Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP.
ABSTRACT 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 beta-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.
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ABSTRACT: Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.Nature Communications 08/2014; 5:4639. · 10.74 Impact Factor
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ABSTRACT: Single gene mutations that primarily affect pancreatic β-cell function account for approximately 1-2% of all cases of diabetes. Overlapping clinical features with common forms of diabetes makes diagnosis of monogenic diabetes challenging. A genetic diagnosis often leads to significant alterations in treatment, allows better prediction of disease prognosis and progression, and has implications for family members. Currently, genetic testing for monogenic diabetes relies on selection of appropriate individual genes for analysis based on the availability of often-limited phenotypic information, decreasing the likelihood of making a genetic diagnosis. We thus developed a targeted next-generation sequencing (NGS) assay for the detection of mutations in 36 genes known to cause monogenic forms of diabetes, including transient or permanent neonatal diabetes mellitus (TNDM or PNDM), maturity-onset diabetes of the young (MODY) and rare syndromic forms of diabetes. A total of 95 patient samples were analyzed: 19 with known causal mutations and 76 with a clinically suggestive phenotype but lacking a genetic diagnosis. All previously identified mutations were detected, validating our assay. Pathogenic sequence changes were identified in 19 out of 76 (25%) patients: 7 of 32 (22%) NDM cases, and 12 of 44 (27%) MODY cases. In 2 NDM patients the causal mutation was not expected as consanguinity was not reported and there were no clinical features aside from diabetes. A 3 year old patient with NDM at 3 months of age who previously tested negative for INS, KCNJ11 and ABCC8 mutations was found to carry a novel homozygous mutation in EIF2AK3 (associated with Wolcott-Rallison syndrome), a gene not previously suspected because consanguinity, delayed growth, abnormal bone development and hepatic complications had not been reported. Similarly, another infant without a history of consanguinity was found to have a homozygous GCK mutation causing PNDM at birth. This study demonstrates the effectiveness of multi-gene panel analysis in uncovering molecular diagnoses in patients with monogenic forms of diabetes.Molecular Genetics and Metabolism 09/2014; · 2.83 Impact Factor
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
Received June 10, 2005; Revised and Accepted August 3, 2005
Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming
subunit of the ATP-sensitive potassium (KATP) 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 Mg21, to isolate the inhibitory effects of ATP on Kir6.2, showed that
both types of mutation reduce channel inhibition by ATP. However, in pancreatic b-cells, KATPchannel
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 KATPchannels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg21
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 KATPchannels in two ways: by increasing MgATP activation and by
decreasing ATP inhibition. They further show that the fraction of unblocked KATPcurrent at physiological
MgATP concentrations correlates with the severity of the clinical phenotype.
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 Kþ
(KATP) 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 b-cells and many brain neurones (12), and SUR2 is
expressed in the heart, smooth muscle, skeletal muscle and
the brain (13,14).
KATPchannels mediate glucose-induced insulin secretion
from pancreatic b-cells by coupling cell metabolism to electri-
cal activity of the plasma membrane (15–17). In the absence
of glucose, KATPchannels 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 b-cell
KATPchannels, and leads to a membrane depolarization that
opens Ca2þchannels, stimulates electrical activity and Ca2þ
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). KATPchannels
are also closed by sulphonylurea and glinide drugs, which
stimulate insulin secretion by binding to the SUR subunit of
the KATP 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 KATPchannels, but among the most
important are the adenine nucleotides, which are believed to
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Human Molecular Genetics, 2005, Vol. 14, No. 18
Advance Access published on August 8, 2005
mediate the metabolic regulation of KATP 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
Mg2þas when a truncated version of Kir6.2 (Kir6.2DC) is
expressed in the absence of SUR the ATP sensitivity of the
channel is the same whether or not Mg2þis present (22). Con-
versely, 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 Mg2þ, KATPchannel activity
is governed by the balance between the stimulatory and inhibi-
tory effects of nucleotides. Functional analysis of cloned KATP
channels has revealed that all KCNJ11 mutations examined to
date produce a marked decrease in the ability of ATP to block
the KATPchannel, 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 Mg2þ, 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 inter-
action 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 KATP channels carrying
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 deve-
lopmental 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 popu-
lation of homomeric and heteromeric channels that results as
The results reported here show that Mg2þproduces a much
greater shift in the ATP sensitivity of heterozygous KATP
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, KATPcurrents at ATP concentrations within
the physiological range are strikingly increased. This is
expected to hyperpolarize the pancreatic b-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]iand disease severity.
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 Mg2þ. As previously reported, the cation
caused a small shift in the IC50for ATP block of wild-type
channels (from 7 mM to 13 mM, Table 1) (22). It is evident
that both homR201C and hetR201C channels are very much
less sensitive to ATP in the presence of Mg2þthan in its
absence (Table 1). In addition, this effect is greater than for
wild-type channels: on addition of Mg2þ, the IC50increased
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 (IC50¼ 307 mM) than in
the absence (11 mM) of Mg2þ. The physiological implications
of this finding are discussed in detail subsequently.
Role of phosphoinositides
One mechanism by which MgATP could enhance KATP
channel activity is via synthesis of membrane phosphoinosi-
tides such as PIP3and PIP2, which enhance the activity and
decrease the ATP sensitivity, of the KATPchannel by interact-
ing with Kir6.2 (31–33). In this case, the greater effect of
MgATP on R201C channels could reflect an enhanced sensi-
tivity to phosphoinositides. We tested this possibility using
the lipid kinase inhibitor LY294002. Figure 1C shows that
by the presence of 10 mM LY294002, which blocks PIP3
production. In the presence of the lipid kinase inhibitor, the
IC50for ATP inhibition was 2.2 + 0.4 mM (n ¼ 6), not signifi-
cantly different from that found in its absence (2.4 mM).
Furthermore, 100 mM LY294002, which blocks both PIP2and
PIP3production, had no effect on KATPchannel activity in
the presence of either 1 or 10 mM MgATP (n ¼ 5).
Role of SUR1
An alternative explanation for the difference in ATP-sensitivity
in the presence and absence of Mg2þis that MgATP hydrolysis
by the nucleotide-binding domains (NBDs) of SUR1 leads to
the generation of MgADP, which stimulates KATPchannel
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 signifi-
cant difference in the IC50for ATP inhibition of Kir6.2-R201C/
SUR1-KAKM channels in the presence and absence of Mg2þ
(Fig. 1D), as expected if these mutations abolish KATP
channel activation by MgATP: the IC50were 231 + 50 mM
(n ¼ 6) and 150 + 27 mM (n ¼ 6), respectively. There was
also no significant difference in the IC50for ATP inhibition
of Kir6.2-R201C/SUR1-KAKM channels in the presence of
Mg2þ(150 + 27 mM, n ¼ 6) from that of Kir6.2-R201C/
SUR1 channels in the absence of the cation (106 + 12 mM,
n ¼ 6). However, in all cases the IC50was substantially less
than that of Kir6.2-R201C/SUR1 channels in the presence of
Mg2þ(2.4 mM). This is consistent with the idea that the large
reduction in the ATP sensitivity of R201C channels produced
by Mg2þis mainly conferred by MgATP binding/hydrolysis
at the NBDs of SUR1. In other words, the mutation enhances
2718Human Molecular Genetics, 2005, Vol. 14, No. 18
the stimulatory effect of MgATP that is mediated via SUR1,
thereby producing an apparent reduction in the inhibitory
effect of ATP when Mg2þis present.
MgATP activation is altered by other gain-of-function
mutations in Kir6.2
We next explored the effect of Mg2þon the ATP-sensitivity of
KATPchannels 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.
Figure 1. (A and B) Mean relationship between [ATP] and KATPconductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for
KATPchannels comprising SUR1 and either wild-type (WT, W, n ¼ 6), hetR201C (*, n ¼ 6) or homR201C (B, n ¼ 5) Kir6.2, in the absence (A) and presence
(B) of Mg2þ. The smooth curves are the best fit of Eq. 1 to the data. For wild-type, IC50¼ 6.6 mM (A), 13 mM (B); h ¼ 1.07 (A), 1.02 (B). For hetR201C,
IC50¼ 10.4 mM (A), 307 mM (B); h ¼ 1.0 (A), 0.76 (B). For homR201C, IC50¼ 102 mM (A), 2.4 mM (B); h ¼ 1.3 (A), 1.0 (B). The grey bars illustrate the
physiological range of [ATP] in b-cells. (C) Mean relationship between [MgATP] and KATPconductance (G), expressed relative to the conductance in the
absence of nucleotide (GC), for Kir6.2-R201C/SUR1 channels in the presence (B, n ¼ 6) and absence (*, n ¼ 5) of 10 mM LY292004 (LY). The smooth
curves are the best fit of Eq. 1 to the data. IC50¼ 2.4 mM (2LY); 2.0 mM (þLY); h ¼ 1.0 (2LY), 1.08 (þLY). (D) Mean relationship between [ATP] and
KATPconductance (G), expressed relative to the conductance in the absence of nucleotide (GC) for Kir6.2-R201C/SUR1 channels measured in the absence
(W, n ¼ 5) and presence (*, n ¼ 5) of Mg2þ, and for Kir6.2-R201C/SUR1-KA/KM channels in the absence ( , n ¼ 6) and presence of Mg2þ(B, n ¼ 6) chan-
nels. The smooth curves are the best fit of Eq. 1 to the data. For Kir6.2-R201C/SUR1, IC50¼ 2.4 mM, h ¼ 1.0 (2 mM Mg2þ) and IC50¼ 102 mM, h ¼ 1.3 (0 mM
Mg2þ). For Kir6.2-R201C/SUR1-KA/KM, IC50¼ 208 mM, h ¼ 1.96 (0 mM Mg2þ) and IC50¼ 147 mM, h ¼ 1.08 (2 mM Mg2þ).
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 Mg2þ. n ¼ 4–11.
MutationPhenotype Homo IC50(mM)
Mg-free MgATP MgATP
7400+1500 ,15% at
1260+300 17+ 6
V59M58 +13 44+7
2400+300 11+ 2
1960+140 11+ 2
Human Molecular Genetics, 2005, Vol. 14, No. 182719
However, the extent of the shift in the IC50varies, 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 par-
ticularly evident for hetV59M and hetV59G channels
(Fig. 3), perhaps because the homomeric channels are so
much less sensitive to MgATP.
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 homo-
MgATP sensitivity determines whole-cell current
In oocytes, whole-cell KATPcurrents are normally blocked by
the resting ATP concentration, but they can be activated by the
metabolic inhibitor azide, which reduces cellular ATP levels.
Figure 2. Mean relationship between [ATP] and KATPconductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for the indicated
homomeric mutant channels in the presence (*) or absence of Mg2þ(W). The smooth curves are the best fit of Eq. 1 to the data. For homR201C, IC50¼ 102 mM,
h ¼ 1.3 (2Mg2þ) and IC50¼ 2.4 mM, h ¼ 1.0 (þMg2þ). For homR201H, IC50¼ 296 mM (2Mg2þ), 1.95 mM (þMg2þ); h ¼ 1.8 (2Mg2þ), 0.8 (þMg2þ). For
homV59M, IC50¼ 60 mM (2Mg2þ), 440 mM (þMg2þ); h ¼ 0.97 (2Mg2þ), 0.77 (þMg2þ). For homV59G, IC50¼ 8.1 mM and h ¼ 0.75 (2Mg2þ); in the pre-
sence of Mg2þthe line was drawn by hand. Data in the absence of Mg2þare the same as those given by Proks et al. (27) (R201C, V59G, Q52R). The number of
patches is indicated by each graph.
2720 Human Molecular Genetics, 2005, Vol. 14, No. 18
Owing to their reduced ATP sensitivity, however, significant
resting currents can be recorded from KCNJ11 mutant chan-
nels. Finally, we compared the magnitude of the resting
KATPcurrent measured in whole-cell recordings with the frac-
tion of KATPcurrent 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
b-cells (36,37). To control for possible differences in KATP
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
KATP 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 KATPchannel.
Our data demonstrate that the ATP sensitivity of KATPchan-
nels containing gain-of-function mutations in Kir6.2 is much
less in the presence of Mg2þthan 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 KATPchannel
Figure 3. Mean relationship between [ATP] and KATPconductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for the indicated
heterozygous mutant channels in the presence (*) or absence of Mg2þ(W). For hetV59M, IC50¼ 17 mM (2Mg2þ), 62 mM (þMg2þ); h ¼ 0.94 (2Mg2þ), 0.9
(þMg2þ). The value of a for hetV59M in the presence of Mg2þwas 0.1. For hetV59G, IC50¼ 26 mM (2Mg2þ), 319 mM (þMg2þ); h ¼ 1.18 (2Mg2þ), 0.8
(þMg2þ); in the absence of Mg2þthe curve was fitted assuming that 1/16 of the heterozygous population are homomeric mutant channels (IC50¼ 8.1 mM
and h ¼ 0.75); in the presence of Mg2þa ¼ 0.3. Data in the absence of Mg2þare the same as those given by Proks et al. (27) (R201C, V59G, Q52R). The
number of patches is indicated by each graph.
Human Molecular Genetics, 2005, Vol. 14, No. 18 2721
to MgATP in two different ways: by decreasing ATP
inhibition, as previously described (1,6,27,28) and by increas-
ing MgATP activation, as shown here.
Mechanism of the Mg-dependent shift in ATP sensitivity
Studies of wild-type KATPchannels have previously shown
that Mg2þdoes not alter the affinity of Kir6.2 for ATP,
because the ATP sensitivity of Kir6.2DC expressed in the
absence of SUR is not affected by Mg2þ(22). Although phos-
phoinositides can shift the ATP sensitivity of KATPchannels
(31–33), our results suggest that an increase in their synthesis
also does not underlie the Mg-dependent shift in ATP sensi-
tivity. Instead, Mg-nucleotide interactions with SUR1 are
primarily responsible for the shift in the ATP concentration-
inhibition curve produced by Mg2þ. This effect is greater for
mutant Kir6.2 channels than for wild-type channels. Further-
more, the fact that the shift in IC50produced by Mg2þis
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 KATPchannel
There are several possible explanations for how a mutation
in Kir6.2 enhances channel activation by MgATP. First, con-
formational 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 transduc-
tion) 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 popu-
lation, 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 het-
erozygous 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
Kir6.2DC 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 (KO) and
closed (KC) states is affected; if KOincreases and KCstays
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 KATPchannels 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 com-
plete 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.
Figure 4. Mean relationship between [MgATP] and KATPconductance (G), expressed relative to the conductance in the absence of nucleotide (GC), for wild-type
channels (W) and the indicated heterozygous (*) and homomeric (B) mutant channels. The data are the same as those in Figures 2 and 3. The grey bars illustrate
the physiological range of [ATP] in b-cells.
2722Human Molecular Genetics, 2005, Vol. 14, No. 18
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 KATPchannels.
Our results demonstrate that the increase in KATP current
at physiologically relevant concentrations of ATP produced
by KCNJ11 mutations is substantially greater in the presence
of Mg2þthan in its absence. This suggests that the shift in
ATP sensitivity and increase in whole-cell KATP current,
required to cause neonatal diabetes may be greater than had
been anticipated from earlier studies carried out in the
absence of Mg2þ(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 physiologi-
cally relevant concentrations of MgATP (1–5 mM), mutations
that cause small increases in KATPcurrent result in transient
neonatal diabetes, whereas larger increases in current cause
neonatal diabetes alone, and an even greater increase is associ-
ated with DEND syndrome (Table 1). The variation in KATP
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
In pancreatic b-cells, an increase in KATPcurrent 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 KATPcurrent, the more severely insulin secretion will be
Table 2. Fraction of unblocked KATPconductance 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.
Mutation PhenotypeFraction unblocked IKATPat MgATP
Table 3. Whole-cell KATPcurrent measured in two-electrode voltage clamp for
KATPchannels 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)
MutationPhenotype Resting currentReferences
23+4 (n ¼ 14)
17+7 (n ¼ 10)
Human Molecular Genetics, 2005, Vol. 14, No. 182723
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 KATPchannel.
Kir6.2 is also expressed in skeletal muscle, cardiac muscle
and neurons throughout the brain (9,10), a distribution consist-
ent 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 KATP
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 KATPcurrent, and which lead to the largest
increases in whole-cell current. There are many possible
reasons why KATPchannels could contribute less to the elec-
trical activity of brain and muscle than pancreatic b-cells.
These include differences in cell metabolism, contributions
to membrane current from other ion channels, a low KATP
channel density and association of Kir6.2 with SUR2 [which
reduces the response to metabolism (43)].
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 KATPcurrent magnitude at physiological
[ATP]i. Because the reduction in ATP sensitivity is much
greater when Mg2þis 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 KATPcurrent at physiological
[ATP]iand 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
KATPcurrent 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
MATERIALS AND METHODS
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 QuickChangeTMXL system
(Stratagene). Wild-type and mutant cDNAs were cloned in
the pBF vector, and capped mRNA prepared using the mMES-
SAGE 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 heterozy-
gous 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 simu-
late the heterozygous state is that the levels of expression
maybe differentfor wild-type
However, the shift in the IC50for ATP inhibition of our het-
erozygous 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 popu-
lation, namely those that have one mutant (or one wild-type)
subunit, and those that have all mutant or all wild-type chan-
nels. This amounts to 63% of the heterozygous channel popu-
lation 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 KATPchannel was altered
when tandem dimers were constructed: the EC50was much
smaller for the dimer than for either homomeric mutant or
wild-type channels. Thus, we believe that simulating the het-
erozygous state by co-injection of wild-type and mutant sub-
units 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 Mg2þ, 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 8C with a solution containing (in mM): 90 KCl,
Figure 5. Relationship between the fraction of unblocked KATPconductance
(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/GCvalue 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.
2724 Human Molecular Genetics, 2005, Vol. 14, No. 18
1 MgCl2, 1.8 CaCl2and 5 HEPES (pH 7.4 with KOH). Meta-
bolic 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 210 mV.
Macroscopic currents were recorded from giant excised
inside-out patches using the patch-clamp technique in
response to 3 sec voltage ramps from 2110 mV to þ100 mV
(holding potential, 0 mV) and 20–22 8C. Currents were fil-
tered 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) solu-
tion 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 mm dia-
meter in which the tip of the patch pipette was inserted.
The macroscopic slope conductance was measured between
2100 and þ10 mV. ATP concentration-response curves were
fit with the Hill equation:
G=Gc¼ a þ ð1 ? aÞ=ð1 þ ð½ATP?=IC50ÞhÞ
where [ATP] is the ATP concentration, IC50is the ATP con-
centration 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, Gcwas taken as the mean of
the conductance in control solution before and after ATP
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 signifi-
cance 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.
We thank the Wellcome Trust, the Royal Society and the EU
(BioSim) for support. F.M.A. is a Royal Society Research
Conflict of Interest statement. None declared.
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