Báo cáo khoa học: Studies of the ATPase activity of the ABC protein SUR1 pptx

Studies of the ATPase activity of the ABC protein SUR1
Heidi de Wet1, Michael V. Mikhailov1, Constantina Fotinou1, Mathias Dreger1, Tim J. Craig1,
´
Catherine Venien-Bryan2 and Frances M. Ashcroft1
1 Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UK
2 Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK

Keywords
ABCC8; ATPase activity; KATP channel;
nucleotide-binding domain; sulfonylurea
receptor
Correspondence
F. M. Ashcroft, University Laboratory of
Physiology, Parks Road, Oxford OX1 3PT,
UK
Fax: +44 1865 285812
Tel: +44 1865 285810
E-mail:
(Received 4 April 2007, revised 8 May 2007,
accepted 14 May 2007)
doi:10.1111/j.1742-4658.2007.05879.x

The ATP-sensitive potassium (KATP) channel couples glucose metabolism
to insulin secretion in pancreatic b-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and ⁄ or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea
receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also
compared the ATPase activity of sulfonylurea receptor 1 with that of the
isolated nucleotide-binding domains (fused to maltose-binding protein to
improve solubility). Electron microscopy showed that nucleotide-binding
domains purified as ring-like complexes corresponding to  8 momomers.
The ATPase activities expressed as maximal turnover rate [in nmol
PiỈs)1Ỉ(nmol protein))1] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea

receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and
a mixture of nucleotide-binding domain 1 and nucleotide-binding
domain 2, respectively. Corresponding Km values (in mm) were 0.1, 0.6,
0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower Km
than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were
found with GTP, but the Km values were lower. Mutation of the Walker A
lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding
domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (Ki 16 lm), but not
MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of
the nucleotide-binding domains. These data demonstrate that the ATPase
activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may
influence the activity of the protein.

ATP-sensitive potassium (KATP) channels couple cell
metabolism to membrane excitability and transmembrane ion fluxes. In pancreatic b-cells, they are of

crucial importance for regulating insulin secretion [1].
At substimulatory glucose concentrations, KATP channels are open and generate a negative potential that

Abbreviations
ABC, ATP-binding cassette; BeF, beryllium fluoride; CFTR, cystic fibrosis transmembrane conductance regulator; DDM, dodecylmaltoside;
DMPC, 1,2-dimyristoyl-sn-glycero-phosphocholine; EM, electron microscopy; KATP, ATP-sensitive potassium; MBP, maltose-binding protein;
MRP1, multidrug resistance protein 1; NBD, nucleotide-binding domain; SUR, sulfonylurea receptor; SUR1F, full-length flagged-tagged SUR1;
WA, Walker A; WB, Walker B.

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H. de Wet et al.

keeps voltage-gated Ca2+ channels closed and abolishes Ca2+ influx. Because a rise in intracellular Ca2+
is needed to stimulate insulin granule release, this prevents insulin secretion. When plasma glucose levels
increase, glucose uptake and metabolism lead to changes in the intracellular concentrations of adenine
nucleotides that close KATP channels, triggering Ca2+
channel opening, Ca2+ influx, elevation of intracellular
Ca2+ and insulin release.
The b-cell KATP channel is a large octameric complex that comprises a central tetrameric Kir6.2 pore
surrounded by four sulfonylurea receptor (SUR) 1
subunits [2]. Both Kir6.2 and SUR1 subunits are
involved in the metabolic regulation of channel activity: ATP binding to Kir6.2 causes channel inhibition
[3], whereas interaction of Mg-nucleotides (MgATP
and MgADP) with SUR1 stimulates channel opening
[4–6]. Impairment of nucleotide interactions with either
subunit can lead to neonatal diabetes or its converse,
congenital hyperinsulinism [1].
SUR belongs to the ATP-binding cassette (ABC)
protein superfamily [7]. It has 17 transmembrane helices and two large cytosolic loops, which contain the
nucleotide-binding domains (NBDs) NBD1 and
NBD2. As in all ABC proteins, each NBD contains a
highly conserved Walker A (WA) and Walker B (WB)
motif involved in ATP binding and hydrolysis, an
invariant ‘signature sequence’, and several other conserved residues. Crystallization of a number of prokaryotic NBDs and ABC proteins indicates that they
associate in a sandwich dimer conformation [8–11], in
which residues from the WA and WB motifs of one
NBD interact with the signature sequence of the other
NBD to form separate ATP-binding sites, with distinct
properties. Each ATP-binding site therefore contains
contributions from both NBD1 and NBD2. Evidence

of physical interaction between the NBDs, and
molecular modeling studies, support the idea that
SUR1 also conforms to the sandwich dimer model
[12,13]. Functional studies demonstrate that formation
of such a sandwich dimer is critical for driving gating
of cystic fibrosis transmembrane conductance regulator
(CFTR) channels [14], but this has not yet been demonstrated for KATP channels.
There are two genes that encode SUR, ABCC8
(SUR1) and ABCC9 (SUR2) [15–17]. The latter exists
in several splice variants, the most important being
SUR2A and SUR2B. Differences in the SUR subunit
contribute to the variable metabolic sensitivities of
KATP channels in different tissues. For example, even
when heterologously expressed in the same cell, recombinant Kir6.2–SUR2A channels open less readily on
metabolic inhibition than Kir6.2–SUR1 channels [18].

ATPase activity of SUR1

It has been suggested that this may relate to differences in the ATPase activity of SUR1 and SUR2 [19].
The ATPase activity of full-length SUR1 has not
been measured directly to date. However, MgATP
hydrolysis has been measured directly for recombinant
proteins in which either NBD1 or NBD2 of SUR was
fused to the maltose-binding protein [19–21]. ATPase
activity of native SUR (i.e. containing both NBDs and
transmembrane domains) has also been inferred by
comparing covalent labeling with 8-azido-[32P]ATP[aP]
and 8-azido-[32P]ATP[cP] [22]. In these studies, however, hydrolysis by NBD2, but not NBD1, was detected. Unlike prokaryotic ABC proteins, NBD1 and
NBD2 of SUR1 show significant sequence differences:
thus, the ATPase activity of the isolated recombinant

NBD homodimers will not necessarily reflect that of
the NBD heterodimer expected for native SUR1. Furthermore, the presence of the transmembrane domains
in SUR1 may influence ATPase activity. We have
therefore purified SUR1 and compared its capacity to
hydrolyze ATP and GTP with that of isolated NBD1,
or NBD2, of SUR1 [fused to maltose-binding protein
(MBP)]. We also measured the ATPase activity of a
mixture of NBD1 and NBD2 proteins. In addition, the
effects of the inhibitors beryllium fluoride (BeF) and
MgADP were explored.

Results
Purification and characterization of SUR1
MBP–NBDs and SUR1F
SDS ⁄ PAGE and Coomassie staining revealed a single
major band following purification of full-length
flagged-tagged SUR1 (SUR1F), MBP–NBD1 and
MBP–NBD2 (Fig. 1A). For simplicity, we refer to
these proteins subsequently as SUR1, NBD1 and
NBD2. MALDI-TOF analysis of total purified proteins confirmed their identities as well as the absence
of any other contaminating ATPases (data not shown).
Additional bands visible on these gels were identified
as degradation products by MALDI-TOF analysis of
gel cut-outs.
Gel filtration of SUR1 yielded two fractions
(Fig. 1Ba). The smaller peak corresponds to the
molecular mass expected for monomeric SUR1 and
the larger peak runs as expected for a mixture of tetrameric and oligomeric species.
Gel filtration revealed that NBD1, NBD2 or a 1 : 1
mixture of NBD1 and NBD2 eluted as a single sharp

peak corresponding to a single oligomeric species
(Fig. 1Bb). Calculated molecular masses gave approximate sizes of  8 monomers for NBD1 and  9 mono-

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H. de Wet et al.

A
kDa

1 2

1

2 3

188

250
150

kDa

SUR1

98
NBD1

100

NBD2

62

75

49

50

38
28

Ba

Bb
Octamer

Oligomer
Tetramer
Monomer

Ca

Fig. 1. Purification of SUR1, NBD1 and

NBD2. (A) Coomassie-stained denaturing
gels of purified SUR1 (left, lane 2), MBP–
NBD1 (right, lane 1), and MBP–NBD2 (right,
lane 2). Molecular mass markers, lane 1
(left) and lane 3 (right). Samples shown are
the purified eluates from affinity resins, and
and were not subjected to further purification by gel filtration. (Ba) Gel filtration analysis of purified SUR1. (Bb) Gel filtration
analysis of purified MBP–NBD1. (Ca) Negative stain electron micrograph of MBP–
NBD1. The scale bar is 100 nm. Black
arrows indicate ring-like structures. The
white arrow points to a stack of rings. (Cb)
Ten different classes of particle. The size of
˚
the boxes is 280 A.

Cb

mers for NBD2. No larger aggregates or other protein
species were detected. SDS ⁄ PAGE analysis of the proteins in the respective gel filtration fractions confirmed
their identities as MBP–NBD1 and MBP–NBD2.
Because gel filtration suggested that NBD1 and NBD2
associate as oligomers, we collected the peak eluates
and examined them by negative stain electron microscopy (EM). This revealed that both proteins formed
ring-like oligomers. For NBD1, the outer diameter of
˚
the projected structure was between 120 and 140 A
˚ (Fig. 1C). A simand the inner diameter was 40–75 A
ilar structure was observed for NBD2 and for a 1 : 1
mixture of NBD1 and NBD2. Oligomerization was
independent of the presence of MgATP (data not

shown). MBP alone did not form ring-like oligomers

3534

(data not shown), suggesting that the interaction is
mediated by the NBD part, rather than the MBP part,
of the MBP–NBD fusion proteins.
Nucleotide hydrolysis by SUR1
Recombinant full-length SUR1 hydrolyzed MgATP
very slowly, with a maximal turnover rate of
0.03 s)1 (Table 1 and Fig. 2A) and a Km of 0.1 mm.
No ATPase activity was detected in the absence of
Mg2+ or from protein purified from cells transfected
with an SUR1 construct lacking the FLAG tag used for
affinity purification (Fig. 2A). Because KATP channels
are stimulated by GTP, via interaction with the NBDs
of SUR1 [32], we also investigated the ability of SUR1

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H. de Wet et al.

ATPase activity of SUR1

Table 1. ATPase activities and kinetic constants.

Construct

Turnover rate

Vmax
(nmol PiỈs)1Ỉ
(nmol PiỈmin)1Ỉ Km
nmol)1 protein) mg)1)
(mM)

SUR1
MBP–NBD1
MBP–NBD2
MBP–NBD1 + 2

0.03
0.03
0.13
0.08

±
±
±
±

0.005
0.003
0.01
0.01

9.0
23.8
103.81
61.22

±
±
±
±

1.7
2.40
8.70
6.78

0.10
0.6
0.65
0.56

n
±
±
±
±

0.03 6
0.09 14
0.13 12
0.11 10

to hydrolyze GTP. Figure 2B shows that GTP was also
hydrolyzed, but with a much higher Km (> 1 mm),
which suggests that GTP binds to SUR1 with a lower

affinity than ATP. The turnover rate, estimated by fitA 12

B
12

MgATP
No FLAG tag
Mg free

10

10

8

nmol Pi/min/mg

nmol Pi/min/mg

ting the data to the Michaelis–Menten equation, was
similar to that of ATP.
Mutation of residues in the Walker motifs of SUR
impairs channel activation by MgATP and MgADP
[5]. Mutating the WA lysine in NBD1 of SUR1 to
alanine (K719A) reduced ATPase activity by approximately 60%, whereas mutating the WA lysine in
NBD2 to methionine (K1385M) inhibited ATPase
activity by about 80% (Fig. 2C) when compared to
wild-type controls assayed in parallel (n ¼ 2). Neither
mutation affected the Km for ATP. These data are
consistent with the idea that these mutations affect

the rate of hydrolysis but do not influence ATP
binding.

6
4
2

8
6
4
2

0
0.01

0.1

1

0
0.01

10

0.1

[ATP] (mM)

D
1.2

0.8

KA
KM
Wt of KA
Wt of KM

0.6
0.4
0.2
0.0
0.01

10

1.4
1.2

Fractional turnover rate

mutant turnover rate/wt turnover
rate

C

1.0

1

[GTP] (mM)

1.0
0.8
0.6
0.4
0.2

0.1

1

10

0.0
0.001

[ATP] (mM)

0.01

0.1

1

0

[inhibitor] (mM)

Fig. 2. ATPase activity of SUR1. (A) ATPase activity in the presence (filled circles, n ¼ 6) or absence of Mg2+ (crosses, n ¼ 2) of purified

SUR1, at 37 °C. Membranes from cells expressing SUR1 lacking a FLAG tag and purified as usual show no ATPase activity (open circles,
n ¼ 1). (B) GTPase activity of SUR1 (n ¼ 4). The line is fitted to the Michaelis–Menten equation, with an estimated Vmax and Km of 10 nmol
PiỈmin)1Ỉmg)1 and 0.86 mM, respectively. (C) ATPase activity of SUR1 containing the mutations K719A (triangles, n ¼ 2) or K1385M (diamonds, n ¼ 2). Data are expressed as a fraction of the turnover rate for wild-type SUR1 assayed in parallel. (D) Inhibition of ATPase activity
at 1 mM MgATP by ADP (open circles, n ¼ 4) and by BeF (filled circles, n ¼ 3). Data are expressed as a fraction of the turnover rate in the
absence of inhibitor.

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H. de Wet et al.

Table 2. Inhibition constants for ADP and BeF. ND, not detected. n indicates the number of different protein preparations.

Construct

IC50(ADP)
(mM)

Ki(ADP)
(mM)

N

IC50(ADP)
(mM)

Ki(BeF)
(mM)

SUR1
MBP–NBD1
MBP–NBD2
MBP–NBD1 + 2

ND
5.7 ± 1.5
2.2 ± 0.7
1.6 ± 0.3

ND
2.12 ± 0.54
0.84 ± 0.25
0.60 ± 0.1

4
3
3
3

0.072
0.087
0.048
0.050

0.016
0.033

0.019
0.020

BeF (BeF3– and BeF42–) is a potent inhibitor of
ATP hydrolysis by many ABC proteins, including
P-glycoprotein [33] and the isolated NBD2 of SUR2A
[20]. It acts by arresting the ATPase cycle in the prehydrolytic conformation [34]. The ATPase activity of
SUR1 was potently and completely inhibited by BeF
(Ki 16 lm; Table 2; Fig. 2D). Previous studies have
shown that MgADP blocks the ATPase activity of
NBD2 of SUR2A [20] by trapping the ATPase cycle in
the posthydrolytic conformation. Unexpectedly, however, no effect of ADP on the ATPase activity of
SUR1 was observed (Fig. 2D).
The ATPase activity of SUR1 is about 10-fold less
than what we previously reported for the complete
octameric KATP channel complex, SUR1–Kir6.2 (turnover rate 0.4 ± 0.03 s)1 calculated from Mikhailov
et al. [2]).

A

±
±
±
±

0.011
0.008
0.002
0.003

3
3
4
4

Nucleotide hydrolysis activities of NBD1
NBD1 displayed low maximal ATPase activity, similar
to that of SUR1, but the Km was about six-fold larger
(0.6 mm, P < 0.005) (Table 1 and Fig. 3A). This suggests that ATP binds more tightly to full-length SUR1
than to the isolated NBD1. GTP was hydrolyzed with
a Km higher than that for ATP (Fig. 3B). There was a
very small, but significant, apparent hydrolysis of ADP
(Table 3, Fig. 3B). Negligible ATP hydrolysis was
observed in the absence of Mg2+ or in protein preparations from cells expressing MBP alone (Fig. 3A).
BeF blocked ATP hydrolysis at NBD1 with a
Ki of 33 lm (Table 2 and Fig. 3C). Unlike with
SUR1, however, inhibition appeared to be incomplete, and the maximal block was 76%. In marked
contrast to SUR1, MgADP inhibited ATP hydrolysis

30
+Mg2+

15

25

2+

–Mg
MBP

nmol Pi/min/mg

20
nmol Pi/min/mg

0.02
0.02
0.01
0.01

B
25

10
5
0
0.01

0.1

1

10

20
15
10

1.2

1.0
0.8
0.6
0.4
0.2

BeF
ADP

0.0
0.001

0.01

0.1
[Inhibitor] (mM)

0
0.01

0.1

1

[Nucleotide] (mM)

C

3536

GTP
ADP

5

[ATP] (mM)

Fractional turnover rate

±
±
±
±

n

1

10

10

Fig. 3. ATPase activity of NBD1. (A) ATPase
activity of NBD1 in the presence (filled
circles, n ¼ 14) or absence (open circles,
n ¼ 2) of Mg2+, at 37 °C. MBP control
(open triangles, n ¼ 2). (B) GTP (filled
circles, n ¼ 3) and ADP (filled triangles, n ¼
3) hydrolytic activity. The line is fitted to the
Michaelis–Menten equation, through the

GTP data points with an estimated Vmax and
Km of 39 nmol PiỈmin)1Ỉmg)1 and 2.6 mM,
respectively. (C) Inhibition of ATPase activity
at 1 mM MgATP by ADP (open circles, n ¼
3) or BeF (filled triangles, n ¼ 3). Data are
expressed as a fraction of the turnover rate
in the absence of inhibitor.

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H. de Wet et al.

ATPase activity of SUR1

Table 3. ADPase activities and kinetic constants.

Construct

Turnover rate
(nmol PiỈs)1Ỉ
nmol)1 protein)

Vmax
(nmol PiỈ
min)1Ỉmg)1)

Km
(mM)

n

MBP–NBD1
MBP–NBD2
MBP–NBD1 + 2

0.005 ± 0.001
0.014 ± 0.003
0.012 ± 0.002

3.83 ± 1.26
11.26 ± 2.40
9.80 ± 2.69

0.17 ± 0.08
0.07 ± 0.02
0.06 ± 0.01

3
3
3

at NBD1, with a Ki of 2.1 mm (Table 2 and
Fig. 2C).
Nucleotide hydrolysis activities of NBD2
The maximal ATPase activity of NBD2 was about
four-fold greater than that of either SUR1
(P < 0.001) or NBD1 (P < 0.001). The Km was similar to that of NBD1 and about six-fold larger than
that of SUR1 (Table 1, Fig. 4A (P < 0.005). These
results suggest that the ATP-binding affinities of

NBD1 and NBD2 are similar, but that the hydrolytic
step occurs more rapidly in NBD2, and that, compared with SUR1, the ATP-binding affinity of NBD2
is less but hydrolysis is faster. GTP was also hydrolyzed, with a Km higher than that for ATP (Fig. 4B).
MgADP was hydrolyzed at very low rate, but this was

about three-fold greater than that of NBD1 (Table 3,
Fig. 4B). Negligible ATP hydrolysis was observed in
the absence of Mg2+.
As found for NBD1, MgATP hydrolysis (1 mm) was
inhibited by both BeF and MgADP (Table 2 and
Fig. 4C). The Ki for BeF inhibition (19 lm) was lower
than that for NBD1, and maximal inhibition was
86%. ATP hydrolysis was also inhibited by MgADP,
with a Ki of 0.84 mm (Table 2).
Nucleotide hydrolysis activities of NBD1 + NBD2
Interactions of the isolated MBP–NBDs of the
CFTR, or the multidrug resistance protein MRP1,
have been demonstrated previously [35,36]. We therefore examined ATP hydrolysis in a 1 : 1 mixture of
NBD1 and NBD2. ATP hydrolysis by the
NBD1 + NBD2 mixture had a maximal turnover
rate intermediate between that of NBD1 and NBD2
alone, and the Km (0.56 mm) was not significantly different from that of either NBD1 or NBD2 (Table 1,
Fig. 5A). Thus, as found for MRP1 [36], mixing the
two NBDs did not have a major impact on the catalytic activity of either NBD.
GTP was hydrolyzed by the NBD1 + NBD2 mixture with Km > 1 mm (Fig. 5B). As observed for the

A

B

nmol Pi/min/mg

120

+Mg

100

-Mg2+

100
80
60
40

0
0.01

Fractional turnover rate

C

GTP
ADP

80
60
40
20

20

Fig. 4. ATPase activity of NBD2. (A) ATPase
activity of NBD2 in the presence (filled circles, n ¼ 12) or absence (open circles, n ¼
2) of Mg2+, at 37 °C. (B) GTP (filled circles,
n ¼ 3) and ADP (filled triangles, n ¼ 3)
hydrolytic activity. The line is fitted to the
Michaelis–Menten equation, with an estimated Vmax and Km of 153 nmol PiỈmin)1Ỉmg)1
and 2.2 mM, respectively. (C) Inhibition of
ATPase activity at 1 mM MgATP by ADP
(open circles, n ¼ 3) or BeF (filled triangles,
n ¼ 4). Data are expressed as a fraction of
the turnover rate in the absence of inhibitor.

120

2+

nmol Pi/min/mg

140

0.1
1
[ATP] (mM)

10

0
0.01

0.1
1
[Nucleotide] (mM)

10

1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.001

BeF
ADP
0.01

0.1

1

10

[Inhibitor] (mM)

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ATPase activity of SUR1

A

H. de Wet et al.

B

80

60
2+

+Mg

50

-Mg2+

nmol Pi/min/mg

nmol Pi/min/mg

60

40

GTP
ADP

40
30
20

20

10
0
0.01

0.1
1
[ATP] (mM)

10

0
0.01

0.1
1
[Nucleotide] (mM)

Fractional turnover rate

C 1.2
1.0

0.8
0.6
0.4
0.2
0.0
0.001

BeF
ADP
0.01

0.1
[Inhibitor] (mM)

1

10

individual domains, the NBD mixture apparently
hydrolyzed MgADP but with a very low turnover rate
(Table 3, Fig. 5B), and no hydrolysis was observed in
the absence of Mg2+.
MgATP hydrolysis was inhibited by BeF with a Ki
of 20 lm (Table 2, Fig. 5C), which is not significantly
different from that of either NBD1 (30 lm) or
NBD2 (19 lm) alone. However, the Ki for MgADP
inhibition of ATP hydrolysis was significantly less
than that of NBD1 (P < 0.05): it was also lower than
that of NBD2, although this difference was not
significant (Table 2). A value intermediate between

those of NBD1 and NBD2 would be expected if
the NBDs were functionally independent: thus, these
data suggest that the NBDs may functionally interact
when mixed. This finding is consistent with the
idea that at least some heterodimers of NBD1
and NBD2 are present in the NBD1 + NBD2 mixture.

Discussion
Nucleotide handling by SUR1
The ATPase activity of SUR1 was 10-fold lower, and
the Km three-fold larger, than that measured for the
purified complete KATP channel (Kir6.2–SUR1) complex [2]. This suggests that ATP binds with lower
3538

10

Fig. 5. ATPase activity of NBD1 + NBD2.
(A) ATPase activity of the NBD1 + NBD2
mixture in the presence (filled circles, n ¼
10) or absence (open circles, n ¼ 2) of
Mg2+, at 37 °C. (B) GTP (circles, n ¼ 3) and
ADP (triangles, n ¼ 3) hydrolytic activity of
NBD1 + NBD2. The line through the GTP
data points is fitted to the Michaelis–
Menten equation, with an estimated Vmax
and Km of 70 nmol PiỈmin)1Ỉmg)1 and
1.7 mM, respectively. (C) Inhibition of
ATPase activity at 1 mM MgATP by ADP
(open circles, n ¼ 3) or BeF (filled triangles,
n ¼ 4). Data are expressed as a fraction of

the turnover rate in the absence of inhibitor.

affinity and the rate of ATP hydrolysis is faster in
the KATP channel complex than in SUR1 alone.
CryoEM analysis revealed that the KATP channel
associates as a large octameric complex in which the
individual SUR1 subunits are tightly packed around
a central Kir6.2 tetrameric pore [2]. The higher
ATPase activity of the KATP channel complex might
therefore result from interactions between adjacent
SUR1 subunits, and ⁄ or between SUR1 and Kir6.2,
that enhance cooperativity and ⁄ or crosstalk between
the NBDs.
The ATPase activity of purified SUR1 (Vmax,
9 nmol PiỈmin)1Ỉmg)1) is at the lower end of the range
found for MRP1: from 5 to 10 nmol PiỈ min)1Ỉmg)1
[37] to 470 nmol PiỈmin)1Ỉmg)1 [38]. It is less than that
reported for CFTR (60 nmol PiỈ min)1 Ỉmg)1 protein
[39]), or P-glcyoprotein (320–3900 nmol PiỈ min)1Ỉmg)1
[40,41]), but higher than that found for ABCR
(1.3 mol PiỈmin)1Ỉmg)1 [42]). It is possible that the
lower ATPase activity of SUR1 is related to the
unique role of this ABC protein as a channel regulator
rather than a transporter. It is worth noting that the
activity of other ABC transporters, including the
closely related MRP1 [38], are stimulated by their
substrates. It is possible that mechanism by which
Kir6.2 stimulates the ATPase activity of SUR1 resembles this substrate activation.

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H. de Wet et al.

The Km for ATP hydrolysis by SUR1 (0.1 mm) was
similar to that reported for purified CFTR and MRP1
(0.1–3 mm) [37–39], but significantly lower than we
measured for the isolated NBDs of SUR1. We speculate that the presence of the transmembrane domains in
SUR1 induces conformational changes in the NBDs, or
in their association, that influences ATP binding. The
Km of the purified KATP channel complex (SUR1F–
Kir6.2) was 0.4 ± 0.2 mm [2], which is not significantly
different from that found for the isolated NBDs, but is
somewhat greater than that of SUR1. Thus, it seems
possible that the presence of Kir6.2 within the KATP
complex may further modify interactions between the
NBDs that occur in SUR1 alone. Both the NBDs and
the transmembrane domains of SUR1 are known to
interact with the cytosolic and transmembrane domains
of Kir6.2, respectively [23,25,43].
Mutation of the WA lysines (K719A, K1385M)
reduced ATPase activity by 70–80%. Mutation of the
equivalent residues in full-length CFTR [44], or the
isolated NBD2 of SUR2A [45] and NBD1 or NBD2
of CFTR [46, 47], also reduces, but does not fully
abolish, ATPase activity. Nevertheless, these mutations
completely ablate the ability of MgADP to stimulate
KATP channel activity [5]. Thus, WA mutations in
SUR1 may also influence nucleotide binding [48]
and ⁄ or the mechanism by which nucleotide binding ⁄ hydrolysis is coupled to channel activity.

ATPase activity of the isolated NBDs
As previously reported, MBP-fusion proteins of isolated NBD1 and NBD2 domains hydrolyzed ATP. The
Km for ATP hydrolysis ( 600 lm) did not vary significantly between the isolated NBDs (NBD1, NBD2 or
the NBD1–NBD2 mixture). Previous studies yielded
somewhat lower values of 290 lm and 350 lm for
NBD1 and NBD2, respectively [19]. For comparison,
values for the NBDS of SUR2A were 220 lm for
NBD1 [19] and ranged from 370 lm [19] to 4.4 mm
[45] for NBD2. The rates of ATP hydrolysis that we
observed are about two-fold (NBD1) and up to fivefold (NBD2) higher than those previously reported for
the isolated NBDs of SUR1 [19]. It is possible that
these differences reflect differences in the sequence of
isolated domains used in the different studies. Mixing
NBD1 and NBD2 did not alter ATPase activity. This
is similar to what is found for MRP1 [36], the ABC
protein most closely related to SUR1, but contrasts
with the NBDs of CFTR, where the activity of NBD1
is enhanced by heterodimerization with NBD2 [35,49].
Like other ABC proteins, including MRP1 [38],
SUR1 and both of its isolated NBDs had a broad nuc-

ATPase activity of SUR1

leotide specificity and hydrolyzed GTP as well as ATP.
There appeared to be a small amount of hydrolysis of
MgADP by both NBD1 and NBD2, which contributed
less than 10% of the ATP hydrolysis rate. It is possible
that SUR1 exhibits adenylate kinase activity, as has
been suggested for CFTR [50,51]. In this case, hydrolysis of ATP generated from ADP (by adenylate kinase
activity) might account for the increase in free phosphate that we observed.

Inhibition of ATPase activity
A decrease in the ATPase activity of SUR1 was
observed when the conserved lysine in the WA motif
was mutated either in NBD1 or NBD2. Mutation of
the WA motif in NBD1 reduced ATPase activity by
about 60%. If we assume that the relative extent of
ATPase activity at NBD1 and NBD2 remains the
same in full-length SUR1 (i.e. that of NBD1 is
 20% of that of NBD2), then the marked inhibition
of ATPase activity of SUR1 suggests that the WA
mutation in NBD1 also reduced hydrolysis at NBD2.
This might indicate possible interactions between the
NBDs. The fact that the same mutations did not
affect MgADP binding to NBD2 [52] suggests that it
is the hydrolytic capacity that is affected. Mutation
of the WA lysine at NBD2 blocked ATPase activity
of SUR1 by about 80%. Although this would be consistent with inhibition of NBD2 alone, it may
also reflect a partial decrease in hydrolysis at both
NBD1 and NBD2.
The ATPase activity of SUR1 was potently inhibited
by BeF, which traps ABC proteins in a prehydrolytic
ATP-bound conformation [33,34]. Inhibition by BeF
(1 mm) has previously only been reported for the isolated NBD2 of SUR2A [20,21].
MgADP also inhibited ATP hydrolysis by isolated
NBDs, albeit with very low affinity. The lowest value
of Ki (0.6 mm) was found for the NBD1 + NBD2
mixture. The inability of ADP to block ATP hydrolysis by SUR1 is surprising: possible explanations for
this finding include a lower ADP affinity for SUR1 or
a higher adenylate kinase activity. We presume that
this effect is ameliorated in the KATP channel complex,

as MgADP stimulates channel activity, and reverses
channel inhibition by ATP, via interaction with the
NBDs of SUR1 [4,5].
Oligomerization of the NBDs
Gel filtration indicated that MBP–NBD1, MBP–NBD2
and a 1 : 1 mixture of the two purified as a multimer
of around eight or nine monomers. When viewed by

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3539


ATPase activity of SUR1

H. de Wet et al.

EM, the proteins formed ring-like structures with an
˚
outer diameter of  120–140 A. This is similar to the
outer diameter of the purified octameric KATP complex
˚
(180 A) [2], and is consistent with the idea that the
ring-like structures represent eight MBP–NBDs that
coassemble as a tetramer of dimers. The inner diameter
˚
of the NBD ring was 40–75 A. This space is expected
to be occupied by Kir6.2 in the native KATP channel
complex. The widest diameter of cytoplasmic domain
of the related Kir channels Kir3.1 and Kir3.2 was

˚
 80 A in the crystal structure [53,54]. Thus, the NBDs
are likely to pack somewhat less tightly in the KATP
complex than in the ring-like structures that we
observed for the isolated NBDs.
These results suggest that the NBDs may be involved
in physical subunit–subunit associations within the
KATP channel complex, and raise the possibility that
they may also be involved in functional interactions
between subunits. Previous studies have also suggested
that NBD1 and NBD2 can physically interact [23,24]
and that purified NBD1 of SUR1 can exist as a tetramer
[26]. Interaction of isolated recombinant NBDs to form
functional heterodimers has also been reported for
several other ABC proteins [35,36,55]. Such heterodimerization enhanced the ATPase activity of some
ABCC proteins (e.g. CFTR) [35,48], attenuated ATPase
activity in others (e.g. ABCR [55]), or was without effect
(e.g. MRP1 [36]), as we found for SUR1.
Implications for channel gating
Unlike those of other ABC proteins, the functional
role of SUR1 is that of a channel regulator, and ATP
hydrolysis by SUR1 plays an important role in the
metabolic regulation of the KATP channel [23]. In
electrophysiologic studies, both MgATP and MgADP
stimulate KATP channel activity [3–6]. However, current evidence suggests that it is the presence of
MgADP at NBD2 that results in KATP channel opening, and that MgATP must be hydrolyzed to MgADP
in order for channel activation to occur [20].
It is difficult to measure the EC50 for MgATP activation of wild-type KATP currents in electrophysiological studies, due to simultaneous inhibition via the
ATP-binding site of Kir6.2. Coexpression of SUR1
with Kir6.2 carrying mutations in the ATP-binding

site, however, suggests that half-maximal channel activation is produced by MgATP concentrations of
around 0.1 mm or greater [6]. This is in agreement
with the results we report here for SUR1 and those
found previously for the KATP complex [2].
Mutation of the WA lysines markedly decreased but
did not completely abolish ATP hydrolysis by SUR1,
3540

in agreement with the electrophysiological data. The
same mutations shifted the IC50 for ATP block of the
KATP channel to a value (13–16 lm) [6]) intermediate
between that seen for wild-type channels in the presence ( 30 lm) [6] and absence (6 lm) [6] of Mg2+.
One might expect that a mutation which abolished
MgATP binding ⁄ hydrolysis would have an IC50 similar
to that found in Mg-free solution for wild-type channels. The fact that this is not the case suggests that
binding ⁄ hydrolysis of MgATP is not entirely abolished
by WA mutations. Interestingly, the same mutations
completely abolished the ability of MgADP to stimulate KATP channel currents [5].

Conclusion
SUR1 is unique among ABC proteins in that it serves
as a channel regulator, forming a tightly associated
octameric KATP channel complex in which four Kir6.2
subunits form a central pore surrounded by four
SUR1 subunits [2]. The fact that the isolated NBDs of
SUR1 associate in tetrameric ring-like structures even
when Kir6.2 is not present suggests that these domains
possess some intrinsic capacity for stable association
and that this may contribute to formation of the octameric KATP channel complex. Here we show that the
ATPase activity of SUR1 alone differs from those of

both the isolated NBDs and of the octameric KATP
channel complex. This suggests that the ATPase activity of the NBDs is influenced both by the presence of
the transmembrane domains of SUR1 and by the
tetrameric Kir6.2 pore. Thus, just as SUR1 influences
the channel activity of Kir6.2, so Kir6.2 appears to
modulate the ATPase activity of SUR. This may be
considered analogous to the way in which substrates
stimulate the activity of other ABC proteins.

Experimental procedures
Protein expression and purification
A FLAG-tag was inserted into the extracellular loop
between transmembrane helices 11 and 12 of rat SUR1
(GenBank L40624), as previously reported [2]. This fulllength construct of SUR1 (SUR1F) was expressed in
insect cells (Sf9) using a baculovirus expression system
(Invitrogen, Paisley, UK), and expression was verified
and quantified by [3H]glibenclamide binding [23]. Cells
were grown and harvested as previously described [2], disrupted using a Stansted TC5W homogenizer (Stansted
Fluid Power Ltd, Stansted, UK) at a pressure of
10 000 lbỈin)2, and centrifuged at 200 g for 10 min using
a Beckman Allegra 6KR centrifuge with S/N02E3297

FEBS Journal 274 (2007) 3532–3544 ª 2007 The Authors Journal compilation ª 2007 FEBS


H. de Wet et al.

rotor. The supernatant was loaded on a step sucrose gradient (10% ⁄ 46%) and centrifuged at 100 000 g for 1 h
using a Beckman L7 centrifuge with SW28 rotor. The
intermediate phase was collected and diluted four times

with 50 mm Tris ⁄ HCl (pH 8.8) and 200 mm NaCl. Dodecylmaltoside (DDM) (0.5% w ⁄ v) was then added, and
membranes were solubilized for 20 min at room temperature, and then centrifuged at 48 400 g for 20 min using a
Beckman Avanti J-20XP centrifuge with JA-25.50 rotor.
Anti-FLAG M2 affinity gel (Sigma, Poole, UK) was
added to the supernatant and incubated for 2 h. The
suspension was washed with 20 volumes of 50 mm
Tris ⁄ HCl (pH 8.8), 150 mm NaCl and 0.1% DDM. Protein was then eluted with 100 lm 3-FLAG peptide
(Sigma), 50 mm Tris ⁄ HCl (pH 8.8), 150 mm NaCl, 0.2%
DDM, 0.05% 1,2-dimyristoyl-sn-glycero-phosphocholine
(DMPC). All procedures were carried out at 4 °C. The
purified protein yield ranged between 50 and 100 lgỈL)1.
The identity and purity of SUR1F was confirmed by
MALDI-TOF MS. All assays were performed on freshly
prepared SUR1F.
Rat NBDs were cloned into the pMAL-c2X vector (New
England Biolabs, Hitchin, UK) to yield MBP-fusion constructs, in which MBP is attached to the N-terminal end of
the NBD. This strategy was employed because the NBDs
alone are known to be poorly soluble [24]. The nucleotide
sequence used for NBD1 was Val608 to Leu1004, and that
used for NBD2 was Lys1319 to Lys1581. Plasmids were
transformed into BL21-CodonPlus Escherichia coli cells
(Stratagene, La Jolla, CA, USA). One liter of Terrific Broth
(Sigma) in baffled flasks was inoculated with 50 mL of
transformed BL21-CodonPlus, grown to a D600 of 1 and
induced with 0.4 mm isopropyl thio-b-d-galactoside. Cells
were harvested after 4 h, at 200 g for 20 min. They were resuspended in 30 mL of buffer A (50 mm Tris ⁄ HCl, pH 7.5,
150 mm NaCl, 2 mm dithiothreitol and 1% protease inhibitors; all Sigma). Cells were lysed by two passages through a
Stansted TC5W homogenizer at 12 000 lbỈin)2 and kept on
ice throughout. Insoluble debris was pelleted by centrifugation for 30 min at 48 400 g using a Beckman Avanti
J-20XP with JA-25.50 rotor, and the supernatant was incubated by rotation for 1 h at 4 °C with 2 mL of amylose resin.

Unbound protein was eluted by washing with 2 · 10 mL of
buffer A, and bound protein was eluted after 15 min of rotating incubation with 4 mL of elution buffer (buffer A with
10 mm maltose and 20% glycerol). The identity of proteins
of expected sizes for NBD1 and NBD2 were confirmed using
antibody to NBD1 [25] and an antibody to MBP (rabbit
polyclonal; New England Biolabs), respectively.
Yields were typically  6 mgỈL)1 for NBD1–MBP and
0.8 mgỈL)1 for NBD2–MBP, and comprised > 95% of
total purified protein. When not used fresh, purified proteins were stored at ) 80 °C in 20% glycerol. Proteins were
separated on 4–12% gradient Bis ⁄ Tris gels and visualized
by Coomassie staining (Invitrogen).

ATPase activity of SUR1

MALDI-TOF MS
MALDI-TOF MS (MS and MS ⁄ MS) was performed using
a Bruker Ultraflex TOF ⁄ TOF mass spectrometer (Bruker
Daltonics, Coventry, UK) equipped with a nitrogen laser.
Gel bands of interest were digested in-gel according to
standard procedures [26] using proteomics-grade Trypsin
(Sigma-Aldrich). MS was performed using a-cyano-4-hydroxycinnamic acid as matrix. Peptide mass fingerprint
spectra were matched against the NCBI nonredundant
protein database using the search engine mascot (Matrix
Science, London, UK) via an in-house license. MS ⁄ MS
spectra were taken using the LIFT method (Bruker Daltonics). The accuracy of MS spectra was typically better
than 50 p.p.m.: the accuracy of MS ⁄ MS-generated fragment ions was in the range of ± 0.2 Da.

Gel filtration
Purified NBD–MBPs were diluted to 0.5 lgỈlL)1, and
300 lL was analyzed on a Superdex-200 gel filtration column

(Tricon, Amersham Biosciences, Little Chalfont, UK) in ATPase buffer (50 mm Tris ⁄ HCl, pH 7.2, 150 mm NH4Cl,
10 mm MgCl2). Relative protein size was calculated using
Bio-Rad Gel Filtration standards (Cat no. 151-1901). Peaks
were collected and analyzed by EM, and for ATPase activity.
Purified SUR1F was mixed with DMPC ⁄ DDM to final
concentrations of 0.05% w ⁄ v for DMPC and 0.1% w ⁄ v for
DDM. It was further concentrated to 1.45 mgỈmL)1, and
250 lL was loaded on a Superdex 200 (10 ⁄ 30) gel filtration
column pre-equilibrated with 50 mL of buffer containing
50 mm Tris (pH 8.8), 150 mm NaCl, 0.05% w ⁄ v DMPC
and 0.1% w ⁄ v DDM.

EM and image processing
For EM, protein samples were diluted to a concentration
of between 0.05 and 0.1 mgỈmL)1, applied to EM grids coated with carbon film and stained with 2% uranyl acetate.
Preparations were examined using a CM120 electron microscope (FEI, Eindhoven, the Netherlands) with an acceleration voltage of 100 kV. Electron micrographs were taken
at a magnification of · 45 000. Selected images were digitized with a step size of 25 lm on a Nikon Super Coolscan
9000 (Nikon, London, UK). The web and spider software
packages [27] were used for all image processing. In total,
524 particles were windowed, subjected to reference-free
alignment, and sorted into 10 classes using the partitional
method (K-means method) of clustering [28].

Nucleotide hydrolysis
ATPase activity was normally measured for proteins purified as above, but without the gel filtration step. These

FEBS Journal 274 (2007) 3532–3544 ª 2007 The Authors Journal compilation ª 2007 FEBS

3541

ATPase activity of SUR1

H. de Wet et al.

preparations were of adequate purity (Fig. 1). The faint
bands seen below the major products are degradation
products of the purified protein as confirmed by
MALDI-TOF analysis. Total protein was calculated from
the major band, representing undegraded protein, using
BSA standards and scion image software. Gel filtration,
especially in the case of SUR1F, diluted the amount of
protein available and was therefore not used routinely.
However, no difference in the ATPase activity of any of
the purified proteins was observed if a gel filtration step
was included.
ATPase (or GTPase) activity was measured using a
colorimetric assay for liberated inorganic phosphate (Pi), as
described previously [2,29]. All assays were performed at
37 °C in ATPase assay buffer (50 mm Tris ⁄ HCl, pH 7.2,
150 mm NH4Cl, 10 mm MgCl2). Pi release was linear over
the time course of the assay, and remained linear in the presence of inhibitors. Because both the ATPase activity and the
Km are rather low, no substrate depletion was observed over
the time period of the experiment. The rate of the reaction
was proportional to the amount of protein used, and there
was a linear relationship between protein concentration and
activity. The protein concentration was 1 lm for BeF inhibition and 3 lm for all other experiments, to ensure that
ligand binding would not significantly alter the concentration of free ligand ⁄ inhibitor. In some experiments, equal
amounts of MBP–NBD1 and MBP–NBD2 (lg ⁄ lg) were
mixed and allowed to interact on ice for 45 min prior to the

hydrolysis assay. To compensate for contaminating phosphate present in all commercial ATP (and GTP) preparations, we included negative controls for each experimental
condition, in which the protein was denatured by 5% SDS
(final concentration) prior to the hydrolysis assay. Absorbance from denatured controls was always subtracted from
the equivalent experimental values. The maximal concentration of MgNTP that could be used without interference
from contaminating (endogenous) Pi was 3 mm.
We used the sodium salts of ATP, GTP and MgADP.
However, no difference in the ATP hydrolysis rate was
observed if the potassium salt of ATP was employed. ATP,
ADP, GTP were obtained from Sigma and were of ‡ 99%
purity.
BeF (BeF3– and BeF42–) was prepared as previously described [30]. Briefly, 300 mm stocks of BeSO4 and NaF were
freshly prepared in Mg2+-free ATPase buffer. NaF was
added to the ATPase buffer to a final concentration of
10 mm. Increasing concentrations of BeSO4 were added
immediately before addition of protein. To avoid the formation of MgF2, free Mg2+ was kept at  100 lm.

Data analysis
Experimental repeats (n) refer to separate protein preparations. Data points from each preparation were done in
duplicate. Values are given as mean ± SEM.

3542

The Michaelis–Menten equation was fitted to concentration–activity relationships and used to obtain the Km.
All activities were expressed as Vmax (nmol Pi releasedỈ
min)1Ỉmg)1 protein) and as maximal turnover rate (nmol Pi
releasedỈs)1Ỉnmol)1 protein). The latter is more appropriate
for direct comparison of enzyme activity of our constructs,
as it takes into account the large differences in protein
size (SUR1 ¼ 181 kDa; MBP–NBD1 ¼ 87 kDa; MBP–
NBD2 ¼ 74 kDa). We also report Vmax to enable direct

comparison with the literature on other ABC proteins.
The IC50 values for MgADP and BeF inhibition were
calculated by fitting the data to the Langmuir equation:
y ẳ B ỵ 1=ẵ1 ỵ ẵI=IC50 ịị
where y is the ATP hydrolysis rate, IC50 is the concentration of inhibitor I at half-maximal inhibition, and B is the
level of remaining ATPase activity at maximal inhibition
(where B ¼ 0 for complete inhibition). Ki values were then
calculated from the IC50 using the equation for competitive
inhibition of Cheng & Prusoff [31]:

Ki ẳ IC50 =1 ỵ ẵẵATP=Km ATPịị
As there was no signicant difference in Km between
NBD1, NBD2 and the NBD1 + NBD2 mixture, these data
were pooled and the mean Km (0.60 ± 0.06, n ¼ 36) was
used to calculate the Ki values of these proteins.

Acknowledgements
We thank the Wellcome Trust (F. M. Ashcroft and
´
C. Venien-Bryan), the EU (F. M. Ashcroft, EuroDiaLSHM-CT-2006-518153) and Servier (F. M. Ashcroft)
for support. F. M. Ashcroft is a Royal Society
Research Professor. T. J. Craig and H. de Wet are
Wellcome Trust Training Fellows. T. J. Craig, M. Dreger and H. de Wet are supported by the Wellcome
Trust OXION Initiative in Ion Channels and Disease.

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FEBS Journal 274 (2007) 3532–3544 ª 2007 The Authors Journal compilation ª 2007 FEBS