The ATPase activities of sulfonylurea receptor 2A and
sulfonylurea receptor 2B are influenced by the C-terminal
42 amino acids
Heidi de Wet, Constantina Fotinou, Nawaz Amad, Matthias Dreger and Frances M. Ashcroft
Department of Physiology, Anatomy and Genetics, University of Oxford, UK
Introduction
ATP-sensitive potassium channels (K
ATP
channels) link
the metabolic state of the cell to its electrical excitabil-
ity [1]. They are involved in the response to cardiac
stress, ischemic preconditioning, vascular smooth mus-
cle tone, skeletal muscle glucose uptake, neuronal
excitability, transmitter release, and insulin secretion
from pancreatic b-cells [2].
The pore of the K
ATP
channel consists of four Kir6.2
subunits, each of which is associated with a regulatory
sulfonylurea receptor (SUR) subunit. There are several
types of the latter: SUR1 in b-cells and neurons, SUR2A
in cardiac and skeletal muscle, and SUR2B in smooth
muscle and some neurons [1]. SUR2A and SUR2B are
encoded by splice variants of a single gene, ABCC9, and
differ only in their C-terminal 42 amino acids.
ATP blocks K
ATP
channel activity by binding to
Kir6.2, whereas the SUR subunit endows the channel
with sensitivity to inhibition by sulfonylurea drugs and
to the stimulatory actions of MgADP and the K

SUR2B was less active than that of SUR2A. We further found that the
NBDs of SUR2B interact, and that the activity of full-length SUR cannot
be predicted from that of either the isolated NBDs or NBD mixtures.
Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted
in ATPase activity resembling that of NBD2 of SUR2A rather than that of
NBD2 of SUR2B: this might indicate that these amino acids are responsi-
ble for the lower ATPase activity of SUR2B and the isolated NBD2 of
SUR2B. We suggest that the lower ATPase activity of SUR2B may result
in enhanced duration of the MgADP-bound state, leading to channel
activation.
Abbreviations
ABC, ATP-binding cassette; AMP-PCP, Adenylyl(b,c-methylene)diphosphonate; DDM, dodecylmaltoside; K
ATP
channel, ATP-sensitive
potassium channel; MBP, maltose-binding protein; MRP1, multidrug resistance protein 1; NBD, nucleotide-binding domain; SUR,
sulfonylurea receptor, TMD, transmembrane domain.
2654 FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS
domains (TMDs) and two intracellular nucleotide-bind-
ing domains (NBDs). It is thought that, as in other
ATP-binding cassette (ABC) proteins [4], the NBDs of
SUR associate in a head-to-tail conformation to form
two dimeric nucleotide-binding sites (site 1 and site 2)
that comprise the Walker A and Walker B motifs of
one NBD and the linker domain of the other.
In the absence of Mg
2+
, there is little difference in
ATP block of Kir6.2 ⁄ SUR2A and Kir6.2 ⁄ SUR2B
channels [5], indicating that SUR2A and SUR2B
do not differentially influence ATP binding to Kir6.2.

Figure 1A shows SDS ⁄ PAGE analysis of purified
fusion proteins consisting of maltose-binding protein
(MBP) linked at its C-terminus to one of the NBDs of
SUR2 (MBP-NBD fusion proteins). Figure 1B,C shows
SDS/PAGE analysis of purified full-length SUR2A and
SUR2B. MALDI-TOF MS analysis confirmed their
identities. For simplicity, we refer to MBP–NBD fusion
proteins hereafter as NBD1, NBD2A (NBD2 of
SUR2A), NBD2B (NBD2 of SUR2B), and NBD2-DC.
ATP hydrolysis by NBDs
NBD1 and NBD2A displayed higher ATPase activity
than NBD2B (Fig. 2A; Table 1), with NBD1 having
the highest rate. K
m
values were similar for NBD1
(647 lm), NBD2B (792 lm), and NBD2A (529 lm).
The different activities of NBD2A and NBD2B could
result from an inhibitory effect of the C-terminal 42
amino acids of NBD2B or a stimulatory effect of the
equivalent amino acids of NBD2A. To determine
which of these hypotheses is correct, we generated a
truncated NBD2 construct, NBD2-DC, which lacked
the last 42 amino acids. Figure 2A and Table 1 show
that the ATPase activity of NBD2-DC was greater
than that of SUR2B but similar to that of NBD2A,
favoring the idea that the last 42 amino acids of
NBD2B reduce its catalytic activity. The K
m
value was
the lowest of all the isolated NBDs (336 lm).

C
SUR2B
21
260
160
110
80
60
50
30
20
40
175.5
kDa
62 kDa
100
50
37
25
20
15
10
75
250
150
56
Fig. 1. Protein purification. Coomassie-stained denaturing gels of
purified MBP–NBDs (A), full-length SUR2A (B) and SUR2B (C).
Numbers adjacent to the gel indicate the molecular masses (kDa).
(A) Lanes: 1, NBD2A; 2, NBD2B; 3, NBD1; 4, molecular mass mark-

and 2.3 · 10
)3
s
)1
, and K
m
val-
ues of 373 and 38 lm, respectively (Fig. 3; Table 1).
No ATPase activity was detected in the absence of
Mg
2+
(Fig. 3A). The activities of SUR2A and SUR2B
were approximately fourfold and 10-fold lower, respec-
tively, than that previously reported for SUR1 (k
cat
of
26.3 · 10
)3
s
)1
[9]), and also lower than that of a mix-
ture of the respective NBDs. However, they were only
three-fold less active than their respective NBD2s. The
difference in ATPase activity between full-length
SUR2A and SUR2B and their isolated NBDs is not a
consequence of the detergent [0.2% dodecylmaltoside
(DDM)] and lipid [0.05% 1,2-dimyristoyl-sn-glycero-
phosphocholine (DMPC)] associated with the full-
length proteins, as this was without effect on the
activity of either isolated SUR2A or SUR2-DC (data

20
25
30
[ATP] (mM)
0.01 0.1 1 10
0
5
10
15
20
25
30
35
nmol P
i
·min
–1
·mg
–1
nmol P
i
·min
–1
·mg
–1
1
2ΔC
2A
2B
Fig. 2. ATPase activity of the NBDs. (A) ATPase activities of NBD1 (1,

**P < 0.005 against NBD1.
Construct
Turnover
rate
(s
)1
· 10
)3
)
V
max
(nmol
P
i
Æmin
)1
Æmg
)1
) K
m
(lM) n
NBD1 33.8 ± 2.4 30.8 ± 2.2 647 ± 110 5
NBD2A 19.3 ± 3.0 21.2 ± 2.8 529 ± 170 7
NBD2B 6.1 ± 1.5** 6.7 ± 1.6 792 ± 151 7
NBD2-DC 24.5 ± 4.1 29.5 ± 4.4 336 ± 30 3
NBD1 + NBD2A 27.0 ± 3.3 27.1 ± 3.3 941 ± 174 4
NBD1 + NBD2B 25.2 ± 3.2* 24.6 ± 3.0 880 ± 308 4
Average for NBD1
and NBD2B
14.0 ± 4.4 15.3 ± 4.8 528 ± 180 4

0.5
1.0
1.5
2.0
2.5
3.0
3.5
nmol P
i
·min
–1
·mg
–1
nmol P
i
·min
–1
·mg
–1
nmol P
i
·min
–1
·mg
–1
Fig. 3. ATPase activity of SUR2. (A) ATPase
activity of purified SUR2A in the presence
(
•
, n = 4) or absence (s, n =1)ofMg

max
of
0.73nmol P
i
ÆminÆmg
)1
and an offset of
0.05 nmol P
i
ÆminÆmg
)1
for SUR2B.
A
[ADP] (mM)
0.01 0.1 1 10
Fractional activity
0.0
0.4
0.8
1.2
0.2
0.6
1.0
B
[ADP] (mM)
0.01 0.1 1 10
Fractional activity
0.0
0.4
0.8

without (gray bars) 3 m
M MgADP (n = 3).
Data are expressed as a fraction of the
turnover rate in the absence of inhibitor.
(A, B) The lines are fitted to Eqn (1), and K
i
values were calculated using Eqn (2).
H. de Wet et al. ATPase activity of SUR2A and SUR2B
FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS 2657
Beryllium fluoride inhibited the ATPase activity of
NBD1, NBD2A and NBD2B with a K
i
of  25 lm
(Fig. 5A; Table 2). Mixing NBD1 with either NBD2A
or NBD2B did not alter the K
i
(Fig. 5B; Table 2).
Discussion
ATP hydrolysis by the NBDs
Previous studies of ATP hydrolysis by the NBDs of
SUR2A have yielded a K
m
of 220 lm for NBD1 [11]
and K
m
values ranging from 370 lm [11] to 4.4 mm
[12] for NBD2A. The values that we obtained for the
isolated NBDs lie within this range (647 lm for
NBD1, and 529 lm for NBD2A).
The rate of ATP hydrolysis of NBD1 was greater

Æmin
)1
per mg protein) [13].
Mixing NBD1 and NBD2 of SUR2A did not alter
ATPase activity, as found for SUR1 [9] and MRP1
[10], but in contrast to a previous study of the NBDs
of SUR2A [8]. This may also reflect construct differ-
ences: our NBD1 is 31 amino acids longer at the
N-terminus, and our NBD2A is 26 amino acids shorter
at the N-terminus, than those of Park et al. [8].
To our knowledge, this is the first time that the
activity of NBD2B or full-length SUR2B has been
reported. Consistent with the fact that full-length
SUR2B has a lower turnover rate than SUR2A,
NBD2B displayed the slowest hydrolytic rate of the
isolated NBDs (k
cat
of 6 · 10
)3
s
)1
, more than three-
fold lower than either NBD1, NBD2A, or NBD2-DC).
The ATPase activity of NBD2-DC, which lacks the
C-terminal 42 amino acids (i.e. Lys1333–Val1502), was
30 nmol P
i
Æmin
)1
per mg protein, within the range of

fluoride (lM) n
NBD1 443 ± 107 3 26.0 ± 4.6 4
NBD2A 368 ± 109 3 25.8 ± 3.3 4
NBD2B 305 ± 52 3 28.3 ± 5.7 4
NBD1 + NBD2A 370 ± 138 3 23.9 ± 1.4 4
NBD1 + NBD2B 352 ± 106 3 22.7 ± 2.3 4
SUR2A No inhibition 3 ND
SUR2B No inhibition 3 ND
A
[Beryllium fluoride] (mM)
0.01 0.1 1 10
Fractional activity
0.0
0.4
0.8
1.2
B
[Beryllium fluoride] (mM)
0.01 0.1 1 10
Fractional activity
0.0
0.4
0.8
1.2
Fig. 5. Inhibition by beryllium fluoride. (A)
Inhibition of ATPase activity at 1 m
M MgATP
by beryllium fluoride for NBD1 (
, n = 4),
NBD2A (s, n = 3), and NBD2B (

per mg protein) and SUR2B (0.8
nmol P
i
Æmin
)1
per mg protein) are significantly less
than that of SUR1 (9 nmol P
i
Æmin
)1
per mg protein)
[9]. They are also less than those of the cystic fibrosis
transmembrane conductance regulator (60 nmol
P
i
Æmin
)1
per mg protein [17]) and MRP1 (5–470 nmol
P
i
Æmin
)1
Æmg
)1
[18,19]), two other members of the
ABCC subfamily. However, the ATPase activity is not
dissimilar from that found for ABCR (1.3 nmol
P
i
Æmin

the catalytic site. Thus, these amino acids may interact
with the NBDs to modulate binding affinity. This
interaction appears to require the TMDs of SUR2, as
the K
m
values of NBD2 and the NBD1 + NBD2B
mixture are much greater than that of full-length
SUR2B.
Effects of inhibitors
MgADP inhibited ATP hydrolysis by the isolated
NBDs, albeit with low affinity (K
i
of 0.3–0.4 mm), as
reported for NBD2 of SUR2A [14]. In contrast,
MgADP did not block ATP hydrolysis by full-length
SUR2A or SUR2B; similar results were found for
SUR1 [9]. A possible explanation is that the ADP
affinity of the full-length proteins is much lower than
that of the isolated NBDs. However, the lack of
MgADP inhibition must somehow be ameliorated in
the K
ATP
channel complex, because MgADP is able to
stimulate channel activity and reverse channel inhibi-
tion by ATP via interaction with the NBDs of SUR2
[12]. Furthermore, MgADP is able to displace azido-
[
32
P]ATP[aP] binding to NBD1 and NBD2 of full-
length SUR2A and SUR2B [22]: the K

2+
. This suggests that
MgATP activation of Kir6.2 ⁄ SUR2A is less than that
of Kir6.2 ⁄ SUR2B [23]. In support of this idea, if K
ATP
channels are preblocked with AMP-PCP, then GTP
(at concentrations that do not interact with Kir6.2)
activates SUR2B-containing channels but blocks
Kir6.2 ⁄ SUR2A channels [5].
It has been proposed that the reduced ability of
MgATP to stimulate Kir6.2 ⁄ SUR2A channels results
from SUR2A being less efficient at hydrolyzing
MgATP than SUR2B [6]. In direct opposition to this
idea, we found that SUR2B hydrolyzes ATP much less
vigorously than SUR2A. We cannot exclude the possi-
bility that the opposite is true when Kir6.2 is present.
However, an alternative explanation is afforded by
previous studies showing that mutations at site 2 that
reduce the ATPase activity of SUR1 can lead to
enhanced activation of Kir6.2 ⁄ SUR1 channels by
MgATP [24].
We speculate that the lower rate of ATP hydrolysis
by SUR2B is associated with prolonged occupancy of
H. de Wet et al. ATPase activity of SUR2A and SUR2B
FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS 2659
site 2 of SUR2B by MgADP. This would lead to
enhanced activation of Kir6.2 ⁄ SUR2B channels and a
reduced turnover rate. Consistent with the idea that
NBD2 of SUR2B remains in the MgADP-bound, acti-
vated state for longer, MgATP first blocks Kir6.2 ⁄ -

(Sigma, Poole, UK). The wash buffer was 150 mm NaCl
and 50 mm Tris ⁄ HCl (pH 8.8), supplemented with 0.2%
(w ⁄ v) DDM and 0.05% (w ⁄ v) DMPC. The elution buffer
was the same as the wash buffer plus 100 lm 3-FLAG pep-
tide. Purified protein averaged 50 lgÆL
)1
. Protein identity
and purity were confirmed by MALDI-TOF MS. All assays
were performed on freshly prepared protein.
Rat SUR2 NBDs were cloned into the pMAL-c2X vector
(New England Biolabs, Hitchin, UK) to yield MBP fusion
constructs. The sequences used were Gln635–Glu889 for
NBD1, Lys1333–Lys1545 for NBD2A, Lys1333–Met1545
for NBD2B, and Lys1333–Val1502 for NBD2-DC. Plasmids
were transformed into BL21-CodonPlus Escherichia coli
cells (Stratagene, La Jolla, CA, USA). Protein expression
and purification were carried out as described previously
for the NBDs of SUR1 [9], but without a gel filtration step.
Briefly, BL21-CodonPlus E. coli cells expressing MBP–
NBDs were lysed under pressure in 150 mm NaCl, 50 mm
Tris ⁄ HCl (pH 7.5), and 10% glycerol. Insoluble protein
and debris were removed by centrifugation at 48 400 g for
30 min. The supernatant was mixed with amylose resin for
1 h at 4 °C (New England Biolabs), washed, and eluted in
the presence of 10 mm maltose. Wash and elution buffers
contained 150 mm NaCl, 50 mm Tris ⁄ HCl (pH 7.5) and
20% glycerol to promote protein stability, but no deter-
gents or lipids. Protein identity and purity were confirmed
by MALDI-TOF MS. Yields were typically  3mgÆL
)1

NBD2 (w ⁄ w) were mixed and allowed to interact on ice for
45 min prior to the hydrolysis assay.
To control for contaminating P
i
in commercial ATP prepa-
rations, we included negative controls for each experimental
condition, in which the protein was denatured by 5% SDS
(final concentration) prior to the hydrolysis assay. Absor-
bance from denatured controls was subtracted from the
equivalent experimental values. The maximal concentration
of MgNTP that could be used without gross interference
from contaminating P
i
was 3 mm. We used the sodium salt of
ATP and the potassium salt of MgADP. ATP and ADP were
from Sigma and of ‡ 99% purity. Beryllium fluoride was
prepared as previously described [9].
Data analysis
Experimental repeats (n) refer to separate protein prepara-
tions. Data points from each preparation were obtained in
duplicate. Values are given as mean ± standard error of
the mean. Significance was tested with Student’s t-test.
The Michaelis–Menten equation was fitted to concentra-
tion–activity relationships to obtain the K
m
. All activities
were expressed as V
max
(nmol P
i

50
values by using the equation for competitive
inhibition of Chen and Prusoff (1973) [25]:
K
i
¼
IC
50
1 þ
½ATP
K
m
ðATPÞ

ð2Þ
Acknowledgement
This work was supported by the Wellcome Trust, the
Royal Society and the European Union (EDICT:
201924).
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