Identification of a Functionally Important Negatively
Charged Residue Within the Second Catalytic Site of the
SUR1 Nucleotide-Binding Domains
Jeff D. Campbell,1,2 Peter Proks,1 Jonathan D. Lippiat,1 Mark S.P. Sansom,2 and Frances M. Ashcroft1
The ATP-sensitive Kⴙ channel (KATP channel) couples
glucose metabolism to insulin secretion in pancreatic
␤-cells. It is comprised of sulfonylurea receptor (SUR)-1
and Kir6.2 proteins. Binding of Mg nucleotides to the
nucleotide-binding domains (NBDs) of SUR1 stimulates
channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. To elucidate the
structural basis of this regulation, we constructed a
molecular model of the NBDs of SUR1, based on the
crystal structures of mammalian proteins that belong to
the same family of ATP-binding cassette transporter
proteins. This model is a dimer in which there are two
nucleotide-binding sites, each of which contains residues from NBD1 as well as from NBD2. It makes the
novel prediction that residue D860 in NBD1 helps coordinate Mg nucleotides at site 2. We tested this prediction experimentally and found that, unlike wild-type
channels, channels containing the SUR1-D860A mutation were not activated by MgADP in either the presence or absence of MgATP. Our model should be useful
for designing experiments aimed at elucidating the
relationship between the structure and function of the
KATP channel. Diabetes 53 (Suppl. 3):S123–S127, 2004
he ATP-sensitive K⫹ channel (KATP channel)
plays a crucial role in insulin secretion by coupling ␤-cell metabolism to insulin secretion (1).
At low glucose levels, the KATP channel is open,
keeping the resting membrane potential hyperpolarized
and preventing electrical activity and insulin secretion (2).
Increased glucose metabolism results in closure of the
KATP channels, leading to a depolarization of the ␤-cell
membrane, initiation of electrical activity, opening of
voltage-gated calcium channels, and an influx of Ca2⫹ ions
that triggers the release of insulin granules.
T
From the 1University Laboratory of Physiology, University of Oxford, Oxford,
U.K.; and 2Laboratory of Molecular Biophysics, University of Oxford, Oxford,
U.K.
Address correspondence and reprint requests to Prof. Frances M. Ashcroft,
University Laboratory of Physiology, University of Oxford, Parks Rd., Oxford,
OX1 3PT, U.K. E-mail: [email protected].
Received for publication 12 March 2004 and accepted in revised form
25 May 2004.
This article is based on a presentation at a symposium. The symposium and
the publication of this article were made possible by an unrestricted educational grant from Servier.
ABC, ATP-binding cassette; CFTR, cystic fibrosis transmembrane regulator;
KATP channel, ATP-sensitive K⫹ channel; Kir channel, inwardly rectifying K⫹
channel; NBD, nucleotide-binding domain; SUR, sulfonylurea receptor; TAP,
transporter associated with antigen processing; TMD, transmembrane domain; WA, Walker-A; WB, Walker-B.
© 2004 by the American Diabetes Association.
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
Two very different protein subunits make up the KATP
channel (3). The pore is composed of four inwardly rectifying K⫹ channel (Kir channel) subunits (Kir6.2), and each
of these is associated with a sulfonylurea receptor (SUR)
subunit (SUR1), which modulates the opening and closing
(gating) of the channel. Metabolically generated changes
in intracellular adenine nucleotides influence channel gating via interaction with both types of subunit. Thus, ATP
(and ADP) closes the channel by binding to Kir6.2, whereas channel opening is mediated by interaction of Mg nucleotides (MgATP and MgADP) with SUR1 (4,5).
SUR belongs to the ATP-binding cassette (ABC) family
of transporter proteins (6). Like other ABC transporters, it
has two sets of transmembrane domains (TMDs), each
containing six membrane spanning helices, and two nucleotide binding domains (NBDs). It also possesses an additional NH2-terminal set of five transmembrane helices (Fig.
1A). The NBDs of all ABC proteins are highly conserved
and contain several motifs involved in ATP binding and
hydrolysis, including a Walker-A (WA) and Walker-B (WB)
motif linked by a “signature” sequence (LLSGGQ) and two
shorter sequences containing conserved glutamine (Glnloop) and histidine (His-loop) residues (Fig. 1A). The
crystal structures of a number of isolated NBDs suggest
that their topology is also highly conserved across both
prokaryotes and eukaryotes. Furthermore, the X-ray structures show that the NBDs form a dimer containing two
nucleotide-binding pockets. These do not correspond to
the NBDs encoded in the primary sequence, however,
because the ATP molecules are sandwiched between the
WA of one NBD and the signature sequence of the opposite
NBD (Fig. 1B). For this reason, we henceforth refer to the
two nucleotide-binding pockets as site 1 (Walker motifs of
NBD1, linker NBD2) and site 2 (Walker motifs of NBD2,
linker NBD1).
Because the sequences of NBD1 and NBD2 of SUR1 are
not identical, the structure of these two sites will also not
be identical. This may provide a structural explanation for
the observed differences in function between the two sites
of SUR1 (8). As described previously (7), it is possible to
equate the properties described for NBD1 in functional
studies with those of site 1 and to equate those described
for NBD2 with site 2 (8). Therefore, site 1 binds nucleotides with higher affinity than site 2 (half-maximal inhibitory concentration [IC50] of 4 and 60 ␮mol/l for ATP and 26
and 100 ␮mol/l for ADP at sites 1 and 2, respectively). In
addition, ATP is preferentially hydrolyzed at site 2.
To examine these differences in nucleotide handling
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KATP CHANNEL REGULATION BY SUR1
FIG. 1. A: Left, the transmembrane topology of SUR1. A
schematic diagram of SUR1 showing the TMDs and the
NBDs. Right, schematic showing the structure of the
NBD of an ABC transporter. B: Model of the NBDs of
SUR1 as viewed from the extracellular surface. The
model shows the nucleotide sandwich dimer and the
conserved motifs. The WA motifs, WB motifs, and signature sequence are yellow, orange, and green, respectively. The ATP molecule is shown in red, and Mg2ⴙ is
shown in gray. NBD1 is blue and NBD1 is purple. Sites 1
and 2 are indicated. C: Expanded view of site 2 of the
SUR1 model showing the proximity of D860 to the ATP
␥-phosphate in Site 2.
further, we built a molecular model of the SUR1-NBD
heterodimer using a combination of mammalian and bacterial crystal structures as templates. We then mutated
residues putatively identified in the model as being involved in nucleotide handling or catalysis and tested their
functional effects on KATP channel activity.
RESEARCH DESIGN AND METHODS
Modeling. The sequences of NBD1 and NBD2 of SUR1 were aligned with
those of CFTR (cystic fibrosis transmembrane regulator) (protein database
[PDB] code 1ROZ) and TAP1 (transporter associated with antigen processing)
(PDB code 1JJ7), respectively, using ClustalW (9) and individual monomers
generated using MODELLER 6 (version 2) (10). The individual NBD models
were then least-squares fitted to the MJ0796 dimer crystal structure (PDB
code 1L2T) (11). Molecular graphics diagrams were generated using VMD
(visual molecular dynamics) (12) and PovRay (http://www.povray.org). Molecular dynamic simulations were performed as previously described (13).
Electrophysiology. Xenopus oocytes were defolliculated and coinjected
with ⬃5 ng each of mRNAs encoding Kir6.2 (GenBank accession no. D50581)
and either wild-type or mutant SUR1 (GenBank accession no. L40624). The
final injection volume was ⬃50 nl per oocyte. Isolated oocytes were maintained as previously described (14), and currents were studied 1– 4 days after
injection. Macroscopic currents were recorded from giant excised inside-out
patches at 20 –24°C (14). The pipette solution contained (in mmol/l) 140 KCl,
1.2 MgCl2, 2.6 CaCl2, and 10 HEPES (pH 7.4 with KOH), and the internal (bath)
solution contained 110 KCl, 1.44 MgCl2, 30 KOH, 10 EGTA, 10 HEPES (pH 7.2
with KOH), and nucleotides as indicated. Currents were recorded with an
Axopatch 200B amplifier (Axon Instruments), filtered at 0.2 kHz and sampled
at 0.5 kHz.
RESULTS AND DISCUSSION
Modeling the SUR1 NBD heterodimer. Because the
sequence of NBD1 differs from that of NBD2, there are
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likely to be slight structural differences between the two
NBDs. To take account of these differences, we used two
different templates to construct a molecular model of the
SUR1 heterodimer: NBD1 was modeled on the crystal
structure of NBD1 of mouse CFTR (15) and NBD2 was
modeled on the X-ray structure of the human TAP1 NBD
(16). The two NBD models were then assembled in the
nucleotide sandwich dimer configuration by least-squares
fitting to the bacterial NBD homodimer MJ0796 (11). The
CFTR-NBD1 and TAP1-NBD sequences share 33 and 26%
sequence identity to the NBD1 and NBD2 of SUR1, respectively, which is the highest homology currently available
for NBDs of known structure. Additionally, the CFTRNBD1 and TAP1-NBD2 structures were cocrystallized with
MgATP and MgADP, respectively. Thus, our model will
represent a structure with ATP docked at NBD1 and ADP
docked at NBD2. It is thought that this corresponds to the
“active” conformation of SUR.
Predictions of the molecular model. Figure 1B shows
the model of the NBD heterodimer of SUR1, with ATP
docked at site 1 and ADP docked at site 2. Given that site
2 shows an enhanced ability to hydrolyze ATP (17), we
searched for negatively charged residues proximal to the
phosphate tail of ATP that could participate in coordination and hydrolysis of the nucleotide. Two negatively
charged residues, D1505 and E1506 in the WB motif of
NBD2, were observed to line site 2. Mutation of either of
these residues abolishes the ability of MgADP to stimulate
channel activity (5,18), and E1506 is of particular interest
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
J.D. CAMPBELL AND ASSOCIATES
FIG. 2. A: Macroscopic currents recorded from oocytes coexpressing Kir6.2 and either SUR1, or SUR2BD860A in response to a series of voltage ramps from
ⴚ110 to ⴙ100 mV. 100 ␮mol/l ADP and 100 ␮mol/l
ATP were added to the intracellular solution as indicated by the bars. All solutions contained Mg2ⴙ. B:
Mean KATP conductance recorded in response to 100
␮mol/l ADP, 100 ␮mol/l ATP, or 100 ␮mol/l ADP ⴙ 100
␮mol/l ATP in patches excised from oocytes coexpressing Kir6.2 and either wild-type SUR1 (䡺) or
SUR1-D860A (f). The slope conductance (G) is expressed as a fraction of the mean of that obtained in
control solution before exposure to MgADP (Gc).
The number of patches is three in each case.
because its mutation to lysine (E1506K) leads to congenital hyperinsulinism in humans (18). Our model also identified a third negatively charged residue, D860, which lies
⬃5 Å from the ␥-phosphate and could potentially interact
with ATP (Fig. 1C). It is noteworthy that, in contrast to the
other negatively charged residues, which line site 2, D860
is contributed by the opposite NBD. The equivalent residue in NBD2 is D1512, which lines site 1. To examine
whether D860 and D1512 are of functional importance, we
mutated them, individually, to alanine.
Effects of the D860A mutation on Mg nucleotide
activation. Figure 2 shows that MgADP stimulates KATP
channel activity approximately twofold in the absence of
other nucleotides, in accordance with previous results
(14). In contrast, when D860 in NBD1 of SUR1 was
mutated to alanine, MgADP blocked rather than stimulated channel activity. The extent of block (62 ⫾ 5%, n ⫽
5) is similar to that seen in the absence of Mg2⫹ for
wild-type channels (⬃60%) (14). This suggests that the
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
mutation abolishes the stimulatory effect of MgADP on the
channel and unmasks the inhibitory effect of ADP that is
mediated via the inhibitory site on Kir6.2 (19).
Addition of 100 ␮mol/l MgATP produced a greater block
of Kir6.2/SUR1-D860A channels than of wild-type channels: 95 ⫾ 1% (n ⫽ 3) compared with 85 ⫾ 5% (n ⫽ 3) (Fig.
2). This has also been observed for mutation of the WA
lysine (20) or WB aspartate (5) in either NBD1 or NBD2. It
can be attributed to loss of MgATP hydrolysis at the NBDs
of SUR1, which, in the case of the wild-type channel,
enhances channel activity and shifts the relationship between channel inhibition and ATP concentration to higher
ATP levels.
MgADP was unable to stimulate the activity of channels
carrying the D860A mutation in the presence of ATP, in
contrast to wild-type channels (Fig. 2). The equivalent
mutation in NBD2 of SUR1 (D1512A) did not result in
functional channels.
Role of D860. Taken together, our results demonstrate a
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KATP CHANNEL REGULATION BY SUR1
A
B
because an equivalent residue (D1512) lines site 1. However, there are likely to be differences in the way that D860
and D1512 function at their respective binding sites. This
is because a nonconserved arginine residue, R1379, which
is NH2-terminal to the WA motif of site 2, lies directly
adjacent to D860 (Fig. 3A), whereas no such positively
charged residue exists in site 1. Mutation of the equivalent
arginine residue in SUR2B (R1344) impaired ADP-induced
channel activation as effectively as mutation of the WA
lysine (21). This suggests both R1379 and D860 play an
important functional role: potentially, R1379 has the ability
to undergo alternating charge-charge interactions with
ATP and D860.
We investigated this hypothesis by molecular dynamics
simulations of our SUR1 NBD model. We ran four simulations: the wild-type model with 1) ATP docked at both
NBDs, or 2) ATP docked at NBD1 and ADP docked at
NBD2, and 3, 4) the equivalent simulations of the D860A
mutant. The wild-type simulations demonstrate that R1379
preferentially interacts with ADP rather than ATP (Fig.
3B). Furthermore, the D860A simulations demonstrate
that removal of the negatively charged aspartate disrupts
the interaction of R1379 with nucleotide; in contrast to the
wild-type simulations, R1379 more strongly interacts with
the ATP than with ADP. The reduction in the predicted
interaction of R1379 with ADP following mutation of D860
is consistent with the impaired ability of MgADP to
stimulate channel activity (Fig. 2).
CONCLUSION
We have demonstrated that an approach that combines
molecular modeling predictions, simulations, and functional analysis is useful for understanding the structural
biology of the NBDs of SUR1. Our results define a new
residue, D860, which contributes to the complex mechanism by which nucleotide binding to these NBDs results in
opening of the KATP channel.
ACKNOWLEDGMENTS
FIG. 3. A: Detailed view of the interaction between R1379 and D860 in
site 1. B: Molecular dynamics simulations of mutant and wild-type SUR
NBD models. The simulation system contained water molecules and
counter ions (not shown) and was simulated for a 1-ns equilibration
run followed by a 1-ns production run from which data were analyzed.
The distance between the R1379 side-chain nitrogens and the ATP
␤-phosphate is plotted for the wild-type model with ATP docked at both
NBDs (blue), the wild-type model with ATP docked at NBD1 and ADP
docked at NBD2 (green), the D860A mutant model with ATP docked at
both NBDs (red), and the D860A mutant model with ATP docked at
NBD1 and ADP docked at NBD2 (black).
critical role for D860 in nucleotide activation of the KATP
channel. Because MgADP activation was abolished, the
effect of this mutation cannot be attributed solely to loss of
MgATP hydrolysis. Instead, it must prevent either binding
of Mg nucleotides to site 2 of SUR1 or transduction to
Kir6.2 of the conformational change consequent on nucleotide binding. Additional experiments are required to
determine which of these possibilities is correct.
Although we cannot say whether D860 simply participates in nucleotide binding or also acts as a hydrolytic
residue, the presence of D860 alone is not responsible for
the functional differences between site 1 and site 2. This is
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We thank the Wellcome Trust and the Royal Society for
support. J.D.C. is a Wellcome Trust Structural Biology
D.Phil student and a Linacre College Canadian National
scholar, and F.M.A. is the GlaxoSmithKline Research
Professor of the Royal Society.
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