence of Mg2+ a=0.3. Data in the absence of Mg2+ are the same as those given by Proks et al. (27) (R201C, V59G, Q52R). The number of patches is indicated by each graph.

 
Figure 4 compares MgATP concentration-inhibition curves for wild-type, heterozygous and homomeric channels for the V59M and V59G mutations (Fig. 1B for R201C). Because MgATP causes much less (apparent) inhibition of mutant channels than ATP, a significant amount of current remains unblocked at ATP concentrations within the physiological range (1–10 mM, grey bars). It is also clear that the magnitude of the heterozygous currents in the presence of physiological concentrations of MgATP is correlated with disease severity, being greatest for V59G, intermediate for V59M and smallest for R201C and R201H. Mean data are given in Table 2, which also gives values published for other Kir6.2 mutations that cause permanent or transient neonatal diabetes (6,27,28). Table 2 also shows data obtained for the corresponding homomeric channels.

View larger version (16K): [in this window] [in a new window]   Figure 4. Mean relationship between [MgATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for wild-type channels () and the indicated heterozygous () and homomeric () mutant channels. The data are the same as those in Figures 2 and 3. The grey bars illustrate the physiological range of [ATP] in ß-cells.

 

View this table: [in this window] [in a new window]   Table 2. Fraction of unblocked KATP conductance measured in excised patches in the presence of 1,3 or 5 mM MgATP for channels containing SUR1 and either wild-type Kir6.2, or homomeric (A) or heterozygous (B) Kir6.2 mutant subunits. Some data are recalculated from refs (6,27,28). In some cases, data at 3 mM or 5 mM ATP were not available. In these cases, we estimated the value from the fitted dose-response curve

 
MgATP sensitivity determines whole-cell current magnitude
In oocytes, whole-cell K_ATP currents are normally blocked by the resting ATP concentration, but they can be activated by the metabolic inhibitor azide, which reduces cellular ATP levels. Owing to their reduced ATP sensitivity, however, significant resting currents can be recorded from KCNJ11 mutant channels. Finally, we compared the magnitude of the resting K_ATP current measured in whole-cell recordings with the fraction of K_ATP current that was not blocked by 3 mM MgATP in the inside-out patch. We chose 3 mM ATP as being close to that measured in Xenopus oocytes (34,35) and within the range of ATP concentrations measured in pancreatic ß-cells (36,37). To control for possible differences in K_ATP channel expression between oocytes, we expressed the whole-cell current in the absence of azide as a fraction of that in the presence of the metabolic inhibitor (Table 3). As Figure 5 shows, there was a linear relationship between the whole-cell and excised patch data: the less sensitive the K_ATP current is to MgATP inhibition the greater is the whole-cell current. This suggests that the whole-cell current magnitude is largely determined by the MgATP sensitivity of the K_ATP channel.

View this table: [in this window] [in a new window]   Table 3. Whole-cell KATP current measured in two-electrode voltage clamp for KATP channels composed of SUR1 and either wild-type Kir6.2, homomeric (A) or heterozygous (B) Kir6.2 mutant subunits. The resting current (in the absence of azide) is expressed as a fraction of that measured after azide application. Some data are recalculated from refs (6,27,28)

 

View larger version (14K): [in this window] [in a new window]   Figure 5. Relationship between the fraction of unblocked KATP conductance (G/GC) in the excised patch in the presence of 3 mM MgATP, and the resting whole-cell current, expressed as a fraction of that in the presence of 3 mM azide. Most data points are the mean of 5–12 experiments; in some cases, when the G/GC value in the excised patch at 3 mM MgATP was not available, we used the extrapolated value from the fitted dose-response curve and the standard error value was taken from the data point at 1 mM MgATP. The line is a linear fit with a slope of 1.75; the regression coefficient is 0.92.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
Our data demonstrate that the ATP sensitivity of K_ATP channels containing gain-of-function mutations in Kir6.2 is much less in the presence of Mg²⁺ than in its absence. This is due to an enhanced stimulatory action of MgATP that is mediated via the nucleotide-binding domains of SUR1. Thus KCNJ11 mutations reduce the overall sensitivity of the K_ATP channel to MgATP in two different ways: by decreasing ATP inhibition, as previously described (1,6,27,28) and by increasing MgATP activation, as shown here.

Mechanism of the Mg-dependent shift in ATP sensitivity
Studies of wild-type K_ATP channels have previously shown that Mg²⁺ does not alter the affinity of Kir6.2 for ATP, because the ATP sensitivity of Kir6.2C expressed in the absence of SUR is not affected by Mg²⁺ (22). Although phosphoinositides can shift the ATP sensitivity of K_ATP channels (31–33), our results suggest that an increase in their synthesis also does not underlie the Mg-dependent shift in ATP sensitivity. Instead, Mg-nucleotide interactions with SUR1 are primarily responsible for the shift in the ATP concentration-inhibition curve produced by Mg²⁺. This effect is greater for mutant Kir6.2 channels than for wild-type channels. Furthermore, the fact that the shift in IC₅₀ produced by Mg²⁺ is similar for homomeric and heterozygous channels suggests that a single mutant Kir6.2 subunit is enough to modify the MgATP activation of the channel. This also suggests, at least for the mutant channels studied, that MgATP activation is transduced by a single SUR subunit in the K_ATP channel complex.

There are several possible explanations for how a mutation in Kir6.2 enhances channel activation by MgATP. First, conformational changes in Kir6.2 induced by the mutation may produce secondary allosteric changes in SUR1 conformation that either enhance MgATP binding and/or hydrolysis at the NBDs of SUR1, or facilitate the transduction of ATP binding/hydrolysis into opening of the Kir6.2 pore. Second, conformational changes in SUR1 induced by MgATP binding/hydrolysis may impair ATP binding (and/or transduction) to mutant Kir6.2. It is less likely that Kir6.2 mutations interfere with the mechanism by which SUR1 increases the ATP sensitivity of Kir6.2 (21) because this effect is not Mg-dependent in wild-type Kir6.2/SUR1 channels.

The existence of a pedestal at high [MgATP]_i, which is particularly obvious for hetV59G and hetV59M channels, indicates that these channels cannot be completely closed, even when all four ATP-binding sites are occupied. In the case of heterozygous channels, one obvious possibility is that there is an excess of mutant subunits in the channel population, which biases the ATP sensitivity towards that of the mutant channel. However, this explanation does not seem to apply in the case of hetV59M channels, as both heterozygous and homomeric V59M channels are blocked to a similar extent by 10 mM ATP (this would imply 100% of subunits in the heterozygous population were homV59M which is incompatible with the difference in IC₅₀ between hetV59M and hom V59M channels). An alternative idea is that the pedestal arises from an impaired efficacy of channel closure, as observed previously for the C166S and L164C mutations in Kir6.2C channels (38,39). This is predicted to increase both the IC₅₀ and the pedestal, as is in fact observed for hetV59G channels. A third possibility is that the ratio of the binding constants for ATP binding to the open (K_O) and closed (K_C) states is affected; if K_O increases and K_C stays the same, there can be a substantial effect on the pedestal but not on the IC₅₀. The data obtained for hetV59M (Figs 3 and 4) and hetI296L (28) channels favour this idea.

Implications for action of therapeutic drugs
Sulphonylureas inhibit K_ATP channels by binding to the SUR subunit of the channel (12,21). At saturating sulphonylurea concentrations, this produces 50–70% block of channel activity (40,41). In addition, they prevent the stimulatory action of MgADP at SUR1 (41). Consequently, the inhibitory effect of MgADP on Kir6.2 is unmasked, and this adds to the inhibitory effect of the sulphonylurea itself to produce a complete block of channel activity (40,41). Thus we would expect reduced tolbutamide block of R201C/H channels because ADP binding (like ATP binding) to Kir6.2 is reduced. However, tolbutamide blocks homR201C and hetR201C channels almost as effectively as wild-type channels (27). This suggests that despite enhancing MgADP activation of Kir6.2/SUR1 currents, the mutation does not interfere with the ability of sulphonylureas to abolish MgADP stimulation; and that loss of MgADP activation contributes substantially to sulphonylurea block of mutant K_ATP channels.

Physiological implications
Our results demonstrate that the increase in K_ATP current at physiologically relevant concentrations of ATP produced by KCNJ11 mutations is substantially greater in the presence of Mg²⁺ than in its absence. This suggests that the shift in ATP sensitivity and increase in whole-cell K_ATP current, required to cause neonatal diabetes may be greater than had been anticipated from earlier studies carried out in the absence of Mg²⁺ (27).

The data also reveal that in the simulated heterozygous state there is a good correlation between the severity of the clinical phenotype and the extent of MgATP inhibition. At physiologically relevant concentrations of MgATP (1–5 mM), mutations that cause small increases in K_ATP current result in transient neonatal diabetes, whereas larger increases in current cause neonatal diabetes alone, and an even greater increase is associated with DEND syndrome (Table 1). The variation in K_ATP current in the presence of MgATP which is observed in excised patches is expected to be paralleled by equivalent differences in the whole-cell current in the absence of metabolic inhibition.

In pancreatic ß-cells, an increase in K_ATP current will lead to a smaller membrane depolarization in response to increased metabolism (42). Consequently, electrical activity and insulin secretion will be diminished, and the greater the increase in K_ATP current, the more severely insulin secretion will be impaired. The ß-cell may be especially sensitive to gain-of-function mutations in Kir6.2 as its metabolism has evolved to be very sensitive to blood glucose levels and the resting potential is largely determined by the K_ATP channel.

Kir6.2 is also expressed in skeletal muscle, cardiac muscle and neurons throughout the brain (9,10), a distribution consistent with the neurological symptoms found in DEND syndrome. It seems possible that in these tissues a greater reduction in ATP sensitivity is required to increase the K_ATP current sufficiently to influence electrical activity. This would explain why neurological symptoms only occur with those mutations that have the greatest effects on the MgATP sensitivity of the K_ATP current, and which lead to the largest increases in whole-cell current. There are many possible reasons why K_ATP channels could contribute less to the electrical activity of brain and muscle than pancreatic ß-cells. These include differences in cell metabolism, contributions to membrane current from other ion channels, a low K_ATP channel density and association of Kir6.2 with SUR2 [which reduces the response to metabolism (43)].

	CONCLUSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
In conclusion, KCNJ11 mutations that cause PNDM or DEND syndrome result in enhanced MgATP activation as well as reduced inhibition by ATP. These two effects combine to produce an increased K_ATP current magnitude at physiological [ATP]_i. Because the reduction in ATP sensitivity is much greater when Mg²⁺ is present, it appears that the stimulatory effect of KCNJ11 mutations on MgATP activation may be of greater physiological importance than the decrease in ATP inhibition. We observed a good correlation between the magnitude of the increase in K_ATP current at physiological [ATP]_i and the disease phenotype, with larger currents being associated with DEND syndrome than with neonatal diabetes alone. Our results are also consistent with the idea that mutations/polymorphisms that cause smaller increases in K_ATP current can lead to monogenic diabetes that manifests in later life [childhood or early twenties (7)] and that they may contribute to the development of polygenic Type 2 diabetes (42).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 
Human Kir6.2 (GenBank accession no. NM000525 with E23 and I377) and rat SUR1 [GenBank accession no. L40624 (29)] were used in this study. Site-directed mutagenesis of Kir6.2 was performed using the QuickChange^TMXL system (Stratagene). Wild-type and mutant cDNAs were cloned in the pBF vector, and capped mRNA prepared using the mMESSAGE mMACHINE large scale in vitro transcription kit (Ambion, Austin, TX, USA), as previously described (24).

Currents were recorded from Xenopus laevis oocytes 1–3 days after injection with 0.8 ng wild-type or mutant Kir6.2 mRNA and 4 ng of SUR1 mRNA (giving a 1 : 5 ratio). For each batch of oocytes, all mutations were injected to enable direct comparison of their effects. To simulate the heterozygous state, SUR1 was coexpressed with a 1 : 1 mixture of wild-type and mutant Kir6.2 (27). A potential problem with using co-injection of wild-type and mutant mRNAs to simulate the heterozygous state is that the levels of expression may be different for wild-type and mutant proteins. However, the shift in the IC₅₀ for ATP inhibition of our heterozygous population was similar to that found by Markworth et al. (44) who addressed this issue quantitatively, which suggests that in fact wild-type and mutant subunits are expressed at similar levels in our studies. It has been argued that an alternative way to simulate the heterozygous state is to construct tandem dimers of wild-type and mutant subunits. However, this approach also has its problems. First, some channel types will be missing from the heterozygous population, namely those that have one mutant (or one wild-type) subunit, and those that have all mutant or all wild-type channels. This amounts to 63% of the heterozygous channel population if the subunits distribute according to binomial theory. Secondly, linking adjacent subunits in tandem may itself modify channel ATP sensitivity, as both the N- and C-terminal domains contribute to the binding site (45). For example, modification of the N-terminus is known to result in altered ATP sensitivity (27,46). Indeed, we observed that the ability of Mg-nucleotides to activate the K_ATP channel was altered when tandem dimers were constructed: the EC₅₀ was much smaller for the dimer than for either homomeric mutant or wild-type channels. Thus, we believe that simulating the heterozygous state by co-injection of wild-type and mutant subunits is the approach least prone to error. It is also important to point out that coexpression of two mRNAs most closely simulates the situation in the patient's cells (where differences in expression may also occur). Further, the main aim of this study is to compare ATP concentration-response data in the absence and presence of Mg²⁺, and this is done under identical conditions of channel composition.

Whole-cell currents were recorded from intact oocytes using the two-electrode voltage-clamp method, filtered at 1 kHz and digitized at 4 kHz. Oocytes were constantly perfused at 20–22°C with a solution containing (in mM): 90 KCl, 1 MgCl₂, 1.8 CaCl₂ and 5 HEPES (pH 7.4 with KOH). Metabolic inhibition was produced by 3 mM Na-azide. Whole-cell currents were monitored in response to voltage steps of ±20 mV from a holding potential of –10 mV.

Macroscopic currents were recorded from giant excised inside-out patches using the patch-clamp technique in response to 3 sec voltage ramps from –110 mV to +100 mV (holding potential, 0 mV) and 20–22°C. Currents were filtered at 0.15 kHz and digitized at 0.5 kHz. The pipette solution contained (mM): 140 KCl, 1.2 MgCl₂, 2.6 CaCl₂ and 10 HEPES (pH 7.4 with KOH). The Mg-free internal (bath) solution contained (mM): 107 KCl, 1 K₂SO₄, 10 EGTA, 10 HEPES (pH 7.2 with KOH) and nucleotides as indicated. The Mg-containing internal solution was the same as the Mg-free solution except that 2 mM MgCl₂ was added and MgATP (instead of ATP) was added as indicated. Rapid exchange of internal solutions was achieved by using a local perfusion system consisting of eight tubes of 200 µm diameter in which the tip of the patch pipette was inserted.

The macroscopic slope conductance was measured between –100 and +10 mV. ATP concentration-response curves were fit with the Hill equation:

1

where [ATP] is the ATP concentration, IC₅₀ is the ATP concentration at which inhibition is half maximal, h is the slope factor (Hill coefficient) and a is the fraction of unblockable current. Except where indicated in text, a was taken as 0. To account for possible rundown, G_c was taken as the mean of the conductance in control solution before and after ATP application.

Data were analysed with pCLAMP8 (Axon Instruments, CA, USA), Origin 6.02 (Microcal Software, Northampton, MA, USA) and Igor (Wavemetrics, Lake Oswego, OR, USA) software, and are given as mean±SEM. Statistical significance was evaluated using an unpaired two-tailed Student's t-test. A probability value of P<0.05 was taken as the criteria for a significant difference.

	ACKNOWLEDGEMENTS

 
We thank the Wellcome Trust, the Royal Society and the EU (BioSim) for support. F.M.A. is a Royal Society Research Professor.

Conflict of Interest statement. None declared.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS REFERENCES

 

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