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Molecular and Genomic |
1 University Laboratory of Physiology, Oxford University, Oxford OX1 3PT, UK
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Abstract |
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(Received 22 September 2005;
accepted after revision 6 December 2005;
first published online 8 December 2005)
Corresponding author F. M. Ashcroft: University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. Email: frances.ashcroft{at}physiol.ox.ac.uk
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Introduction |
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KATP channels are hetero-octamers of four pore-forming Kir6.x subunits and four regulatory sulphonylurea receptor (SUR) subunits (Clement et al. 1997). In most tissues, Kir6.2 forms the tetrameric pore (Sakura et al. 1995) but it may associate with different SUR subunits, thereby endowing the KATP channel with different sensitivities to modulation by metabolism and therapeutic drugs. The SUR2A isoform is found in cardiac myocytes (Inagaki et al. 1996; Morrissey et al. 2005), SUR2B in vascular smooth muscle (Isomoto et al. 1996) and SUR1 in most other tissues, including pancreatic ß-cells and neurones (Aguilar-Bryan et al. 1995). Coexpression of Kir6.2 with SUR1 gives rise to KATP channels with properties resembling those of pancreatic ß-cells (Sakura et al. 1995; Inagaki et al. 1996; Gribble et al. 1997), whereas coexpression of Kir6.2 with SUR2A produces channels with properties resembling those of the sarcolemmal KATP channels (Babenko et al. 1998; Gribble et al. 1998b).
Metabolic regulation of KATP channel activity is achieved by changes in the intracellular concentrations of adenine nucleotides. Binding of ATP (or ADP), in a Mg2+-independent manner, to Kir6.2 closes the pore (Tucker et al. 1997), whereas interaction of MgATP or MgADP with the nucleotide-binding domains (NBDs) of SUR activates KATP channels (Nichols et al. 1996; Tucker et al. 1997). Thus increased metabolism, by producing an increment in [ATP]i and a concomitant fall in [ADP]i, closes KATP channels, promoting membrane depolarization and electrical activity. Conversely, metabolic inhibition favours KATP opening and membrane hyperpolarization, thereby suppressing electrical activity.
Naturally occurring mutations in KCNJ11, which encodes Kir6.2, can cause neonatal diabetes in humans (Gloyn et al. 2004; Sagen et al. 2004; Ashcroft, 2005; Hattersley & Ashcroft, 2005). Some mutations cause permanent neonatal diabetes mellitus alone, which we refer to here as PNDM. Other mutations are associated with a more severe clinical phenotype, characterized by developmental delay, epilepsy, muscle weakness and neonatal diabetes, which has been termed DEND syndrome (Hattersley & Ashcroft, 2005). Insulin secretion in response to glucose was impaired in both PNDM and DEND patients, but could be stimulated by sulphonylureas in PNDM patients (Sagen et al. 2004; Ashcroft, 2005; Hattersley & Ashcroft, 2005). Functional studies showed that mutations causing PNDM (e.g. R201C), and DEND syndrome (e.g. Q52R), are gain-of-function mutations which result in impaired inhibition of Kir6.2/SUR1 channels by ATP (Proks et al. 2004; Ashcroft, 2005; Tammaro et al. 2005). In pancreatic ß-cells, the reduced ATP sensitivity is expected to increase the whole-cell KATP current, thereby causing membrane hyperpolarization, reduced Ca2+ influx via voltage-gated channels and impairment of insulin secretion (Gloyn et al. 2004). Mutations associated with DEND syndrome produced a greater reduction in ATP sensitivity, and a larger increase in the KATP current, than those causing neonatal diabetes alone (Proks et al. 2004). It has been hypothesized that this leads to hyperpolarization of neurones, and possibly also muscle cells, and so accounts for the neurological symptoms associated with DEND syndrome (Ashcroft, 2005).
Although Kir6.2 is expressed in the heart, no obvious abnormalities were observed in the electrocardiogram (ECG) of patients with PNDM mutations (Gloyn et al. 2004; Ashcroft, 2005). Likewise, transgenic mice in which a mutated Kir6.2 subunit, with reduced ATP sensitivity, was over-expressed under the control of a cardiac-specific promoter, had normal ECGs and their heart rate was reduced by only 
15% (Koster et al. 2001). The cardiac action potential of these mice was of normal duration and the rate at which the cardiac KATP current was activated by metabolic inhibition did not differ from wild-type. Yet excised membrane patches revealed that cardiac KATP channels were far less sensitive to ATP (IC50 of 2.7 mM compared with 50 µM for wild-type, when measured in the absence of Mg2+).
In this paper, we compare the functional effects of the R201H mutation in Kir6.2, which causes PNDM alone, and of the Q52R mutation, which causes DEND syndrome, on recombinant cardiac and ß-cell KATP channels. Because all patients identified to date are heterozygous, we coexpressed wild-type and mutant Kir6.2 to simulate the heterozygous state and we refer to the resulting population of channels containing various mixtures of wild-type and mutant subunits as heterozygous channels.
Our results demonstrate that although both mutations cause a similar shift in the ATP sensitivity of heterozygous ß-cell (SUR1) and cardiac (SUR2A) KATP channels in the absence of Mg2+, when measured in the presence of Mg2+ the reduction in ATP sensitivity is very much greater for ß-cell channels than cardiac channels. This difference may explain why the mutations cause impaired glucose homeostasis and neurological symptoms but have little effect on the heart.
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Methods |
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0.8 ng of wild-type or mutant Kir6.2 mRNA, or 0.8 ng of a 1: 1 mixture of wild-type and mutant mRNA, together with 4 ng of mRNA encoding either SUR1 or SUR2A. The final injection volume was 50 nl per oocyte. 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. The advantages of this approach are discussed in the online Supplemental material. Isolated oocytes were maintained in Barth's solution and studied 1–4 days after injection (Gribble et al. 1997). Whole-cell currents were recorded from intact oocytes using a two-electrode voltage clamp (Gribble et al. 1997) in response to voltage steps of ±20 mV from a holding potential of –10 mV, filtered at 1 kHz and digitized at 4 kHz (Fig. 5). Oocytes were superfused with a solution containing (mM): 90 KCl, 1 MgCl2, 1.8 CaCl2, and 5 Hepes (pH 7.4 with KOH). Metabolic inhibition was produced by 3 mM sodium azide.
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Single-channel currents were recorded at –60 mV from inside-out patches, filtered at 5 kHz and sampled at 20–50 kHz (Fig. 2). Open probability was determined from single-channel patches as the fraction of time spent in the open state for recordings of > 1 min duration. Patches were excised from at least three separate batches of oocytes.
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Results |
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We first compared the ATP sensitivity of recombinant cardiac (Kir6.2/SUR2A) and ß-cell (Kir6.2/SUR1) KATP channels in the absence of Mg2+, to avoid the stimulatory effects of MgATP mediated by SUR (Gribble et al. 1998a). When coexpressed with SUR2A, homomeric (hom) Q52R and R201H channels were much less sensitive to ATP than wild-type channels, being half-maximally blocked (IC50) by 352 µM and 175 µM ATP, respectively, compared with 10 µM for Kir6.2/SUR2A (Fig. 1, Table 1). Similarly, homQ52R/SUR1 and homR201H/SUR1 channels showed reduced ATP sensitivity (Fig. 1, Table 2), as previously described (Proks et al. 2004). Heterozygous channels were also less sensitive to ATP inhibition whether coexpressed with SUR2A or SUR1 (Fig. 1). It is noteworthy that in the absence of Mg2+ only small differences in ATP sensitivity were observed between channels composed of different SUR (Tables 1 and 2).
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The molecular mechanism underlying the reduced ATP sensitivity produced by the Q52R and R201H mutations could include altered ATP binding, an impaired ability to translate ATP binding into pore closure, or an indirect effect that is secondary to changes in the intrinsic (unliganded) gating of the channel. Previous studies have shown that mutations which stabilize the intrinsic open state of the channel (Po(0)) result in a reduced sensitivity to ATP (Trapp et al. 1998; Enkvetchakul et al. 2000; Proks et al. 2004; see also Fig. 3 in Supplemental material). We therefore recorded single-channel currents from wild-type, Q52R and R201H channels coexpressed with SUR2A, and compared these to data obtained previously for the equivalent SUR1 channels (Proks et al. 2004). To assess intrinsic gating, recordings were carried out in the absence of both ATP and Mg2+.
Neither mutation had any effect on the single-channel current amplitude (Fig. 2; Table 3). In the case of R201H, the open probability in the absence of ligand (Po(0)) was also unaffected, for both SUR2A channels and SUR1-containing channels (Table 3; Proks et al. 2004). This suggests that the R201H mutation decreases KATP channel ATP sensitivity by impairing ATP binding and/or efficacy, and is consistent with its putative location in the ATP-binding site (Antcliff et al. 2005).
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C) that expresses in the absence of SUR. Strikingly, there was no difference in either the single-channel kinetics (Fig. 2) or the IC50 for ATP inhibition of Q52R-Kir6.2C and Kir6.2C. Mean Po(0) values were 0.17 ± 0.03 (n= 5) and 0.16 ± 0.02 (n= 6) and mean IC50 values were 247 ± 15 µM (n= 5) and 194 ± 10 µM (n= 6) for Q52R-Kir6.2C and Kir6.2C, respectively. This suggests that the altered gating of Q52R/SUR channels is conferred by SUR. The magnitude of this effect appears to be independent of the SUR subtype (Table 3). Effects on ATP sensitivity in the presence of Mg2+
In native cells, KATP channel activity is regulated by the balance between ATP inhibition at Kir6.2, and Mg2+-nucleotide activation mediated by SUR (Tucker et al. 1998). We therefore next examined the ATP sensitivity of wild-type and mutant channels in the presence of Mg2+. Under these conditions, differences in ATP sensitivity between mutant Kir6.2/SUR1 and Kir6.2/SUR2A channels were far more dramatic (Fig. 3; Tables 1 and 2).
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This difference in MgATP sensitivity was even more striking for heterozygous channels (Fig. 3; Tables 1 and 2). When coexpressed with SUR1, the IC50 for MgATP inhibition of hetR201H was 12-fold greater than wild-type and that of hetQ52R was 49-fold greater. In the presence of SUR2A, however, the decrease in MgATP sensitivity was substantially less: 2.5-fold for hetR201H and 1.8-fold for hetQ52R channels. This can be explained by the fact that Mg2+ causes a much greater shift in the IC50 for ATP inhibition of heterozygous SUR1 channels than of SUR2A channels (Fig. 2 in Supplemental material).
As a consequence, in the physiological range of [MgATP]i (1–5 mM), there is a significant KATP current for hetSUR1 channels but much less current for hetSUR2A, and an even smaller amount for wild-type SUR1 or SUR2A channels (Table 2). At 1 mM ATP, the current was 2%, 11% and 10% of maximal for wild-type, hetR201H and hetQ52R SUR2A channels, respectively (Table 1). However, for SUR1 channels, the equivalent values were 1.3% (wild-type), 22% (hetR201H) and 42% (hetQ52R). Thus, for a mutation that causes PNDM (R201H) the current is 2-fold larger, and for one causing DEND syndrome (Q52R) 4-fold larger, for ß-cell (SUR1) than cardiac (SUR2A) KATP channels.
Mechanism of enhanced MgATP sensitivity in SUR2A-containing channels
There are several possible explanations for the smaller difference in the IC50 for MgATP block of wild-type and mutant Kir6.2 channels containing SUR2A than for wild-type and mutant SUR1 channels. These include a reduced affinity of SUR2A for MgATP, impaired ATP hydrolysis by SUR2A, a reduced efficacy of the hydrolytic product of MgATP (MgADP) to activate SUR2A channels, defective coupling between MgATP binding/hydrolysis at SUR2A and Kir6.2 opening, or some combination of these.
To investigate the stimulatory effects of Mg-nucleotides on SUR1 and SUR2A independently of their inhibitory effect on Kir6.2, we used MgGTP and MgGDP, as these nucleotides cause less block at Kir6.2 than ATP (or ADP) but potently activate SUR (Trapp et al. 1997). We confined our analysis to R201H channels because the open probability of Q52R channels is already maximal and it is not possible to enhance it further. This precludes examination of the effect of the Q52R mutation on the stimulatory effect of Mg-nucleotides.
We first quantified the effects of GDP on wild-type and homR201H channels containing either SUR1 or SUR2A. In the absence of Mg2+, GDP blocked both wild-type and mutant channels, albeit less potently than ATP. However, there was no significant difference in the IC50 obtained for SUR1 and SUR2A channels (Fig. 4A). Markedly different results were obtained in the presence of Mg2+. Wild-type Kir6.2/SUR2A and Kir6.2/SUR1 channels were activated by MgGDP, with 1 mM MgGDP eliciting a 2.3-fold activation in both cases (Fig. 4C and E). At this concentration, GDP blocks Kir6.2 by 40%, as indicated by the data obtained in the absence of Mg2+. This suggests that the overall stimulatory effect of MgGDP is actually closer to 3-fold for wild-type channels. When Kir6.2-R201H was coexpressed with SUR2A, 1 mM MgGDP also stimulated channel activity 3-fold (Fig. 4C). Since this GDP concentration produces very little block of homKir6.2-R201H/SUR2A channels in the absence of Mg2+, the R201H mutation does not appear to alter MgGDP-dependent activation by SUR2A. In contrast, activation of homR201H-SUR1 channels was greatly enhanced, 1 mM MgGDP producing a 10-fold increase in current (Fig. 4E). Thus, these results suggest that the R201H mutation in Kir6.2 facilitates the mechanism by which Mg-nucleotide handling at SUR1 (but not SUR2A) is translated into opening of the Kir6.2 pore.
To determine if the effects of nucleoside triphosphates are also affected by the Kir6.2 mutation we explored the effects of MgGTP (Fig. 4D and F). In the absence of Mg2+, GTP blocked wild-type SUR2A and SUR1 channels to a similar extent. Although the nucleotide was less effective at blocking R201H channels, it again blocked SUR2A and SUR1 channels to a similar extent. In the presence of Mg2+, both wild-type SUR2A and SUR1 channels were blocked by MgGTP, with IC50 of 1.0 ± 0.4 mM (n= 4) and 2.2 ± 0.8 mM (n= 5), respectively. However, there were striking differences between the effects of MgGTP on homR201H channels. Thus SUR2A channels were still blocked by MgGTP (although to a lesser extent) whereas SUR1 channels were activated by MgGTP.
Resting KATP currents
Finally, we assessed if the different ATP sensitivities of SUR1 and SUR2A mutant channels lead to differences in the amplitude of resting KATP currents, when expressed in oocytes. Wild-type Kir6.2/SUR1 channels are closed at rest due to the high intracellular ATP concentration ([ATP]i), and open only in response to metabolic inhibitors, such as azide, which lower [ATP]i (Fig. 5; Gribble et al. 1997). In contrast, wild-type SUR2A channels are activated only poorly by azide (Fig. 5). This is not due to differences in channel expression, as equivalent current amplitudes were recorded from patches excised from oocytes expressing Kir6.2/SUR2A (5.0 ± 0.8 nA at –100 mV, n= 5) or Kir6.2/SUR1 (5.5 ± 0.5 nA, n= 5). Thus, Kir6.2/SUR2A channels are less sensitive to metabolic inhibition than Kir6.2/SUR1 channels when expressed in Xenopus oocytes, as previously reported (Ashcroft & Gribble, 1998).
When mutant Kir6.2 was coexpressed with SUR2A, the resulting homomeric channels exhibited significant resting currents, although these were not as large as for channels containing SUR1 (Fig. 5). The greater extent of activation of R201H/SUR2A channels on metabolic poisoning, compared with R201H/SUR1 channels, may be because the open probability (Po) of R201H/SUR2A channels was lower initially (as R201H/SUR2A will be less activated by resting levels of MgADP in the oocyte) which provides greater scope for activation. This idea is supported by the fact that both Q52R/SUR2A and Q52R/SUR1 channels, which have a high intrinsic Po, showed only a small increase in current on azide application (Fig. 5).
Resting currents through hetR201H/SUR2A channels were not significantly different from wild-type, but those of hetQ52R/SUR2A were slightly larger (Fig. 5). However, for both mutations, resting currents were much larger for heterozygous SUR1 channels than SUR2 channels, and they were increased by azide to a greater extent. This is harmonious with the much smaller currents observed in the presence of 1 mM MgATP in excised patches found for heterozygous mutant SUR2A than mutant SUR1 channels.
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Discussion |
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Our results are consistent with previous studies which suggest that mutations at R201H reduce ATP inhibition at Kir6.2 by affecting ATP binding and/or transduction (Gloyn et al. 2004). The location of R201 within the ATP-binding site (John et al. 2003; Antcliff et al. 2005) is consistent with this idea.
As previously described (Proks et al. 2004), the Q52R mutation enhanced the intrinsic gating of Kir6.2/SUR1 channels and concomitantly reduced their ATP sensitivity. This suggests that the mutation alters ATP inhibition indirectly, by stabilizing the open state of the channel. A similar argument can be applied in the case of Kir6.2-Q52R/SUR2A. However, although the Q52R mutation increased the open probability of both SUR1 and SUR2A channels, it had no effect on Kir6.2C expressed in the absence of SUR. This indicates that the mutation does not alter the intrinsic gating of Kir6.2 but instead influences the interaction of Kir6.2 with SUR. This seems plausible given that Q52 is predicted to lie on the outer surface of the tetramer in a model of Kir6.2 (Antcliff et al. 2005), a position that would facilitate interactions with SUR.
It is evident that the Q52R mutation causes a greater shift in the IC50 for ATP block of SUR2A channels (352 µM) than of SUR1 channels (84 µM). However, the intrinsic open probability was increased to a similar extent. There are two possible explanations for these findings. First, the mutation may differentially influence the interaction of Kir6.2 and SUR. For example, the increased ATP sensitivity produced by the SUR subunit (Tucker et al. 1997) may be abolished when Kir6.2-Q52R is coexpressed with SUR2A, but not when expressed with SUR1. Second, it may reflect differences in the channel kinetics. Because of the steep relationship between Po(0) and the IC50 for ATP inhibition at high Po(0) (Enkvetchakul et al. 2000; see also Fig. 3 in Supplemental material), differences in open probability too small to be experimentally measurable can produce large changes in ATP sensitivity.
Molecular mechanism of reduced ATP sensitivity in Mg2+-containing solution
Our results reveal that the ability of ATP to inhibit homomeric mutant channels is strikingly less in the presence of Mg2+. Furthermore, for both R201H and Q52R mutations, ß-cell-type (SUR1) channels were 3–4 times less sensitive than cardiac-type (SUR2A) channels.
In the presence of Mg2+, nucleotides not only block the channel by binding to Kir6.2 but also increase channel activity by promoting nucleotide binding/hydrolysis at the NBDs of SUR (Zingman et al. 2002b). Although Kir6.2/SUR1 and Kir6.2/SUR2A channels were activated by MgGDP to similar extents, there was a striking difference in the stimulatory effect of MgGDP on homR201H channels containing SUR1 or SUR2A. Thus, activation of R201H/SUR2A channels was similar to that of wild-type channels, whereas activation of R201H/SUR1 channels was > 2-fold larger. This suggests that the R201H mutation enhances the mechanism by which binding of MgGDP (or MgADP) to SUR1 (but not SUR2A) is translated into opening of the Kir6.2 pore. Although the nature of this pathway remains obscure, our results imply that different residues are involved in this process in SUR1 and SUR2A. The fact that MgGTP blocked R201H/SUR2A channels but activated R201H/SUR1 channels can also be explained by enhanced transduction, although we cannot exclude the possibility that the mutation also enhances nucleotide hydrolysis at the NBDs of SUR1 but not SUR2A.
Taken together, our results demonstrate that the reduced MgATP sensitivity of R201H/SUR1 is due to a combination of decreased ATP binding/transduction at Kir6.2 and enhanced activation by Mg-nucleotides at SUR1. In the case of R201H/SUR2A, only ATP binding/transduction at Kir6.2 is affected, resulting in a greater overall ATP sensitivity of cardiac channels than ß-cell channels. A quantitative analysis of the stimulatory action of Mg-nucleotides on Q52R channels was prevented by the high intrinsic open probability of the channel. Nevertheless, the much greater reduction in the overall ability of ATP to inhibit mutant SUR1 channels than SUR2A channels in the presence of Mg2+, despite a similar block in the absence of Mg2+, suggests that MgATP activation may also be enhanced to a greater extent for Kir6.2-Q52R/SUR1 than Kir6.2-Q52R/SUR2A.
Differences between wild-type SUR1 and SUR2 channels
It is striking that whereas Kir6.2/SUR1 channels are potently activated by metabolic inhibition, Kir6.2/SUR2A channels are not (Fig. 5; Ashcroft & Gribble, 1998). Our results suggest that this is because Kir6.2/SUR2A is less sensitive to changes in adenine nucleotides. Decreasing the overall MgATP sensitivity of the channel 28-fold, as in homR201H channels, increases both resting and azide-activated currents. It may appear surprising that although Kir6.2/SUR2A is less sensitive to metabolic inhibition, the extent of ATP block and Mg-nucleotide activation is similar to that of Kir6.2/SUR1, and the IC50 for MgATP inhibition is actually higher (Tables 1 and 2). However, a possible explanation is that the ability of MgADP to activate channels preblocked by ATP is less for SUR2 than SUR1 (Matsuoka et al. 2000; Masia et al. 2005), a difference which may be related to the lower affinity of the NBDs of SUR2A for adenine nucleotides (Matsuo et al. 2000) and/or to the ability of NBD2 of SUR1 to hydrolyse MgATP more effectively than that of SUR2A (Masia et al. 2005).
Physiological relevance
Our results reveal that the magnitude of KATP current in excised patches exposed to physiological levels of MgATP (1–5 mM) is very different when heterozygous Kir6.2 mutations are coexpressed with SUR2A than when coexpressed with SUR1: for example, at 1 mM MgATP, KATP currents are 2-fold greater for hetR201H/SUR1 and 4-fold greater for hetQ52R/SUR1 channels than for their SUR2A equivalents. Electrophysiological studies suggest that cardiac KATP channels are composed of SUR2A (Babenko et al. 1998; Gribble et al. 1998b; Rainbow et al. 2004) and ß-cell channels of SUR1 (Inagaki et al. 1995; Sakura et al. 1995). Thus, PNDM mutations are expected to cause a significantly greater increase in the whole-cell KATP current in the ß-cells than in cardiac myocytes. This may result in ß-cell hyperpolarization and impaired insulin secretion, yet be insufficient to produce changes in cardiac membrane potential or action potential duration. Thus the lack of obvious cardiac effects in patients with PNDM mutations may be attributable to differences in Mg2+-nucleotide handling by SUR1 and SUR2A when coexpressed with mutant Kir6.2. Differences in metabolism between ß-cells and heart may also contribute to reduced activation of cardiac KATP currents by PNDM mutations.
Our data also suggest that the differences in MgATP sensitivity may underlie the paradoxical finding that mice carrying gain-of-function mutations in Kir6.2 under the control of a cardiac-specific promoter showed little evidence of enhanced KATP currents in cardiac myocytes, or cardiac problems despite a marked reduction in the ATP sensitivity of the cardiac KATP channel (Koster et al. 2001). In these experiments, the inhibitory effect of ATP was investigated in the absence of Mg2+. Our experiments raise the possibility that a greater difference in the ATP sensitivity of cardiac and ß-cell channels might be found if the experiments were carried out in the presence of Mg2+.
In addition to the heart, SUR2A is expressed in skeletal muscle (Inagaki et al. 1996). Patients with mutations that cause severe reductions in KATP channel ATP sensitivity experience muscle weakness in addition to neonatal diabetes (DEND and intermediate DEND syndrome; Ashcroft, 2005). The smaller effect of Kir6.2 mutations on the ATP sensitivity of SUR2A channels we observed compared with SUR1 channels, suggests that muscle weakness may not be caused by activation of KATP channels in skeletal muscle. Rather, it may result from reduced transmitter release resulting from overactivity of KATP channels in the presynaptic nerve terminals or motor cortex (Deist et al. 1992; Lee et al. 1996; Allen & Brown, 2004). This could be demonstrated in the clinical situation if muscle weakness was reversible with SUR1-specific types of sulphonylureas such as gliclazide (Gribble & Reimann, 2003). Conversely, if muscle weakness was unaffected by gliclazide but reversed by sulphonylureas that interact with both SUR1 and SUR2A (e.g. glibenclamide) this would argue that muscle weakness is due to overactivity of KATP channels in skeletal muscle.
Finally, our results reveal that mutations in Kir6.2 may not simply affect properties intrinsic to this channel subunit, but also exert allosteric effects on properties conferred by the sulphonylurea receptor. Consequently, to fully understand how PNDM mutations give rise to the disease phenotype it is necessary to characterize the properties of the KATP channel in detail, and to explore how the mutation affects the stimulatory, as well as inhibitory, action of nucleotides.
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Footnotes |
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References |
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), or SUR1 (
) and for Kir6.2-R201H coexpressed with SUR2A (
) or SUR1 (•). The lines are the best fits of eqn (1), with IC50 of 1.4 mM (wild-type) and 7.8 mM (R201H) for GDP and of 0.89 mM (wild-type) and 5.8 mM (homR201H) for GTP. Mean values are given in Tables 1a and 2a of the Supplemental material. C and D, relationship between [MgGDP] (C) or [MgGTP] (D) and KATP conductance for Kir6.2/SUR2A (•) and homKir6.2-R201H/SUR2A (
