Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on June 21, 2007
Human Molecular Genetics 2007 16(16):2011-2019; doi:10.1093/hmg/ddm149

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Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome

Peter Proks, Kenju Shimomura, Tim J. Craig, Christophe A.J. Girard and Frances M. Ashcroft^*

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

^* To whom correspondenceshould be addressed. Tel: +44 1865285810; Fax: +44 1865285813; Email: frances.ashcroft{at}physiol.ox.ac.uk

Received April 23, 2007; Revised June 10, 2007; Accepted June 10, 2007

	ABSTRACT

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Activating mutations in the genes encoding the ATP-sensitive potassium (K_ATP) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. 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. This mutation causes severe developmental delay, epilepsy and permanent neonatal diabetes (DEND syndrome). We show that the F132L mutation reduces the ATP sensitivity of K_ATP channels indirectly, by altering the intrinsic gating of the channel. Thus, the open probability is markedly increased when Kir6.2 is co-expressed with mutant TMD0 alone or with mutant SUR1. The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Our results explain how a mutation in an accessory subunit can produce enhanced activity of the K_ATP channel pore (formed by Kir6.2). They also provide further evidence that interactions between TMD0 of SUR1 and Kir6.2 are critical for K_ATP channel gating and identify a residue crucial for this interaction at both physical and functional levels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ATP-sensitive potassium (K_ATP) channels are weak inwardly rectifying potassium channels that couple cell metabolism to electrical activity. They are found in a wide variety of tissues, including skeletal muscle, cardiac muscle, smooth muscle, brain and endocrine cells (1). In heart and brain they are involved in the response to metabolic stress, in smooth muscle they regulate vascular tone and in pancreatic beta-cells they link changes in blood glucose concentration to insulin secretion (2).

The beta-cell K_ATP channel is an octameric complex comprising two types of protein, Kir6.2 and SUR1, which co-assemble in 4:4 stoichiometry (3). Kir6.2 forms the ion pore, and ATP binding to this subunit closes the channel (4). The sulphonylurea receptor (SUR1) has a regulatory role: interaction of Mg-nucleotides with this subunit enhances channel opening (5,6). This dual regulation by adenine nucleotides means that an increase in beta-cell metabolism inhibits K_ATP channels and leads to membrane depolarization, electrical activity and insulin secretion, whereas a decrease in metabolism opens K_ATP channels and arrests electrical activity. Sulphonylurea drugs, which are used to treat type 2 diabetes, bypass metabolism and stimulate insulin exocytosis by direct closure of K_ATP channels (7).

Heterozygous activating mutations in KCNJ11, which encodes Kir6.2, are a common cause of neonatal diabetes (ND) (8,9). Some of these mutations lead to a more severe syndrome that also includes developmental delay, muscle weakness and epilepsy, a condition termed DEND syndrome (9). All KCNJ11 mutations studied to date decrease the ability of ATP to inhibit channel activity when recombinant channels are expressed heterologously (8,10–15). They may also enhance the ability of Mg-nucleotides to stimulate channel activity (16). Both these effects lead to an increase in the magnitude of the K_ATP current. This is expected to produce beta-cell hyperpolarization, thereby inhibiting electrical activity, calcium influx and insulin secretion. Currents of larger magnitude are considered to hyperpolarize other cells in which Kir6.2 is expressed, such as muscle and brain, thereby accounting for the DEND phenotype (8).

Most Kir6.2 mutations are located within the ATP-binding site, or in regions of the proteins that are involved in channel gating—opening and closing of the pore (8). The former appear to affect ATP binding directly, whereas the latter are thought to influence ATP sensitivity indirectly, via their effect on the channel kinetics (11–13). The severity of the disease appears to correlate with the extent to which the K_ATP channel ATP sensitivity is reduced, with DEND mutations showing the greatest reduction in ATP inhibition (11,12,15).

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). A further eight mutations associated with ND without neurological complications have been described subsequently (18,19). SUR1 is a member of the ATP-binding cassette (ABC) protein superfamily (20). It has 17 transmembrane helices arranged in three groups of five, six and six, which are known as transmembrane domains TMD0, TMD1 and TMD2. Whereas all ABC transporters possess TMD1 and TMD2, TMD0 is unique to the SUR and MRP subfamilies (20). Residue F132 lies within the second intracellular loop of TMD0 of SUR1 (17). Like other ABC proteins, SUR1 has two larger intracellular loops, NBD1 and NBD2, that are involved in Mg-nucleotide binding and hydrolysis.

Neither Kir6.2 nor SUR1 reaches the surface membrane in the absence of their partner subunit (21). However, deletion of the last 26–36 residues of Kir6.2 (Kir6.2C) enables its independent surface expression (4). When co-expressed with Kir6.2C, TMD0 modulates channel gating (22) and this region of SUR is responsible for the different kinetics of channels composed of different SUR isoforms (23). In addition, biochemical studies have shown that TMD0 of SUR1 physically interacts with Kir6.2 (22,24). 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. However, this remains to be explored. Furthermore, changes in gating were not observed for the L225P mutation that also lies within TMD0 (19).

In this paper, we examine the molecular mechanism by which the F132L mutation in SUR1 influences K_ATP channel inhibition by ATP. We show that this mutation alters ATP inhibition indirectly, by stabilizing of the open state of the channel. We further demonstrate that the mutation impairs the ability of TMD0 to couple physically to Kir6.2.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
F132L alters the intrinsic gating of K_ATP channels
The aim of this paper is to determine the molecular mechanism by which the F132L mutation reduces the ATP sensitivity of the K_ATP channel. To address this question, we compared the properties of wild-type and mutant K_ATP channels expressed in Xenopus oocytes. We first compared the effect of the F132L mutation in SUR1 on the kinetics of single K_ATP channel currents. Experiments were carried out in inside-out membrane patches exposed to nucleotide-free solution, where intrinsic gating can be assessed. In the absence of nucleotides, the gating of K_ATP channels consists of bursts of openings separated by long closed intervals (Fig. 1). The F132L mutation dramatically increased the burst duration and reduced the time spent in the interburst intervals. The burst duration increased 3-fold from 19.9 ± 2.3 ms (n = 6) to 59.5 ± 3.8 ms (n = 6) for wild-type and homomeric (hom)F132L channels, respectively. There was no obvious effect on intraburst closed times or on the burst duration of interburst intervals wild-type = 9.7 ± 1.2 ms (n = 6) and homF132L = 9.9 ± 1.5 ms (n = 6). The intrinsic open probability (P_O) of SUR1-F132L channels was significantly greater (P < 0.05) than that of wild-type channels, being 0.72 ± 0.03 (n = 8) for SUR1-F132L, compared with 0.26 ± 0.03 (n = 6) for SUR1 channels. These differences in channel kinetics suggest that the SUR1-F132L mutation influences K_ATP channel ATP sensitivity indirectly, via changes in channel gating, as is found for some Kir6.2 mutations.

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Single-channel currents recorded at –60 mV from inside-out membrane patches excised from oocytes expressing Kir6.2/SUR1, Kir6.2/SUR1-F132L, Kir6.2C, Kir6.2C/TMD0 and Kir6.2C/TMD0-F132L. The dashed lines indicate the zero current level.

 
Because all patients carrying the F132L mutation are heterozygotes, their pancreatic beta-cells will contain a mixture of wild-type and mutant SUR1. We therefore simulated the heterozygous state, by coinjecting a 1:1 mixture of mutant and wild-type SUR1 together with Kir6.2. This will produce a mixed population of homomeric wild-type channels, homomeric mutant channels and heteromeric channels containing between 1 and 3 mutant subunits. We refer to this channel population as heterozygous (het) channels. Because the channels in the heterozygous population will have different P_O, we compared the mean P_O of the heterozygous channel population with that of homomeric F132L channels (see Materials and Methods for details). The mean P_O of hetF132L channels (0.50 ± 0.03; n = 10) was significantly less than that of homF132L channels (0.69 ± 0.01; n = 10).

The gating of Kir6.2C channels expressed in the absence of SUR is characterized by a dramatic reduction in the burst duration (25,26) (Fig. 1). As shown previously (22), co-expression of TMD0 of SUR1 with Kir6.2C restored the normal bursting pattern of K_ATP channels: the intrinsic P_O increased from 0.09 ± 0.03 (n = 6) for Kir6.2C to 0.64 ± 0.03 (n = 6) for Kir6.2C/TMD0 (P < 0.001). Interestingly, Kir6.2C/TMD0 channels had a higher P_O (0.64) than Kir6.2/SUR1 channels (0.26), suggesting that addition of TMD0 alone does not fully recapitulate the intrinsic gating of the wild-type channel. There was a further increase in P_O when the F132L mutation was introduced into TMD0 (to 0.82 ± 0.02; n = 6). 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.

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 K_ATP channels composed of Kir6.2 and either wild-type or mutant SUR1. We first carried out experiments in the absence of intracellular Mg²⁺, in order to isolate the effects of the mutation on the interaction of ATP with Kir6.2 (Fig. 2A). Mutant channels were substantially less sensitive to ATP than wild-type channels: the IC₅₀ for ATP inhibition increased from 7 µM (wild-type) to 30 µM for hetF132L and 51 µM for homF132L channels (Table 1). The shift in the ATP sensitivity of hetF132L channels is similar to that predicted for a mutation that affects channel gating and in which each subunit (wild-type or mutant) contributes additively to the gating of heteromeric channels.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. ATP sensitivity of macroscopic KATP channels containing full-length SUR1 recorded in the absence (A) or presence (B) of Mg2+. Top: KATP currents recorded in response to successive voltage ramps from –110 to +100 mV in an inside-out patch excised from an oocyte expressing Kir6.2/SUR1 or homKir6.2/SUR1-F132L channels, as indicated. The dashed line indicates the zero current level. The bar indicates application of 100 µM ATP (A) or 1 mM MgATP (B). 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. The smooth curves are the best-fit of Eq. (1) to the mean data. For wild-type channels, IC50 = 6.8 µM, h = 1.0. For hetF132L, IC50 = 39 µM, h = 1.0. For homF132L, IC50 = 50 µM, h = 1.3. (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. The smooth curves are the best-fit of Eq. (2) to the mean data. For wild-type channels, IC50 = 14 µM, h = 1.0, a = 0. For hetF132L, IC50 = 117 µM, h = 0.75, a = 0.05. For homF132L, IC50 = 516 µM, h = 0.78, a = 0.16.

 

View this table: [in this window] [in a new window]   Table 1. Values of IC50 for ATP inhibition of various KATP channels in both Mg2+-containing and Mg2+-free solutions

 
The ATP sensitivity of SUR1-F132L mutant channels was further decreased in the presence of 2 mM Mg²⁺ (Fig. 2B). The IC₅₀ was 14 µM for wild-type channels, 122 µM for hetF132L and 910 µM for homF132L channels (Table 1). Thus, in both the absence and presence of Mg²⁺, there was a marked difference in the ATP sensitivity of heterozygous and homomeric channels. There was also a marked increase in the amplitude of the unblocked current in the presence of 3 mM MgATP, which was 0.8 ± 0.4% (n = 7) for wild-type, 13 ± 2% (n = 10) for hetF132L and 31 ± 3% (n = 10) for homF132L. We refer to this unblocked current as the ‘pedestal’. Similar results were reported previously for the effect of the mutation on the channel sensitivity to MgATP (17).

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. In contrast, MgGDP enhances channel activity by interaction with the NBDs of SUR1 but produces substantially less block at Kir6.2 (27,28). It can therefore be used to isolate the effects of Mg-nucleotides on SUR1. 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. We therefore tested the effect of guanine nucleotides on channels pre-blocked by ATPAA, an ATP analogue that binds specifically to Kir6.2 and not to SUR1 (29).

ATPAA (200 µM) reduced homF132L currents to 20% of their value in the absence of nucleotide. This is approximately equivalent to the inhibitory effect of ATP, tested in the absence of Mg²⁺ (Fig. 2A), and is consistent with the idea that ATPAA interacts only with Kir6.2. Subsequent addition of 100 µM MgGDP almost completely restored channel activity blocked by ATPAA (Fig. 3). This concentration of GDP has little inhibitory effect on wild-type K_ATP channels, as demonstrated by its lack of effect on K_ATP currents in the absence of Mg²⁺ (28). Thus the 5-fold increase in current represents the full stimulatory action of MgGDP. HetF132L currents were blocked more effectively by ATPAA than homF132L currents, but addition of MgGDP in the presence of ATPAA also increased hetF132L currents 5-fold. Wild-type K_ATP channels are stimulated up to 2.5-fold by MgGDP (27,28). Thus, these data suggest that MgGDP activation may be enhanced by the F132L mutation in SUR1. A similar effect was found for PNDM mutations in the Kir6.2 subunit (16).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Mean hetKir6.2/SUR1-F132L and homKir6.2/SUR1-F132L currents recorded in the presence of 200 µM ATPAA and 200 µM ATPAA+100 µM MgGDP, as indicated. Currents are expressed as a fraction of the current in nucleotide-free solution. Data represent the mean ± SEM of six patches.

 
Effects of F132L mutation on the ATP sensitivity of TMD0 channels
We next compared the ATP sensitivity of Kir6.2C/TMD0 channels with heterozygous and homomeric TMD0-F132L channels (Fig. 4). As previously reported (22), the ATP sensitivity of Kir6.2C was reduced when this subunit was co-expressed with TMD0. In Mg-free solution, the IC₅₀ for ATP block of Kir6.2C/TMD0 was 700 µM compared with 100–200 µM for Kir6.2C alone (4,30). This effect can be attributed to the increase in the intrinsic P_O that is observed when Kir6.2C is co-expressed with TMD0 (Fig. 1). The F132L mutation further reduced the ATP sensitivity of Kir6.2C/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).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. ATP sensitivity of KATP channels comprising Kir6.2 and TMD0, recorded in the absence (A) or presence (B) of Mg2+. Top: KATP currents recorded at –60 mV in an inside-out patch excised from an oocyte expressing Kir6.2C/TMD0 or homozygous Kir6.2C/TMD0-F132L channels. The dotted line indicates the zero current level. The bar indicates application of 1 mM ATP (A) or 1 mM MgATP (B). Bottom: (A) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2C /TMD0 (open circle, n = 6) and heterozygous (filled circle, n = 6) or homomeric (filled square, n = 6) Kir6.2C /TMD0-F132L channels. The smooth curves are the best-fit of Eq. (2) to the mean data. For wild-type channels, IC50 = 240 µM, h = 0.92, a = 0.09. For hetF132L, IC50 = 762 µM, h = 1.1, a = 0.1. For homF132L, IC50 = 973 µM, h = 1.1, a = 0.1. (B) Mean relationship between [MgATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2C /TMD0 (open circle, n = 5) and heterozygous (filled circle, n = 7) or homomeric (filled square, n = 10) Kir6.2C /TMD0-F132L channels. The smooth curves are the best-fit of Eq. (2) to the mean data. For wild-type channels, IC50 = 500 µM, h = 1.0, a = 0.06. For hetF132L, IC50 = 1.5 mM, h = 1.1, a = 0.14. For homF132L, IC50 = 2.0 mM, h = 1.1, a = 0.14.

 
There was no significant difference in the ATP sensitivity of Kir6.2C/TMD0 channels when measured in the presence of 2 mM Mg²⁺ (Fig. 4B and Table 1). This was not only true for wild-type TMD0, but also for hetTMD0-F132L and homTMD0-F132L (Table 1). This confirms that the shift in ATP sensitivity of channels containing full-length SUR1 observed in the presence of Mg²⁺ is due to the stimulatory effect of MgATP at the NBDs of SUR1 (which are lacking in TMD0).

In contrast to channels containing full-length SUR1 (Fig. 2), those containing TMD0 exhibited significant unblocked current at high ATP concentrations in Mg²⁺-free solution (Fig. 4A). 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.

The F132L mutation disrupts the interaction between TMD0 and Kir6.2
Our results suggest that the F132L mutation may influence K_ATP channel gating by altering the interaction between SUR1 and Kir6.2. We next explored if the mutation influences the physical (as opposed to functional) interaction between these two subunits by co-immunoprecipitation studies. A haemagglutinin (HA) tag was inserted in Kir6.2C and a FLAG tag in TMD0, for affinity purification. Xenopus oocytes were co-injected with Kir6.2C-HA and either wild-type TMD0, TMD0-F132L or a mixture of both (to simulate heterozygosity). Following detergent extraction of oocyte membranes, Kir6.2 was immunoprecipitated with an anti-HA antibody, and bound TMD0 was detected by western blotting with an anti-FLAG antibody and quantified by densitometry (Fig. 5A). The F132L mutation reduced the binding of TMD0 to Kir6.2 by 90% (P < 0.001), indicating that F132 is crucial for this interaction. 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 = 0.28). TMD0 was not detected either when Kir6.2C was not co-expressed, or if the HA-antibody was omitted from the co-immunoprecipitation (data not shown).

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. (A) Co-immunoprecipitation of FLAG-tagged TMD0 and Kir6.2C, using wild-type or F132L TMD0 and wild-type Kir6.2C. Co-immunoprecipitation was performed using anti-HA monoclonal antibodies and protein G–Sepharose, as described. Top: Quantitative densitometry of TMD0 detected in western blots. Data were corrected both for the total amount of TMD0 input and the amount of Kir6.2C pulled down in each assay and are expressed as a percentage of the binding of wild-type TMD0 to Kir6.2C. Data are the mean ± SEM of four separate experiments. Bottom: Representative examples of single co-immunoprecipitation experiments, showing the same gel immunoblotted with FLAG- and HA-antibodies (left), and the input fraction (to show expression levels), also blotted with FLAG- and HA-antibodies (right). Blots are cropped to save space; the cropped areas contained no bands. The higher molecular weight bands in the co-immunoprecipitate blotted with FLAG are IgG light chain/protein G: they are also present when the co-immunoprecipitation is performed in buffer without oocyte lysate (data not shown). The lower molecular weight band is a breakdown product of TMD0 (and is not observed in oocytes not injected with TMD0). (B) Co-immunoprecipitation of TMD0 and Kir6.2C, using wild-type or C166S Kir6.2C and wild-type TMD0. Top and bottom as in (A). Data are the mean ± SEM of three separate experiments.

 
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. To test this idea, we examined the effect of another mutation that increases the open-state stability of the K_ATP channel: C166S in Kir6.2. As Figure 5B shows, this mutation did not influence co-precipitation of TMD0 and Kir6.2. This is consistent with the idea that the interaction is not affected by whether the channel is in the open or closed state and further suggests that F132 has a more specific role in the interaction between K_ATP channel partner subunits.

Interactions between the transmembrane domains of Kir6.2 and SUR1 are involved in trafficking of the protein to the plasma membrane. Substitution of TMD0 of SUR1 with that of MRP1 abolishes surface expression (31), implicating TMD0 in this interaction. Furthermore, mutations in TMD0 have been shown to cause protein misfolding, resulting in defective trafficking and reduction in channel density in the plasmalemma (32,33). Thus, we next examined whether the F132L mutation altered surface expression of the K_ATP channel complex. In agreement with previous studies (21), very little Kir6.2 reached the surface membrane in the absence of SUR1, whereas significantly more Kir6.2 was expressed at the cell surface following truncation of the last 26 amino acids of the protein (Fig. 6). Co-expression with SUR1/TMD0 enhanced surface expression of both Kir6.2 and Kir6.2C: however, this was unaffected by the F132L mutation (Fig. 6).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 6. Surface expression of the indicated KATP channels, expressed as a percentage of that found for the wild-type channel. (A) Full-length Kir6.2 was co-expressed with wild-type or mutant SUR1. (B) Kir6.2C was co-expressed with wild-type or mutant TMD0.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Molecular mechanism of action
Single-channel analysis revealed that the F132L mutation dramatically enhances the burst duration and the intrinsic open probability (P_O) of both homomeric Kir6.2/SUR1-F132L and Kir6.2/TMD0-F132L channels. Thus, F132L is a gating mutation that alters the ATP-sensitivity of the K_ATP channel indirectly, via stabilization of the channel open state (30,34).

Residue F132 is located in TMD0 of SUR1, a region previously implicated in channel gating (22,23). Co-expression of TMD0 with Kir6.2C resulted in functional K_ATP channels with burst kinetics resembling those of Kir6.2/SUR1 channels instead of the isolated openings found when Kir6.2C is expressed alone. However, the P_O of Kir6.2C/TMD0 channels was considerably higher than that of Kir6.2/SUR1 channels, as previously reported (22,23). This is consistent with the demonstration that the additional presence of the cytosolic linker (CL3) between TMD0 and TMD1 was necessary to recapitulate the intrinsic gating of Kir6.2/SUR1 (23).

Physical association of Kir6.2 and TMD0
We found that Kir6.2 physically binds to TMD0, as previously reported (22,23). Our results further show that the F132L mutation impairs this physical association. Co-immunoprecipitation will detect only high-affinity interactions that are not disturbed by detergent extraction of the K_ATP channel complex. Many lower affinity interactions between Kir6.2 and SUR1 presumably occur, which may also have a functional role, but would not be identified by co-immunoprecipitation. These interactions may well be sufficient for subunit association and surface trafficking. Thus, although mutation of F132 did not prevent surface expression of the protein, this does not exclude the posibility that it is involved.

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. It has become clear that SUR1 has two effects of Kir6.2 gating: one stimulatory and one inhibitory (22–24). Thus the mutation could either enhance the stimulatory effect of SUR1 or reduce its inhibitory action. The fact that the P_O is much higher when Kir6.2 is co-expressed with TMD0 rather than SUR1 indicates that TMD0 confers a stimulatory effect. Because the F132L mutation increases the P_O 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. This would be harmonious with the fact that the mutation also reduces the physical interaction. Our data are consistent with the idea that there are multiple interaction sites between Kir6.2 and TMD0. 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).

The physical association of TMD0 and Kir6.2 is consistent with current views of the location of TMD0 in the K_ATP channel complex. Single-particle analysis of the purified K_ATP channel, combined with molecular modelling, suggests that TMD0 sits at the interface between adjacent SUR1, next to Kir6.2 and close to the ATP-binding site on Kir6.2 (35). 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.

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. This is consistent with the idea that wild-type and mutant SUR1 subunits are binomially distributed within the heterozygous channel population and that they exhibit free energy additivity in their effects on channel gating, and thereby ATP sensitivity. A similar argument has been made for gating mutations in the Kir6.2 subunit (11,36).

Mg²⁺ produced a dramatic reduction in the ATP sensitivity of homKir6.2/SUR1-F132L and hetKir6.2/SUR1-F132L channels. No such effect was observed when Kir6.2 was co-expressed with mutant TMD0, consistent with the idea that the effect of Mg²⁺ is mediated via interaction of Mg-nucleotides with the NBDs of SUR1, which TMD0 lacks.

Mg²⁺ caused a 2-fold shift in the IC₅₀ for ATP block of wild-type channels, a 4-fold shift for hetF132L channels and an 18-fold shift for homF132L channels. 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. Experiments with MgGDP support this idea. The fact that MgGDP activated hetF132L and homF132L channels by a similar amount (5-fold) is consistent with the idea that nucleotide binding to several SUR1 subunits is needed to open the Kir6.2 pore. A similar conclusion was reached, using a different approach, for channels containing a Kir6.2 mutation (F333I) associated with ND (14).

Comparison with other gating mutations causing ND
Previous studies have identified a number of mutations in Kir6.2 that reduce K_ATP channel ATP sensitivity indirectly, via an increase in the intrinsic P_O (11–16). These mutations can cause both ND and DEND syndrome, depending on the extent to which the mutation decreases the magnitude of the K_ATP current at physiological concentrations of ATP (8,16). In inside-out patches exposed to 3 mM MgATP, the magnitude of the hetF132L currents is 13% of maximal. Interestingly, this is smaller than that reported to date for other mutations associated with DEND syndrome, which lie between 28 and 40% of maximal. The whole-cell resting currents recorded for both hetF132L channels reported previously (17) are also smaller than those found for other DEND syndrome mutations and more closely resemble those observed for the R201C and V59M mutations (16) which are associated with intermediate DEND syndrome (9). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Molecular biology
Human Kir6.2 (GenBank accession no. NM000525 with E23 and I337) and rat SUR1 (GenBank accession no. L40624) were used in this study. In some experiments, we used a Kir6.2 construct in which the C-terminal 36 amino acids were deleted (Kir6.2C), which enables it to express independently of SUR1 (4). We also used a construct (TMD0) consisting of residues 1–196 of SUR1. For electrophysiological studies, site-directed mutagenesis of SUR1 or TMD0, preparation of mRNA and isolation of Xenopus laevis oocytes were performed as described previously (17,37). Oocytes were injected with 0.8 ng of wild-type or mutant Kir6.2 mRNA and 4 ng of SUR1 mRNA (giving a 1:5 ratio). To simulate the heterozygous state, SUR1 was co-expressed with a 1:1 mixture of wild-type and mutant Kir6.2 (16). Currents were recorded 1–3 days after injection.

For co-immunoprecipitation studies, an N-terminal FLAG tag (DYKDDDDK) was attached to TMD0 of SUR1. An HA tag (YPYDVPDYA) was inserted after residue 102 of Kir6.2 (21), and the last 26 amino acids were deleted in order to facilitate surface expression in the absence of SUR1 (4). All constructs were inserted into the pBF vector, and mRNA was prepared as described (37).

Electrophysiology
Macroscopic currents were recorded from giant inside-out patches using the patch-clamp technique in response to 3 s voltage ramps from –110 to +100 mV (holding potential, 0 mV) at 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, 10 HEPES (pH 7.4 with KOH). For most experiments, the internal (bath) solution contained (mM): 107 KCl, 1 K₂SO₄, 10 EGTA, 10 HEPES (pH 7.2 with KOH). However, for experiments in the presence of Mg²⁺, we used the same internal solution but K₂SO₄ was omitted and 2 mM MgCl₂ and Mg-nucleotides (as indicated) were added. ATP--4-azido-anilide (ATPAA, TEA salt) was purchased from Affinity Technologies (Lexington, KY, USA).

The macroscopic slope conductance was measured between –100 and +10 mV. ATP concentration–response curves of SUR1 containing channels in the absence of Mg²⁺ were fit with the Hill equation:

(1)

where [ATP] is the ATP concentration, IC₅₀ the ATP concentration at which inhibition is half-maximal and h is the slope factor (Hill coefficient). ATP concentration–response curves of TMD0-containing channels and SUR1-containing channels in the presence of Mg²⁺ were fit with a modified Hill equation:

(2)

where a is the fraction of unblocked current (pedestal) at ATP concentrations at which all ATP-binding sites on the K_ATP channel are occupied by the nucleotide.

To control for possible rundown of channel activity, G_c was taken as the mean of the conductance in control solution before and after ATP application. Concentration–response curves were fitted individually for each patch and the mean ± SEM of the individual parameters are given in the text. The curves shown in the figures are the best-fit of Eq. (1) [or (2)] to the mean data.

Single-channel currents were recorded at –60 mV, filtered at 5 kHz and digitized at 50 kHz. Burst durations were determined using a criterion for the critical time as described by Magleby and Pallotta (38).

Open probability (P_O) was measured in the absence of ATP in three ways for homomeric (wild-type and mutant) channels. First, from single-channel recordings in patches containing one or several channels as previously described (12). Secondly, by noise analysis from patches containing many channels (n>10) according to:

(3)

where I is the mean K_ATP current in the absence of ATP (measured over a 1 s interval), ² the current variance and i the single-channel current. The latter was determined in separate experiments from amplitude histograms recorded in the presence of 10 mM ATP, where only a few channels are active. There was no significant difference between the P_O estimated by noise analysis or single-channel recordings. Finally, the heterozygous population contains channels with a variable number of mutant subunits. In this case, the mean P_O was estimated simply from patches containing many channels (n>10) using:

(4)

where I_max is the maximum K_ATP current measured during a 1 s interval. For homomeric channels, P_O calculated in this way was virtually identical to that obtained from Eq. (3).

All 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 P-value of <0.05 was taken as the criteria for a significant difference.

Co-immunoprecipitation of TMD0 and Kir6.2
Oocytes were injected with 5 ng of Kir6.2C mRNA and 5 ng of either TMD0, mutant TMD0 (F132L or C166S) or a 50:50 mix of wild-type and mutant TMD0 mRNAs. Typically 20 oocytes were used per condition per experiment. Lysis of oocytes and immunoprecipitations were performed after 48 h as described by Chan et al. (22), with the following modifications: first, immunoprecipitations and detergent extractions of lysed oocytes were performed in IP buffer [150 mM NaCl, 20 mM HEPES (pH 7.4), 1% dodecylmaltoside, protease inhibitors]; second, the antibody used for immunoprecipitation of Kir6.2 was rat monoclonal anti-HA (Roche); third, bound protein was detected with a mouse monoclonal anti-FLAG antibody (Sigma); fourth, bands on western blots were quantified using Quantity One software (Bio-Rad). Crude lysates (inputs) were also run on western blots to enable data to be corrected for differences in expression levels.

Surface expression assay
Surface expression assays were performed using the protocol of Zerangue et al. (21) on Xenopus laevis oocytes expressing HA-tagged Kir6.2 full-length or Kir6.2 missing the final 26 amino acids (C, as used in the co-immunoprecipitation assays), together with either TMD0 or full-length SUR1. Surface expression was detected with rat monoclonal anti-HA antibody, clone 3F10 (Roche) and HRP-conjugated anti-rat secondary antibody (Jackson Laboratories). Bound antibody was detected by Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce) in a luminometer.

	ACKNOWLEDGEMENTS

 
Financial support was provided by the Wellcome Trust, the Royal Society and the European Union (EuroDia). F.M.A. is a Royal Society Research Professor. T.J.C. is a Wellcome Trust Integrative Physiology Training Fellow (OXION).

Conflict of Interest statement. None declared.

	FOOTNOTES

 
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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