The EMBO Journal (2005) 24, 229–239 |& 2005 European Molecular Biology Organization | All Rights Reserved

rved 0261-4189/05
www.embojournal.org THE
EMBO
JOURNAL
Functional analysis of a structural model of the
ATP-binding site of the KATP channel Kir6.2 subunit

Jennifer F Antcliff1,3, Shozeb Haider2,3, of KATP channel activity is mediated by changes in the
Peter Proks1,3, Mark SP Sansom2 intracellular concentrations of adenine nucleotides, which
and Frances M Ashcroft1,* interact with both Kir6.2 and SUR subunits. Binding of ATP
1
or ADP to Kir6.2 produces channel inhibition (Tucker et al,
University Laboratory of Physiology, Parks Road, Oxford, UK and
2
Laboratory of Molecular Biophysics, Department of Biochemistry,
1998; Tanabe et al, 2000), whereas interaction of Mg nucleo-
University of Oxford, Oxford, UK tides with the nucleotide-binding domains (NBDs) of SUR
stimulates channel activity (Nichols et al, 1996; Gribble et al,
ATP-sensitive potassium (KATP) channels couple cell me- 1997). Loss-of-function mutations in both Kir6.2 and SUR1
tabolism to electrical activity by regulating K þ flux across genes cause congenital hyperinsulinism (Dunne et al, 2004).
the plasma membrane. Channel closure is mediated by Conversely, mutations in Kir6.2 that lead to reduced ATP
ATP, which binds to the pore-forming subunit (Kir6.2). sensitivity of the channel cause permanent neonatal diabetes,
Here we use homology modelling and ligand docking to which in some cases is associated with muscle weakness,
construct a model of the Kir6.2 tetramer and identify the developmental delay and epilepsy (Gloyn et al, 2004).
ATP-binding site. The model is consistent with a large To understand how ATP inhibits the KATP channel and how
amount of functional data and was further tested by mutations in Kir6.2 impair this process, it is necessary to
mutagenesis. Ligand binding occurs at the interface be- identify the location and structure of the ATP-binding site.
tween two subunits. The phosphate tail of ATP interacts The structure of this inhibitory site is unknown, but is likely to
with R201 and K185 in the C-terminus of one subunit, and differ from that of classical ATP-binding sites, because it has
with R50 in the N-terminus of another; the N6 atom of the several unusual properties. For example, Mg2 þ is not required
adenine ring interacts with E179 and R301 in the same for the inhibitory action of the nucleotide (Ashcroft and Kakei,
subunit. Mutation of residues lining the binding pocket 1989); the site is extremely selective for the adenine base
reduced ATP-dependent channel inhibition. The model (Tucker et al, 1998), and addition of bulky groups to the end of
also suggests that interactions between the C-terminus of the phosphate chain does not abolish the inhibitory effect of
one subunit and the ‘slide helix’ of the adjacent subunit ATP (Ämmälä et al, 1991; Tanabe et al, 2000). Many experi-
may be involved in ATP-dependent gating. Consistent with ments suggest that the ATP-binding site involves residues from
a role in gating, mutations in the slide helix bias the both the N- and C-terminal domains (N and C domains) of
intrinsic channel conformation towards the open state. Kir6.2 (Drain et al, 1998; Proks et al, 1999; Tucker et al, 1998;
The EMBO Journal (2005) 24, 229–239. doi:10.1038/ Reimann et al, 1999; Cukras et al, 2002; John et al, 2003;
sj.emboj.7600487; Published online 13 January 2005 Ribalet et al, 2003; Tsuboi et al, 2004). In addition, although
Subject Categories: structural biology; membranes & transport the primary site at which ATP mediates channel inhibition lies
Keywords: ATP binding; KATP channel; Kir6.2 on Kir6.2, coexpression with SUR enhances the potency of ATP
block about 10-fold (Tucker et al, 1997).
Attempts to crystallize Kir6.2, or its C domains, have
proved unsuccessful. However, the crystal structures of a
Introduction putative bacterial Kir channel, KirBac1.1 (Kuo et al, 2003),
ATP-sensitive potassium (KATP) channels play key roles in and of the N and C domains of Kir3.1 (Nishida and
many tissues by linking cell metabolism to electrical activity. MacKinnon, 2002), have recently been solved. We have
They are involved in glucose sensing in pancreatic islets, gut therefore used these structures to construct a molecular
L cells and the brain; in the regulation of vascular smooth model of the Kir6.2 tetramer, and ligand docking to identify
muscle tone; in the response to ischaemic stress in heart and residues interacting with ATP. The model is consistent with a
brain; and in seizure protection (Seino and Miki, 2003). large amount of functional data and was further tested by
KATP channels are octameric complexes of two different additional site-directed mutagenesis. Our results indicate the
proteins (Clement et al, 1997). Four inwardly rectifying K þ location and structure of the four ATP-binding sites, and
channel subunits form a tetrameric pore: in almost all tissues suggest a possible model for how binding of ATP is trans-
except vascular smooth muscle, Kir6.2 serves this role. Each duced to transmembrane elements of the channel and thus to
Kir subunit is associated with a regulatory sulphonylurea closure of the pore.
receptor (SUR) subunit, which belongs to the ATP-binding
cassette family of transporter proteins. Metabolic regulation Results
*Corresponding author. Laboratory of Physiology, University of Oxford, Construction of a molecular model of Kir6.2
Parks Road, Oxford OX1 3PT, UK. Tel.: þ 44 1865 285810; A homology model of Kir6.2 (GenBank D50581) was con-
Fax: þ 44 1865 272469; E-mail: [email protected] structed based on the X-ray crystal structures of KirBac1.1
3
These authors contributed equally to this work
(Kuo et al, 2003) and the intracellular (IC) domains of Kir3.1
Received: 2 September 2004; accepted: 27 October 2004; published (Nishida and MacKinnon, 2002). KirBac1.1 was used as a
online: 13 January 2005 template for the proximal N domain and the transmembrane

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 229
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

by the position of residue 63 in the Kir3.1 crystal structure

(Figure 1A). The residues corresponding to positions 47–50 of
Kir6.2 were not resolved in the crystal structure of Kir3.1:
therefore, they were modelled as a loop and energy mini-
mized. The minimization procedure did not significantly alter
the position of the backbone of residue 50 in Kir6.2. The Ca
r.m.s.d. between the N domain of Kir3.1 (template) and the N
domain of Kir6.2 was 0.4 Å. The C domain of Kir6.2 was built
as described previously (Trapp et al, 2003). The Ca r.m.s.d.
between the Kir3.1 C-domain (template) and the Kir6.2
C-domain (model) was 1.3 Å and that between the TMs of
KirBac (template) and Kir6.2 (model) was 1.7 Å. These values
lie within the range expected for proteins sharing 25%
sequence identity (0.7–2.3 Å) (Russel et al, 1997).
The position of the ATP-binding site was predicted using
ligand docking. ATP was randomly positioned on the C-
domain model and 25 dockings were carried out (Trapp
et al, 2003). No information about the potential binding site
obtained from mutagenesis studies was specified in the dock-
ing procedure. In 19/25 runs (76%), the b-phosphate of ATP
interacted with K185 (which is suggestive of an energy
minimum on the potential energy landscape). The rest of
the molecule lay in an identical position in five runs, and
close to one or more of a consensus set of residues in all
cases. In similar studies, ADP found the same set of residues
as ATP 16/25 times (64%), but GTP, azido-ATP, ADP-ribose
and ATP-ribose never localized to the same position.
The ligand-docking program used did not allow for re-
orientation of side chains in response to the presence of ATP.
It is clear that this would occur, however, because of the strong
negative charge of the phosphate tail. Thus, the side chains of
R201 and K185 were manually oriented to point towards the
ATP molecule and then minimized. However, the backbone
was not altered. The positions of the side chains of other
residues, including R50, were not adjusted. Automated dock-
Figure 1 (A) Alignment of Kir3.1, Kir6.2 and KirBac1.1 sequences. ing on the modified model revealed that the b-phosphate of
Regions boxed in red and black show residues in Kir3.1 and
KirBac1.1, respectively, used to construct the model. (B, C) Model ATP found K185 in all 25/25 runs. The Kir6.2–ATP complexes
of the Kir6.2 tetramer viewed from the side (B) or above (C). (B) were then evaluated based on interaction energies between
Residues are shown in ribbon format, with different colours repre- ATP and the protein, in order to determine the conformation of
senting individual subunits. (C) TMs are shown in backbone format the docked ATP and the residues that contribute to its binding
and IC domains in ribbon format. Different colours indicate indivi-
dual subunits, with dark and light shades representing the N and C site. ATP was then superimposed in the same spatial orienta-
domains, respectively. tion in the other subunits within the tetrameric model.
It is likely that the crystal structures of both KirBac1.1
and Kir3.1 represent the closed state of the channel. First,
(TM) domains of Kir6.2, whereas the distal N domain and KirBac1.1 is clearly closed, as bulky side-chain residues
the C domain were modelled on the IC domains of Kir3.1 occlude access of hydrated K þ to the intracellular mouth
(Figure 1A). This was because, over the region modelled, the of the pore (Kuo et al, 2003). Second, Kir3.1 is activated by
C domains of Kir3.1 and Kir6.2 exhibit a greater sequence binding Gbg proteins, and the IC domains were crystallized in
identity (48%) than those of Kir6.2 and KirBac1.1 (27%). the absence of ligand. Thus, we assume that these structures
Further, the crystal structure of the IC domain of Kir3.1 was form a reasonable template for the closed state of Kir6.2, in
determined at a resolution higher (2.0 Å) than that of which ATP is bound.
KirBac1.1 (3.6 Å). Each of the three segments of the model The KATP channel comprises both Kir6.2 and SUR subunits,
(TMs, N and C domains) were constructed separately, and and it is possible that SUR imposes conformational changes
then joined together. The spatial orientation of the IC domain, on Kir6.2. Thus, our model corresponds most closely to
with respect to the TMs, was determined from the location of Kir6.2DC, a truncated form of Kir6.2 that expresses in the
conserved residues in the IC domain of KirBac1.1. absence of SUR1 (Tucker et al, 1997). Thus, in our discus-
The structure of the N domain of Kir3.1 was resolved sions, we distinguish functional data obtained for Kir6.2DC
between residues 43 and 57 (Nishida and MacKinnon, from that measured for Kir6.2/SUR1 channels.
2002), which correspond to residues 32–46 of Kir6.2
(Figure 1A). The position of these residues with respect to Overall structure of Kir6.2
the C domain of Kir6.2 is fixed by the crystal structure of Figure 1B and C give the overall structure of the tetrameric
the IC domain of Kir3.1. Residue 51 in Kir6.2 is also fixed, model of Kir6.2. It can be divided into two main regions, with

230 The EMBO Journal VOL 24 | NO 2 | 2005 & 2005 European Molecular Biology Organization
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

the ‘slide helix’ (residues 54–66) lying at their interface. The TM2, exhibits the characteristic signature sequence of K þ
TM region is 44 Å long (measured as the Ca–Ca distance channels, except that GYG is replaced by GFG. The r.m.s.d.
between residues P109 and F60) and the cytosolic (IC) between the TMs of the present model and that of an earlier
domain is 63 Å long (D65–A358). When viewed from model of the TMs alone based on KcsA (Capener et al, 2003)
above, the TM tetramer forms a square with dimensions of was 2.2 Å. Most of the variation resides in the extracellular
46 Å  46 Å between the furthest points on adjacent subunits loops that connect TM1 with the pore helix (residues 92–116).
(E104–P102). It sits on a pedestal-like IC domain, which If these are omitted, the r.m.s.d. between the two models is
measures 55 Å  55 Å between D323 and D323 of adjacent excellent: B0.8–1.1 Å.
subunits. The cytosolic pore that permeates the IC domain
ranges from 13 to 50 Å in diameter. The IC domain. The IC domain shows extensive interactions
between subunits: thus, the C domain of one subunit inter-
The TM domains. Kir6.2 has two membrane-spanning acts both with the N domain of the adjacent subunit on one
a-helices (TM1 and TM2) per subunit. TM1 and TM2 are side, and with the C domain of the adjacent subunit on the
connected via loop segments and a short a-helix (the pore other side (Figure 1C). The region of the N domain involved
helix) that loops partway into the membrane. The selectivity is consistent with protein–protein interaction studies, which
filter, which lies within the loop linking the pore helix to implicate residues 30–46 as binding to the C domain (Tucker
and Ashcroft, 1999).
The model predicts several interactions within the IC
domain of Kir6.2, which may help to stabilize the tetrameric
structure. These include a p-stacking interaction between F35
in the N domain of one subunit and Y326 in the C domain
of the adjacent subunit (Figure 2). Two intersubunit ion pairs,
R32–E321 and R34–E308, also connect these domains
(Figure 2). Several electrostatic interactions are found be-
tween the C domains of adjacent subunits (Figure 2). These
include three ion pairs: E229 with R314, R301 with E292 and
R192 with E227.

Interactions between the IC and TM domains. There are three

main sites of interaction between the IC and TM domains.
The N domain is linked directly to the slide helix (Figures 1B
and 3). The C domain connects directly to TM2, but also
Figure 2 Interactions between adjacent subunits. The C domain of
one subunit is shown in grey and its N domain in yellow. The C
appears to be linked to the slide helix of the adjacent subunit
domain of the adjacent subunit is shown in green. Residues are by two loops that span residues 204–209 (loop 1) and 289–
shown in cpk colours. 299 (loop 2) (Figure 3A and B). A web of interactions link

Figure 3 (A, B) Relationship between the N domain of one subunit (yellow) and the C domain of another (green), showing the relation of
loops 1 and 2 to the slide helix. (C) Interactions between loops 1 and 2 and the C domain of one subunit and the slide helix of the adjacent
subunit. (D) Web of predicted interactions between the C linker, loops 1 and 2 of the C domain of one subunit (yellow) and the slide helix of the
adjacent subunit (blue). Coloured lines connect interacting residues, or indicate interactions with ATP.

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 231
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

loops 1 and 2 both to the slide helix and to the ATP-binding

pocket (Figure 3C and D).
As homology models of the TMs of Kirs have been con-
structed and evaluated previously (Loussouarn et al, 2001;
Capener et al, 2003), in this paper we focus on the IC domain,
and the ATP-binding site in particular. We also consider the
slide helix which links the TM and IC domains, as its position
suggests that it may be involved in gating of the pore, both in
the absence and presence of ATP.

The ATP-binding site

There are four ATP-binding pockets, one per subunit, as
predicted by functional studies (Markworth et al, 2000).
Each is located in the upper part of the IC domain, about
17 Å below the plane of the membrane. The position of this
pocket on the outer face of the protein (Figure 4A and B) may
facilitate access of cytosolic ATP to its binding site.
The main binding pocket lies at the interface between the
N and C domains of the same subunit. In addition, the N
domain of the adjacent subunit loops across the C-terminal
part of the binding pocket, such that the side chain of R50 lies
to one side of the entrance to the pocket, while K185 from the
other subunit lies at the opposite side (Figure 5A and B).
Docking of ATP was conducted in the absence of the
N-terminal residues, but it is clear that the location of R50
in the complete, tetrameric, homology model would restrict
access of ATP to its binding site. Thus, either the position of
R50 is incorrect, or the N-domain must move to enable ATP to
enter the binding pocket.
The overall size of the ATP-binding pocket is sufficient
to accommodate the ATP molecule (volume B1070 Å3). The
Ca–Ca distance from A300, which forms the innermost
residue of the hydrophobic pocket, to G334, which forms
the outer boundary, is about 17 Å. The width of the pocket,
measured from K39 to T180, is B10 Å and its height, mea-
sured between residues E179 and F183, is B8 Å.
In all, 17 residues lie within 4.5 Å of ATP in the model:
K38, K39, G40 in the N domain, E179, T180, L181, I182, F183,
S184, K185, R201, A300, R301, T302, F333 and G334 in the C
domain, and R50 from the N domain of the adjacent subunit.
The adenine ring and the ribose sugar of the nucleotide are
positioned on one side of a b-sheet and the phosphate tail on
the other, which effectively separates the charged and un-
Figure 4 (A) Side view of the ATP-binding site. For clarity, the TMs
charged groups of ATP. of only two subunits and the IC domains of two separate subunits
The adenine ring of ATP lies within a mainly hydrophobic are illustrated. ATP (yellow) is docked into its binding sites. (B)
pocket, lined by the side chains of E179, T180, L181, I182, Kir6.2 tetramer, viewed from above, with the TMs removed (resi-
dues 64–177). ATP (yellow) is docked into its binding sites. The N
and the backbone atoms of K38, K39, G40 and R301. The side
domain is shown in ribbon format and the C domain in backbone
chain of E179 and the carbonyl oxygen atom of the backbone format. Different colours represent individual subunits.
of R301 make hydrogen bonds with the N6 atom in the
adenine ring (Figure 5A). The adenine ring is accommodated Although the residue equivalent to R50 is absent in the crystal
tightly within the ATP-binding pocket (Figure 5B), and addi- structure of the IC domains of Kir3.1, on which this model is
tion of an azido group at the 80 position would cause a steric based, the adjacent residue (equivalent to E51) is not. Thus, the
clash with the ribose moiety of ATP and with the side chain of position of E51 is fixed, which also restrains the predicted
T180 (Figure 5C). This explains why 80 -azido-ATP fails to position of R50 and brings it close enough to interact with the
dock into the ATP-binding site of the model or to inhibit the g-phosphate of ATP, as predicted by ATP-binding (Tanabe et al,
channel (Tanabe et al, 1999). 1999) and thiol modification (Trapp et al, 2003) studies. The g-
No interactions are predicted with the ribose moiety of phosphate is not buried in the protein, as in many ATP-binding
ATP, but S184 lies under the ribose and K38 alongside it. sites, but points out into the extracellular solution (Figure 4).
The phosphate tail of ATP makes putative electrostatic
interactions with three positively charged residues: R201 Predictive power of the model
with the a-phosphate, K185 with the b-phosphate and R50 The model accounts for a large amount of published data
(from the adjacent subunit) with the g-phosphate (Figure 5A). and enables previously puzzling findings to be explained

232 The EMBO Journal VOL 24 | NO 2 | 2005 & 2005 European Molecular Biology Organization
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

Figure 5 (A) Interactions between ATP and residues lining the ATP-binding pocket. The subunit origin (A or D) of the residue is indicated.
Predicted hydrogen bonds are indicated by dashed lines and hydrophobic interactions by sunbursts. Residues are shown in cpk colours. (B)
Space-filling model of the ATP-binding pocket. Different subunits are indicated in blue and pink. The electron shells of R50 and K185, and of
ATP, are shown in transparency. (C) Close-up of the binding pocket with 8-azido-ATP placed in the same position as ATP. The dashed lines
indicate where the azido group makes a steric clash with T180 and the ATP molecule itself.

(see Discussion). This provides significant validation of the E179 to glutamine has no effect, whereas mutation to methio-
model, as none of this information was used in its construc- nine reduced the IC50 two-fold (Figure 6, Table I). However,
tion. In addition, we made new mutations to test the pre- mutation to leucine caused a small but insignificant shift in
dictive power of the model. IC50, which does not support an important role for H bond-
ing. None of these mutations altered the intrinsic Po (Table I).
ATP-binding site. Of the 17 residues that lie within 4.5 Å of Mutation of E179 to asparagine caused an B7-fold reduction
ATP in the model, most have already been mutated in in the IC50 for ATP inhibition, but also increased the intrinsic
Kir6.2DC or Kir6.2/SUR1. The effects of these mutations are Po (Figure 6, Table I). This change in Po could account for the
discussed below. However, residues A300 and F333 have not reduced ATP sensitivity (Shyng et al, 1997; Trapp et al, 1998;
been mutated in either construct, nor have L181, F183, R201, Cukras et al, 2002) and this mutation therefore cannot be
A300, R301, T302 and F333 been mutated in Kir6.2DC. To test used to address the role of H bonding. The relatively small
the model, we examined the effects of mutating (in Kir6.DC) effect of mutating E179 may explained by the fact that the N6
residue R201, which is predicted to make an electrostatic atom is also stabilized by H bonding to the backbone of R301
interaction with the a-phosphate of ATP, residues A300 and (Figure 5A), and further suggests that R301 may be more
F333, which lie close to the b-phosphate of ATP, and residue important in co-ordinating the adenine moiety. That E179
E179. In addition to measuring concentration-inhibition and ATP are slightly out of plane, and so likely to form only a
curves for ATP, we also examined the channel open prob- weak H bond, whereas R301 and ATP lie in the same plane, is
ability (Po) in the absence of ATP (which we define as the harmonious with this idea.
intrinsic Po) by single-channel recording. This is because The a-phosphate of ATP is predicted to form an electro-
mutations that increase intrinsic Po indirectly reduce KATP static interaction with the side chain of R201 (Figure 5A).
channel ATP sensitivity (Shyng et al, 1997; Trapp et al, 1998; Consistent with this prediction, mutation of R201 to cysteine
Cukras et al, 2002). Thus, we only considered mutations that results in permanent neonatal diabetes in humans (Gloyn
alter ATP sensitivity without affecting intrinsic Po as influen- et al, 2004), and causes a marked reduction in the ATP
cing ATP binding and/or transduction. sensitivity of Kir6.2DC (Figure 7): the IC50 could not be
The side chain of E179 is predicted to make a hydrogen measured accurately because even as much as 10 mM ATP
bond with the N6 atom in the adenine ring of ATP (Figure 5A). only inhibited the channel by 4375% (n ¼ 6), compared
To test this, we mutated E179 to a range of residues, some with 9671% (n ¼ 5) for the wild-type channel. The IC50
of which are capable of forming H bonds (Q, N), and some of was estimated to be 14 mM, 70-fold larger than the wild-
which are not (M, L). Consistent with this idea, mutation of type channel (Table I).

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 233
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

We next tested the effect of mutating F333, which is increased the IC50 for channel inhibition—from 17 to 514 mM
predicted to lie within 3 Å of the a-phosphate of ATP (Figure 8B). We also tested the effect of mutating Y330, which
(Figure 8A). In this experiment, we used full-length Kir6.2. lies at some distance from ATP in the model, to leucine. This
As full-length Kir6.2 does not express by itself, it was coex- had no effect (Figure 8B). The model predicts that A300 lies
pressed with SUR1: to avoid the stimulatory effects of Mg within 4.5 Å of the adenine ring of ATP (Figure 8A).
nucleotides on SUR1, we used a mutant SUR1 (SUR1-KA/ Unfortunately, mutation of A300 to either D or L failed to
KM) that is not modulated by adenine nucleotides (Gribble yield functional currents.
et al, 1997). This ensures that only ATP binding to Kir6.2 The model predicts that addition of an azido group at the 20
influences channel activity. The F333L mutation markedly position on the adenine ring will impair ATP binding, due to a
steric clash with the backbone of K38 and K39. Consistent
with this prediction, 2-azido-ATP blocked Kir6.2DC currents
by 674% at 100 mM and by 4772% at 1 mM (n ¼ 6).
Assuming a Hill coefficient of 1, the IC50 of was 1.2 mM,
B10-fold greater than for ATP.

Figure 6 (A) KATP currents elicited by voltage ramps from 110 to

þ 100 mV to an inside-out patch excised from a Xenopus oocyte
expressing Kir6.2DC or Kir6.2DC-E179M. The dotted line indicates
the zero current level. (B) Mean relationship between [ATP] and Figure 7 (A) Kir6.2DC and Kir6.2DC-R201H currents evoked by
KATP conductance (G), expressed relative to the conductance in the voltage ramps from 110 to þ 100mV. (B) Mean relationship
absence of nucleotide (Gc) for Kir6.2DC with E179 mutated to N ( , between [ATP] and KATP conductance (G), expressed relative to
n ¼ 5), M ( , n ¼ 5), L ( , n ¼ 5) and Q ( , n ¼ 7). The curves are the conductance in the absence of nucleotide (Gc) for Kir6.2DC-
the best fit to equation (1), using values for IC50 and h given in R201C (n ¼ 5). The curve is the best fit to equation (1) using the
Table I. The dashed line indicates the wild-type data. values given in Table I. The dotted line indicates the wild-type data.

Table I Mean parameters for ATP inhibition and open probability of wild-type and mutant channels
Mutation IC50(n) h Po

Kir6.2DC (wt) 194710 mM (n ¼ 5) 0.9670.04 0.0870.02 (n ¼ 6)

Kir6.2DC-V59G 1.770.1 mM*** (n ¼ 5) 1.170.1 0.7870.03*** (n ¼ 6)
Kir6.2DC-E179Q 240736 mM (n ¼ 7) 0.8870.5 0.1070.01 (n ¼ 7)a
Kir6.2DC-E179L 299747 mM (n ¼ 5) 0.897 0.06 0.1470.01 (n ¼ 5)
Kir6.2DC-E179M 515751 mM** (n ¼ 5) 1.0870.17 0.0970.02 (n ¼ 5)
Kir6.2DC-E179N 1.470.23 mM*** (n ¼ 6) 1.370.1 0.5870.03*** (n ¼ 8)
Kir6.2DC-R201C 147l mMb*** (n ¼ 6) 0.8170.05b 0.1270.02 (n ¼ 6)
IC50 ¼ ATP concentration at which inhibition is half-maximal. h, Hill coefficient. Values indicate the means of the individual fits to equation (1),
for n patches. aFrom Tucker et al (1998). bEstimated by fitting line through data points that extend to 10 mM. Statistical significance was
determined using Student’s t-test. **Po0.01; ***Po0.001.

234 The EMBO Journal VOL 24 | NO 2 | 2005 & 2005 European Molecular Biology Organization
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

caused a marked increase in the intrinsic Po, which was

accompanied by a 10-fold reduction in ATP-sensitivity
(Table I).

Discussion
Subunit interactions in the IC domains
Evidence for a physical interaction between the N and C
domains of Kir6.2 has been obtained from protein–protein
interaction studies (Tucker and Ashcroft, 1999; Jones et al,
2001), which showed that residues 30–46 in the N domain
interact with residues in three separate regions of the C
domain of Kir6.2. The same N-terminal region contributes
to the interaction of the N and C domains of our model (R32,
R34 and F35). Furthermore, the p-stacking interaction pre-
dicted between F35 in the N domain of one subunit and Y326
in the C domain of the adjacent subunit is also observed in
Kir3.1 (Nishida and MacKinnon, 2002). Indeed, hydrophobic
residues at both these positions are conserved across Kir6.x,
2.x and 3.x channels. Naturally occurring mutations at resi-
dues F35 (to V or A) and E322 (to K, which would be
expected to disrupt the adjacent E321-R32 ion pair) cause
permanent neonatal diabetes mellitus (PNDM) in humans
(Sagen et al, 2004; Vaxillaire et al, 2004). This supports the
idea that interactions between the N and C domains of
adjacent subunits are important for KATP channel function.
A number of electrostatic interactions occur between ad-
jacent C domains. Mutagenesis studies suggest that residues
E229 and R314 form an ion pair, since a single charge
neutralization at either position induces channel ‘inacti-
vation’ after patch excision, which can be rescued by a
double charge reversal (E229R/R314E) (Lin et al, 2003).
Furthermore, if E229 and R314 are both mutated to cysteine,
Figure 8 (A) Model of the ATP-binding pocket of Kir6.2 illustrating they can be reversibly crosslinked in the presence of an
the proximity of F333 and Y330 to the phosphate tail of ATP. (B)
Mean relationship between [ATP] and KATP conductance (G), ex-
oxidizing agent, indicating that the residues must lie o4 Å
pressed relative to the conductance in the absence of nucleotide apart. Tandem dimer constructs showed that this ion pair is
(Gc) for Kir6.2-Y330L/SUR1-KA/KM (K, n ¼ 5), and Kir6.2-F333L/ likely to be an intersubunit interaction.
SUR1-KAKM (&, n ¼ 6) channels. The smooth curves are the best Two residues previously suggested as candidates for ion
fit to equation (1). For Kir6.2-Y330L, IC50 ¼16.971.9 mM,
h ¼ 1.370.1. For Kir6.2-F333L, IC50 ¼ 514793 mM, h ¼ 1.270.1.
pair formation (R192, R301), on the basis of the fact that their
The dashed line indicates the control data (IC50 ¼17 mM, h ¼ 0.99; mutation to alanine enhances channel inactivation (Lin et al,
Gribble et al, 1997). 2003), are also predicted to form ion pairs in our model:
residue 192 with E227, and R301 with E292, in adjacent
subunits. Both ion pairs lie in close proximity and are highly
conserved. A glutamate is found at the position equivalent to
The ribose moiety of ATP does not make a direct interac-
E227, an arginine at the equivalent position to R301 and a
tion with the channel (Figure 5A). However, it lies within 4 Å
negatively charged residue at the E292 position, in all Kir
of K185, which may explain why substitution of ribose with
channels. R192 is also highly conserved. This conservation of
20 -deoxyribose or ribose 20 ,30 -dialdehyde reduces the ATP
sequence identity suggests an important role. Further, the
sensitivity of native skeletal muscle KATP channels (Spruce
cluster of acidic and basic residues in this region suggests that
et al, 1987). At a concentration of 1 mM, 20 -deoxyribose ATP
different combinations of electrostatic interactions may occur
inhibited Kir6.2DC by 6172% (n ¼ 11) as compared with
during channel gating.
8273% for ATP (n ¼ 16).

Slide helix mutations. The position of the slide helix suggests Slide helix
that it may be involved in channel gating (Kuo et al, 2003). Our results provide evidence that the slide helix plays a role
With the exception of R54 at the N-terminal end (Cukras et al, in gating, since the V59G stabilizes the open state. In con-
2002; Schulze et al, 2003), no mutations in the Kir6.2 slide trast, mutation of R54 reduces the intrinsic Po of Kir6.2/SUR
helix have yet been studied: thus, we tested this idea by channels; however, this may be secondary to changes in PIP2
mutagenesis of Kir6.2DC. We chose to mutate V59 to glycine or acyl CoA modulation (Cukras et al, 2002; Schulze et al,
(V59G), because this mutation produces neonatal diabetes 2003). Its amphipathic nature suggests that the slide helix
with developmental delay, muscle weakness and epilepsy slides along the membrane interface during channel opening,
(Gloyn et al, 2004). Figure 9 shows that the V59G mutation without substantial rotational movement.

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 235
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

Figure 9 (A) Slide helix of Kir6.2, showing the location of V59. The slide helix of one subunit is shown in yellow and the TM domain of the
adjacent subunit in green. (B) Kir6.2DC and Kir6.2DC-V59G currents evoked by voltage ramps from 110 to þ 100 mV in inside-out patches.
(C) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for
Kir6.2DC-V59G (n ¼ 6). The curve is the best fit to equation (1) using the values given in Table I. The dashed line indicates the wild-type data.
(D) Single Kir6.2DC and Kir6.2DC-V59G channel currents recorded at –60 mV. The arrow indicates the zero current level.

ATP-binding pocket et al, 2000; John et al, 2003; Ribalet et al, 2003). These
The ATP-binding site suggested by the model involves con- studies reveal that mutation of R201 to a negatively charged,
tributions from the N and C domains of adjacent subunits. or uncharged, residue weakens ATP inhibition, and in the
This is supported by a substantial amount of data. First, case of Kir6.2-R201E/SUR1 the current could not be comple-
mutations in both these regions impair channel inhibition by tely blocked even at millimolar ATP levels. Furthermore,
ATP (Drain et al, 1998; Proks et al, 1999; Tucker et al, 1998; inhibition by ATP, ADP and AMP are all affected, consistent
Reimann et al, 1999; Cukras et al, 2002; John et al, 2003; with the idea that R201 interacts with the a-phosphate of ATP.
Ribalet et al, 2003). Further, when mutations in the N and C Mutation of R201 to histidine also reduces the ATP sensitivity
domains that affect ATP sensitivity are combined, the ATP of the Kir6.2/SUR1 channel, and causes PNDM (Gloyn et al,
sensitivity is further reduced, as expected if the N and C 2004). It is interesting that the shift in ATP sensitivity
domains cooperate to influence channel closure by ATP produced by the R201C mutation is greater in the absence
(Proks et al, 1999). Second, protein–protein interaction stu- of SUR1: the IC50 increased B120-fold for Kir6.2DC (Table I)
dies demonstrate a physical interaction between the N and C and 15-fold for Kir6.2/SUR1 (John et al, 2003). This suggests
domains of Kir6.2 (see above). Importantly, the model pre- that SUR1 may modify the binding pocket at the level of R201,
dicts that each N domain contributes to two ATP-binding consistent with previous work (Dabrowski et al, 2004).
sites: that of the same and the adjacent subunit (Figure 4). There is considerable experimental evidence to support the
This is harmonious with FRET experiments which show that interaction of the b-phosphate of ATP with K185 predicted by
the physical relationship of the N and C domains of adjacent the model (Figure 5A). First, mutation of K185 to a negative
subunits is influenced by ATP binding (Tsuboi et al, 2004). charge strongly impairs ATP inhibition, whereas mutation to
SUR enhances the ATP sensitivity of Kir6.2 about 10-fold arginine has only a small effect, in both Kir6.2DC and Kir6.2/
(Tucker et al, 1997). The exposed position of the ATP-binding SUR1 (Tucker et al, 1997, 1998; Reimann et al, 1999; John
site in Kir6.2 suggests that one way this might be achieved et al, 2003). Similar results are found when K185C is mod-
would be if SUR embraced the outer part of the binding site, ified by charged thiol regents (Trapp et al, 2003). This
perhaps by contributing additional residues that stabilize ATP supports an electrostatic interaction between K185 and ATP.
binding, or by allosterically altering the structure of the Furthermore, these modifications impair ATP and ADP in-
binding site. hibition, but not that by AMP, consistent with an interaction
Our studies demonstrate that mutation of R201 causes a with the b-phosphate of ATP (John et al, 2003; Trapp et al,
marked reduction in the ATP sensitivity of Kir6.2DC. Similar 2003). In addition, binding of 8-g-32P-azido ATP to Kir6.2
results have been found for Kir6.2/SUR1 channels (Shyng is impaired by the K186Q mutation (Tanabe et al, 1999).

236 The EMBO Journal VOL 24 | NO 2 | 2005 & 2005 European Molecular Biology Organization
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

Mutation of residues S184 and H186, which flank K185, capable of hydrogen bonding, is able to block the channel
reduces the ATP sensitivity of Kir6.2DC (Tucker et al, 1998); (Dabrowski et al, 2004).
mutation of S184 also impairs the ATP sensitivity of Kir6.2/ Residues T180, L181 and I182 provide a hydrophobic
SUR1, and introduction of charged thiol reagents at either lining to the binding pocket. Extensive mutagenesis has
position in Kir6.2/SUR1 shifts the channel ATP sensitivity as been carried out on I182 in Kir6.2DC (Li et al, 2000).
expected if the residue lies close to the phosphate tail of ATP Substitution with a variety of amino acids markedly reduces
(Trapp et al, 2003). channel inhibition by ATP, the IC50’s increasing from
The g-phosphate is not buried in the protein, as in many B200 mM (I) to B5 mM (E, M, Y, P) and 410 mM (K, R, S,
ATP-binding sites, but points out into the external solution W), without altering intrinsic gating. I182 is accessible to
(Figure 4). This may explain why Mg2 þ is not required for cysteine-modifying agents, but modification with a negatively
ATP binding to Kir6.2 (Tanabe et al, 1999, 2000), and why charged thiol reagent was without effect on the ATP sensitiv-
large groups can be added to the phosphate tail without ity of I182C, indicating that this residue does not lie close to
substantially reducing ATP block. For example, AP4A and the phosphate tail of the nucleotide (Trapp et al, 2003). This
ATP block both Kir6.2DC (Dabrowski et al, 2004) and native is consistent with the predicted location of I182 adjacent to
KATP channels (Jovanovic et al, 1997) with similar affinity, the adenine ring. The threonine at position 180 is interesting
as does the photo-activatable ATP analogue DMNPE-ATP as mutations at this position affect the intrinsic Po of the
(Ämmälä et al, 1991). Likewise, azido-anilino ATP blocks channel (Light et al, 2000), as observed for the asparagine
Kir6.2DC channels with an IC50 comparable to that for ATP mutation at E179 (Table I). Kir6.2/SUR1 containing either
(Tanabe et al, 2000). All these data are consistent with the L181C or F183C mutations did not express functional chan-
open-ended structure of the ATP-binding site predicted by the nels (Trapp et al, 2003).
model. It is worth noting that this is in contrast to most ATP- The model predicts that the binding pocket is lined by
binding sites, which are closed. backbone atoms of K38, K39 and G40 in the N domain
Our model predicts that R50 lies close to the g-phosphate (Figure 5A and B). Consistent with this structure, mutation
of ATP. This is in good agreement with the fact that the ATP of K39 to alanine in Kir6.2/SUR1 reduced ATP sensitivity
sensitivity of Kir6.2/SUR1 channels is reduced by substitu- indirectly, by altering the open state stability rather than
tion of a cysteine at R50, and that this effect is reversed by impairing ATP binding (Cukras et al, 2002).
modification with a positively charged thiol reagent but is
further decreased by modification with a negatively charged How does ATP binding lead to closure of the pore?
thiol agent (Trapp et al, 2003). This suggests that ATP is able ATP binding occurs at the interface between two subunits,
to sense the charge at residue 50 electrostatically, and must as is common in ligand-gated channels (Brejc et al, 2001).
therefore lie in close proximity. As ADP block was unaffected Moreover, subunit interactions indicate that each monomer is
by thiol modification of R50, it appears that the g-phosphate connected to not one, but two other subunits (Figure 4). This
of ATP is involved. However, mutation of R50 in Kir6.2DC or suggests that ATP binding to one subunit will influence the
Kir6.2/SUR1 (Proks et al, 1999; John et al, 2003) is not relative conformations of the two adjacent subunits, and may
consistent with the interaction of R50 and ATP being entirely help to explain why binding of a single ATP molecule is
electrostatic: for example, the IC50 for ATP block of Kir6.2DC sufficient to close the pore. As the binding lies at the interface
is greater for R50G, than for R50E (although there is no between subunits, it also seems possible that the overall
change in intrinsic Po). Thus, mutations at R50 may simply conformation of each monomer will not undergo a dramatic
perturb the protein structure, and thereby access of ATP to its change upon ATP binding: rather, nucleotide binding may
binding site. primarily influence the relationship between the monomers.
G334, which is located far from K185 in the primary
sequence, lies close to both the b-phosphate of ATP (3.3 Å) Conclusions
and to K185 in the three-dimensional model structure In conclusion, by constructing a molecular model of the
(Figure 8). This rationalizes the previously puzzling finding Kir6.2 tetramer, we have identified the putative location of
that mutation of G334 to aspartate markedly decreases ATP the ATP-binding site. Residues that form the ATP-binding site
sensitivity (Drain et al, 1998). As our model shows, this are not well conserved across Kir channel subfamilies, in
mutation would be expected to impede nucleotide binding contrast to those implicated in intra-subunit interactions.
both sterically and electrostatically. Residues F333 and Y330 This suggests that, while the ATP-binding site is unique, Kir
also lie close to the phosphate tail of the ATP molecule. That channels may share a common structural basis for subunit
PNDM is caused by mutations at either of these residues assembly and that the mechanism by which ligand binding is
(F333I, Y330C) is indicative of their importance for channel linked to opening and closing of the pore may also be similar.
function (Sagen et al, 2004; Vaxillaire et al, 2004). The model is in good agreement with a large body of
The model prediction that the N6 atom in the adenine ring mutagenesis data, offers an explanation for the relatively low
of ATP hydrogen bonds with residues lining the binding affinity of Kir6.2 for other nucleotide triphosphates, and
pocket is harmonious with the known efficacy of trinucleo- suggests that SUR1 might enhance ATP sensitivity by embra-
tide phosphates at inhibiting KATP currents: GTP, ITP, UTP cing the outer part of Kir6.2. Our results leads us to suggest
and purine triphosphate (PTP) are all ineffective (Tucker et al, that gating of Kir6.2 by ATP probably involves changes in
1998; Dabrowski et al, 2004). A common feature of these inter-subunit interactions and, further, to speculate that flex-
other nucleotide triphosphates is that, unlike ATP, they lack ible loops in the C-domain (204–209 and 289–299) may help
an NH2 group at the 60 position of the adenine ring and are translate conformational changes induced by ATP binding
therefore unable to form a hydrogen bond. In support of this into movement of the slide helix and thereby closing of the
idea, methyl ATP, which has an NCH3 group and is therefore channel. The model also makes specific suggestions for

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 237
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

subunit–subunit interactions and ligand–protein interactions hydrogen atoms were generated and partial charges assigned using
InsightII (www.accelrys.com). A grid box of 100 120 100 points
that provide a solid basis for further mutagenesis and simula-
was created with a grid spacing of 0.375 Å around the protein to
tion studies. define the interaction space between the ligand and the protein.
In all, 11 rotatable bonds in the ligand were allowed to be flexible.
A total of 25 dockings were carried out with 5000 cycles per run.
Materials and methods Interaction energies were calculated for favourable ligand–protein-
binding sites. Dockings were ranked according to the interaction
Molecular modelling energies between the protein and the ligand. The final conformation
All sequence alignments were carried out using ClustalX. Compara- of the docked ATP was based on a combination of interaction
tive modelling was used to construct the molecular models using energies, functional data obtained from physiological studies and
MODELLER v6.2 (Sali and Blundell, 1993; Fiser et al, 2000). Several chemical intuition. Once residues contributing to the binding site
models were constructed, with the final one being selected on the were identified, ATP was superimposed in the same spatial
basis of low energy function and Ca r.m.s.d. values after super- orientation in the remaining subunits. The quality and the
imposition on the template. The models were constructed in three stereochemical properties of the model were assessed using
segments. PROCHECK v3.4.4. (Morris et al, 1992).
The percent identity between the IC domains of Kir3.1 and Kir6.2
is 48%, and that between the KirBac1.1 and Kir6.2 is 32% for the Functional studies
TMs and slide helices, and 27% for the IC domains. The slide helix Mouse Kir6.2 (Genbank D50581) and rat SUR1 (Genbank L40624)
and TMs of Kir6.2 (residues 55–172) were modelled as a tetramer, were used in this study. Site-directed mutagenesis of Kir6.2 and
using the crystal structure of KirBac1.1 as a template (Kuo et al, preparation of mRNAs was as described (Trapp et al, 1998).
2003). Residues that connect the TMs with the C domain (residues Xenopus laevis oocytes were injected with 0.1 ng full-length wild-
173–176) were modelled as loops. The consensus secondary type or mutant Kir6.2 and B2 ng of SUR1 mRNAs (giving a 1:20
structure predictions for these loops were obtained from the ratio), and studied 1–4 days later (Trapp et al, 1998).
programs PHD (www.cubic.bioc.columbia.edu) and JPRED Macroscopic currents were recorded from giant inside-out
(www.compbio.dundee.ac.uk). The C domain of Kir6.2 (residues patches at a holding potential of 0 mV and 20–241C. Currents were
177–358) was modelled as a tetramer, based on the 2.0 Å crystal evoked by 3 s voltage ramps from 110 to þ 100 mV, recorded with
structure of the C domain of Kir3.1 (Trapp et al, 2003). The an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster
orientation of the C domain with respect to the TMs was obtained City, CA, USA), filtered at 0.15 kHz, digitized at 0.5 kHz using a
by superimposing the conserved stretches of residues in Kir6.2 Digidata 1200 Interface and analysed using pClamp software (Axon
on KirBac1.1. Once the orientation of the segments had been Instruments). The pipette solution contained (mM): 140 KCl, 1.2
established, they were joined using the modelled loops. The loops MgCl2, 2.6 CaCl2 and 10 HEPES (pH 7.4 with KOH). The internal
were then subjected to energy minimization, while keeping the solution contained (mM): 107 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA (free
position of the atoms of the core model frozen. Mg2 þ B600 nM), 10 HEPES (pH 7.2 with KOH) and nucleotides as
The N domain of Kir6.2 (residues 32–46) was modelled based on indicated. Rapid exchange of internal solutions was achieved using
the N-terminal (residues 43–57) structure of Kir3.1 (Nishida and a sewer-pipe system. Single-channel currents were recorded from
MacKinnon, 2002). The remaining residues that contribute to the small inside-out membrane patches at 60 mV, filtered at 5 kHz and
N domain of Kir3.1 are not resolved in the crystal structure, except sampled at 20 kHz. Open probability was determined from records
for E63. This residue is conserved in both Kir3.1 and Kir6.2 (E51). of B1 min duration as I/iN, where I is the macroscopic current,
The remainder of the N-domain residues in Kir6.2 (47–54) were i is the single-channel current amplitude and N is the number of
modelled using KirBac1.1 as a template. As this region is a loop in channels (maximum number of open levels).
KirBac1.1, we also confirmed that residues 47–50 and 52–54 are The macroscopic slope conductance was obtained by fitting a
loops, based on the predictions of the programs above. The position straight line to the I–V relation between 20 and 100 mV (Trapp
of E63 in the crystal structure of Kir3.1 was taken as a reference to et al, 1998). To control for possible rundown, the control
obtain the spatial position and orientation for the N-terminal loops. conductance (Gc) was taken as the mean of that in control solution
The equivalent residue in Kir6.2 (E51) was fixed and the loops on before and after ATP application. ATP concentration–response
either side were subjected to restrained energy minimization curves were fitted by the Hill equation:
procedures using GROMACS v3.1.4 (www.gromacs.org). The N
domain was modelled as a monomer, and docked onto the G=Gc ¼ 1=ð1 þ ð½ATP=IC50 Þh Þ ð1Þ
tetrameric model of the C domain, using the fixed points described where [ATP] is the ATP concentration, IC50 is the concentration at
above. which inhibition is half-maximal and h is the Hill coefficient. All
The Ca–Ca r.m.s.d. between the templates and the model was data are given as mean7s.e.m. Statistical significance was tested by
0.4 Å for the N domain, 1.7 Å for the slide helix and the TMs, and Student’s t-test or ANOVA, as appropriate.
1.3 Å for the C domain.
Kir6.2 is a homotetramer with four potential ATP-binding sites
(one per subunit) that do not interact (Markworth et al, 2000): for Acknowledgements
simplicity, only a monomer was used to dock ATP to the C domain
(Trapp et al, 2003). Docking was carried out using AUTODOCK 3.0.5 We thank the Wellcome Trust and the Royal Society for support.
(Goodsell et al, 1996), which allows for ligand flexibility. Monte JA is a Scholar of Christ Church College, Oxford. FMA is the Royal
Carlo simulated annealing was used to perform the dockings. The Society GlaxoSmithKline Research Professor.

References
Ämmälä C, Bokvist K, Galt S, Rorsman P (1991) Inhibition of ATP- Clement JP, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U,
regulated K+-channels by a photoactivatable ATP-analogue in Aguilar Bryan L, Bryan J (1997) Association and stoichiometry of
mouse pancreatic beta-cells. Biochim Biophys Acta 1092: 347–349 KATP channel subunits. Neuron 18: 827–838
Ashcroft FM, Kakei M (1989) ATP-sensitive K-channels: modulation Cukras CA, Jeliazkova I, Nichols CG (2002) The role of NH2-
by ATP and Mg2+ ions. J Physiol 416: 349–367 terminal positive charges in the activity of inward rectifier KATP
Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, channels. J Gen Physiol 120: 437–446
Smit AB, Sixma TK (2001) Crystal structure of an ACh-binding Dabrowski M, Tarasov A, Ashcroft FM (2004) Mapping the archi-
protein reveals the ligand-binding domain of nicotinic receptors. tecture of the ATP-binding site of the KATP channel subunit Kir6.2.
Nature 411: 269–276 J Physiol 557: 347–354
Capener CE, Proks P, Ashcroft FM, Sansom MS (2003) Filter Drain P, Li L, Wang J (1998) KATP channel inhibition by ATP requires
flexibility in a mammalian K channel: models and simulations distinct functional domains of the cytoplasmic C terminus of the
of Kir6.2 mutants. Biophys J 84: 2345–2356 pore-forming subunit. Proc Natl Acad Sci USA 95: 13953–13958

238 The EMBO Journal VOL 24 | NO 2 | 2005 & 2005 European Molecular Biology Organization
 14602075, 2005, 2, Downloaded from https://www.embopress.org/doi/10.1038/sj.emboj.7600487 by INASP/HINARI - PAKISTAN, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Interaction of ATP with Kir6.2
JF Antcliff et al

Dunne MJ, Cosgrove KE, Shepherd RM, Aynsley-Green A, Lindley Reimann F, Ryder TJ, Tucker SJ, Ashcroft FM (1999) An investiga-
KJ (2004) Hyperinsulinism in infancy: from basic science to tion of the role of lysine 185 in Kir6.2 in inhibition of the KATP
clinical disease. Physiol Rev 84: 239–275 channel by ATP. J Physiol 520: 661–669
Fiser A, Do R, Sali A (2000) Modeling of loops in protein structures. Ribalet B, John SA, Weiss JN (2003) Molecular basis for Kir6.2
Prot Sci 9: 1753–1773 channel inhibition by adenine nucleotides. Biophys J 84: 266–276
Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland Russel RB, Saqi MA, Sayle RA, Bates PA, Sternberg MJ (1997)
AS, Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Recognition of analogous and homologous protein folds: analysis
Frayling TM, Temple IK, Mackay D, Shield JP, Sumnik Z, van of sequence and structure conservation. J Mol Biol 269: 423–439
Rhijn A, Wales JK, Clark P, Gorman S, Aisenberg J, Ellard S, Sagen JV, Raeder H, Hathout E, Shehadeh N, Gudmundsson K,
Njolstad PR, Ashcroft FM, Hattersley AT (2004) Activating muta- Baevre H, Abuelo D, Phornphutkul C, Molnes J, Bell GI, Gloyn
tions in the ATP-sensitive potassium channel subunit Kir6.2 gene AL, Hattersley AT, Molven A, Sovik O, Njolstad PR (2004)
are associated with permanent neonatal diabetes. New Engl J Med Permanent neonatal diabetes due to mutations in KCNJ11 encod-
350: 1838–1849 ing Kir6.2: patient characteristics and initial response to sulfony-
Goodsell DS, Morris GM, Olson AJ (1996) Automated docking of lurea therapy. Diabetes 53: 2713–2718
flexible ligands: applications of AutoDock. J Mol Recog 9: 1–5 Sali A, Blundell TL (1993) Comparative protein modelling by
Gribble FM, Tucker SJ, Ashcroft FM (1997) The essential role of the satisfaction of spatial restraints. J Mol Biol 234: 779–815
Walker A motifs of SUR1 in K-ATP channel activation by MgADP Schulze D, Krauter T, Fritzenschaft H, Soom M, Baukrowitz T
and diazoxide. EMBO J 16: 1145–1152 (2003) Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation
John SA, Weiss JN, Xie LH, Ribalet B (2003) Molecular mechanism of ATP and pH sensitivity in Kir channels. A tale of an active and a
for ATP-dependent closure of the K+ channel Kir6.2. J Physiol silent PIP2 site in the N terminus. J Biol Chem 278: 10500–10505
552: 23–34 Seino S, Miki T (2003) Physiological and pathophysiological roles of
Jones P, Tucker S, Ashcroft FM (2001) Multiple sites of interaction ATP-sensitive K+ channels. Prog Biophys Mol Biol 81: 133–176
between the intracellular domains of an inwardly-rectifying po- Shyng S, Ferrigni T, Nichols CG. (1997) Control of rectification and
tassium channel, Kir6.2. FEBS Lett 508: 85–89 gating of cloned KATP channels by the Kir6.2 subunit. J Gen
Jovanovic A, Alexseev AE, Terzic A (1997) Intracellular diadenosine Physiol 110: 141–153
phosphates. A novel family of inhibitory ligands of the ATP- Shyng SL, Cukras CA, Harwood J, Nichols CG (2000) Structural
sensitive K+ channel. Biochem Pharm 54: 219–225 determinants of PIP2 regulation of inward rectifier KATP channels.
Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, J Gen Physiol 116: 599–608
Cuthbertson J, Ashcroft FM, Ezaki T, Doyle DA (2003) Crystal Spruce AE, Standen NB, Stanfield PR (1987) Studies of the unitary
structure of the potassium channel KirBac1.1 in the closed state. properties of adenosine-50 -triphosphate-regulated potassium
Science 300: 1922–1926 channels of frog skeletal muscle. J Physiol 382: 213–236
Li L, Wang J, Drain P (2000) The I182 region of K(ir)6.2 is closely Tanabe K, Tucker SJ, Ashcroft FM, Proks P, Kioka N, Amachi T,
associated with ligand binding in KATP channel inhibition by ATP. Ueda K (2000) Direct photoaffinity labeling of Kir6.2 by
Biophys J 79: 841–852 [g-32P]ATP-[g]4-azidoanilide. Biochim Biophys Res Commun
Light PE, Bladen C, Winkfein RJ, Walsh MP, French RJ (2000) 272: 316–319
Molecular basis of protein kinase C-induced activation of ATP- Tanabe K, Tucker SJ, Matsuo M, Proks P, Ashcroft FM, Seino S,
sensitive potassium channels. Proc Natl Acad Sci USA 97: Amachi T, Ueda K (1999) Direct photoaffinity labeling of the
9058–9063 Kir6.2 subunit of the ATP-sensitive K+ channel by 8-Azido-ATP.
Lin YW, Jia T, Weinsoft AM, Shyng SL (2003) Stabilization of J Biol Chem 274: 3931–3933
the activity of ATP-sensitive potassium channels by ion Trapp S, Haider S, Jones P, Sansom MSP, Ashcroft FM (2003)
pairs formed between adjacent Kir6.2 subunits. J Gen Physiol Identification of residues contributing to the ATP-binding site of
122: 225–237 Kir6.2. EMBO J 22: 2903–2912
Loussouarn G, Phillips LR, Masia R, Rose T, Nichols CG (2001) Trapp S, Proks P, Tucker SJ, Ashcroft FM (1998) Molecular analysis
Flexibility of the Kir6.2 inward rectifier K+ channel pore. Proc of KATP channel gating and implications for channel inhibition by
Natl Acad Sci USA 98: 4227–4232 ATP. J Gen Physiol 112: 333–349
Markworth E, Schwanstecher C, Schwanstecher M (2000) ATP4 Tsuboi T, Lippiat JD, Ashcroft FM, Rutter GA (2004) ATP-dependent
mediates closure of pancreatic beta-cell ATP-sensitive potassium interaction of the cytosolic domains of the inwardly rectifying K+
channels by interaction with 1 of 4 identical sites. Diabetes 49: channel Kir6.2 revealed by fluorescence resonance energy trans-
1413–1418 fer. Proc Natl Acad Sci USA 101: 76–81
Morris AL, MacArthur MW, Hutchinson EG, Thornton JM (1992) Tucker SJ, Ashcroft FM (1999) Mapping of the physical interaction
Stereochemical quality of protein structure coordinates. Proteins between the intracellular domains of an inwardly rectifying
12: 345–364 potassium channel, Kir6.2. J Biol Chem 274: 33393–33397
Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement IV JP, Tucker SJ, Gribble FM, Proks P, Trapp S, Ryder TJ, Haug T, Reimann
Gonzalez G, Aguilar-Bryan L, Permutt MA, Bryan J (1996) F, Ashcroft FM (1998) Molecular determinants of KATP channel
Adenosine diphosphate as an intracellular regulator of insulin inhibition by ATP. EMBO J 17: 3290–3296
secretion. Science 272: 1785–1787 Tucker SJ, Gribble FM, Zhao C, Trapp S, Ashcroft FM (1997)
Nishida M, MacKinnon R (2002) Structural basis of inward rectifi- Truncation of Kir6.2 produces ATP-sensitive K-channels in the
cation: cytoplasmic pore of the G protein-gated inward rectifier absence of the sulphonylurea receptor. Nature 387: 179–183
GIRK1 at 1.8 Å resolution. Cell 111: 957–965 Vaxillaire M, Populaire C, Busiah K, Cave H, Gloyn AL, Hattersley
Proks P, Gribble FM, Adhikari R, Tucker SJ, Ashcroft FM (1999) AT, Czernichow P, Froguel P, Polak M (2004) Kir6.2 mutations are
Involvement of the N-terminus of Kir6.2 in the inhibition of the a common cause of permanent neonatal diabetes in a large cohort
KATP channel by ATP. J Physiol 514: 19–25 of French patients. Diabetes 53: 2719–2722

& 2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 2 | 2005 239