The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids

Article abstract of DOI:10.1111/j.1742-4658.2010.07675.x

Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K _m for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of 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 responsible 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.

Full text of DOI:10.1111/j.1742-4658.2010.07675.x

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
Keywords
Unusually among ATP-binding cassette proteins, the sulfonylurea receptor
(SUR) acts as a channel regulator. ATP-sensitive potassium channels are
ATP-binding cassette transporter; KATP
channel; sulfonylurea receptor; SUR2A;
octameric complexes composed of four pore-forming Kir6.2 subunits and
four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8)
and SUR2 (ABCC9), with the latter being differentially spliced to give
SUR2B
Correspondence
SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids.
ATP-sensitive potassium channels containing these different SUR2 iso-
forms are differentially modulated by MgATP, with Kir6.2 ⁄ SUR2B being
activated more than Kir6.2 ⁄ SUR2A. We show here that purified SUR2B
has a lower ATPase activity and a 10-fold lower Km for MgATP than
SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of
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.
F. M. Ashcroft, Department of Physiology,
Anatomy and Genetics, Parks Road, Oxford,
OX1 3PT, UK
Fax: +44 1865 285812
Tel: +44 1865 285810
E-mail: frances.ashcroft@dpag.ox.ac.uk
(Received 23 January 2010, revised 26
March 2010, accepted 8 April 2010)
doi:10.1111/j.1742-4658.2010.07675.x
Introduction
ATP-sensitive potassium channels (KATP 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 KATP 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 KATP 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 KATP
channel openers [1,3]. SUR has multiple transmembrane
Abbreviations
ABC, ATP-binding cassette; AMP-PCP, Adenylyl(b,c-methylene)diphosphonate; DDM, dodecylmaltoside; KATP channel, ATP-sensitive
potassium channel; MBP, maltose-binding protein; MRP1, multidrug resistance protein 1; NBD, nucleotide-binding domain; SUR,
sulfonylurea receptor, TMD, transmembrane domain.
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H. de Wet et al.
ATPase activity of SUR2A and SUR2B
A
MBP–SUR2 NBDs
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.
5
6
1
2
3
4
250
150
250
150
100
75
100
75
68 kDa
66 kDa
62 kDa
50
37
50
37
In the absence of Mg2+, 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.
In the presence of Mg2+, however, ATP inhibits
Kir6.2 ⁄ SUR2B less than Kir6.2 ⁄ SUR2A [5]. This sug-
gests MgATP has a greater stimulatory action on
Kir6.2 ⁄ SUR2B than on Kir6.2 ⁄ SUR2A, leading to an
apparent reduction in ATP inhibition. In support of
this idea, when an ATP-insensitive Kir6.2 mutation
was used to remove the effects of ATP on Kir6.2,
MgATP activated KATP channels containing SUR2B
subunits but blocked those composed of SUR2A [6].
The current consensus is that channel opening is
enhanced by MgADP occupation of site 2 and that acti-
vation by MgATP requires its hydrolysis to MgADP. At
least in the case of SUR2, the prehydrolytic state does
not promote channel opening [7]. Because MgADP acti-
vates Kir6.2 ⁄ SUR2B and Kir6.2 ⁄ SUR2A to similar
extents [5], it appears that they bind MgADP with
similar affinities and transduce this binding into channel
opening with similar efficacies. This has led to the
proposal that ability of MgATP to stimulate the activity
of Kir6.2 ⁄ SUR2B channels more than Kir6.2.SUR2A
channels is attributable to greater ATP hydrolysis by
SUR2B than by SUR2A [6]. In this study, we tested this
hypothesis explicitly, by measuring the ATPase activity
of full-length SUR2A and SUR2B, and that of their
isolated NBDs.
25
20
25
15
20
15
10
10
B
SUR2A
1
C
SUR2B
2
1
2
260
175.5
kDa
190
160
179 kDa
110
80
60
50
40
82
40
31
16
30
20
7
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-
ers; 5, SUR2-DC; 6, molecular mass markers. (B) Lanes: 1, SUR2A;
2, molecular mass markers. (C) Lanes: 1, molecular mass markers;
2, SUR2B. Samples are purified eluates from affinity resins without
further purification (A) or eluates from the gel filtration column (B, C).
the highest rate. Km 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 Km value was
the lowest of all the isolated NBDs (336 lm).
Results
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.
We next examined ATP hydrolysis in a 1 : 1 mixture
of NBD1 and either NBD2A or NBD2B (Fig. 2B).
The estimated maximal turnover rate was similar in
both cases. For the NBD1 + NBD2A mixture, kcat
ATP hydrolysis by NBDs
NBD1 and NBD2A displayed higher ATPase activity
than NBD2B (Fig. 2A; Table 1), with NBD1 having
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ATPase activity of SUR2A and SUR2B
H. de Wet et al.
35
30
25
20
15
10
5
A
B
1
30
25
20
15
10
5
2ΔC
2A
2B
10
0
0
0.01
0.1
1
0.01
0.1
1
10
[ATP] (mM)
[ATP] (mM)
Fig. 2. ATPase activity of the NBDs. (A) ATPase activities of NBD1 (1, , n = 5), NBD2-DC (2DC, 4, n = 3), NBD2A (2A, , n = 7)
•
and NBD2B (2B, s, n = 7). The lines are fitted to the Michaelis–Menten equation with estimated Vmax values of 37, 26, 8 and
31 nmol PiÆminÆmg)1, and Km values of 769, 556, 882 and 340 lM, respectively. (B) ATPase activity of a mixture of NBD1 and either
NBD2A (s, n = 4) or NBD2B ( , n = 4). The solid lines are fitted to the Michaelis–Menten equation with estimated Km values of 995
•
and 878 lM, and Vmax values of 31 and 27 nmol PiÆmin)1Æmg)1 protein, respectively. The dashed line is the average of the ATPase activities
of NBD1 and NBD2B.
rates of 6.1 · 10)3 s)1 and 2.3 · 10)3 s)1, and Km val-
ues of 373 and 38 lm, respectively (Fig. 3; Table 1).
No ATPase activity was detected in the absence of
Mg2+ (Fig. 3A). The activities of SUR2A and SUR2B
Table 1. ATPase activities and kinetic constants. n, number of
preparations. *P < 0.01 against the average for NBD1 + NBD2B.
**P < 0.005 against NBD1.
Turnover
were approximately fourfold and 10-fold lower, respec-
tively, than that previously reported for SUR1 (kcat 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
not shown).
rate
(s)1 · 10)3
Vmax (nmol
PiÆmin)1Æmg)1
Construct
)
)
Km (lM)
n
NBD1
33.8 2.4
19.3 3.0
30.8 2.2
21.2 2.8
6.7 1.6
647 110
529 170
792 151
336 30
5
7
7
3
4
4
4
NBD2A
NBD2B
6.1 1.5**
24.5 4.1
27.0 3.3
25.2 3.2*
NBD2-DC
NBD1 + NBD2A
NBD1 + NBD2B
29.5 4.4
27.1 3.3
24.6 3.0
15.3 4.8
941 174
880 308
528 180
Average for NBD1 14.0 4.4
and NBD2B
SUR2A
SUR2B
6.1 2.3
2.3 0.3
2.6 0.8
0.8 0.1
373 93
38 11
4
3
was intermediate between that of the individual NBDs,
and the Km was not significantly different from that for
either NBD alone (Fig. 2B; Table 1). This differs from
previous observations on SUR2A [8], but is in agree-
ment with studies of SUR1 [9] and multidrug resistance
protein 1 (MRP1) [10], where mixing the NBDs did not
have a major impact on their catalytic activity.
SUR2B showed a 10-fold lower Km than SUR2A,
suggesting that it binds ATP more tightly than
SUR2A. The Km values of all four isolated NBDs were
significantly larger than that of SUR2B.
Inhibition of ATP hydrolysis by MgADP and
beryllium fluoride
In contrast, the maximal turnover rate of the
NBD1 + NBD2B mixture was very different from the
average of the activities of NBD1 and NBD2B (Fig. 2B;
Table 1), suggesting that these NBDs interact. However,
the Km remained unchanged, at ꢀ 1 mm ATP.
MgADP inhibited ATP hydrolysis by NBD1, NBD2A
and NBD2B with a Ki of 305–443 lm (Fig. 4A;
Table 2). Inhibition was unchanged by mixing NBD1
and NBD2 (Fig. 4B; Table 2). In contrast to those of
the isolated NBDs, the ATPase activities of full-length
SUR2A and SUR2B were unaffected by 3 mm
MgADP (Fig. 4C).
ATP hydrolysis by SUR2A and SUR2B
We next examined the ATPase activity of the full-
length proteins. Recombinant SUR2A and SUR2B
hydrolyzed MgATP slowly, with maximal turnover
Beryllium fluoride is a potent inhibitor of ATP hydro-
lysis of many ABC proteins that acts by arresting
the ATPase cycle in the prehydrolytic conformation.
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H. de Wet et al.
ATPase activity of SUR2A and SUR2B
A
B
SUR2A
SUR2B
1.2
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.001 0.01
0.1
1
10
0.001
0.01
0.1
1
10
[ATP] (mM)
[ATP] (mM)
Fig. 3. ATPase activity of SUR2. (A) ATPase
C
activity of purified SUR2A in the presence
, n = 4) or absence (s, n = 1) of Mg2+
.
3.5
(
•
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(B) ATPase activity of purified SUR2B in
the presence ( , n = 3) or absence (s,
•
n = 1) of 10 mM Mg2+. (C) ATPase activi-
ties of SUR2A and SUR2B plotted on the
same scale. The lines are fitted to the
Michaelis–Menten equation using a Km of
460 lM, a Vmax of 2.52 nmol PiÆminÆmg)1
and an offset of 0.1 nmol PiÆminÆmg)1 for
SUR2A, and a Km of 41 lM, a Vmax of
0.73nmol PiÆminÆmg)1 and an offset of
0.05 nmol PiÆminÆmg)1 for SUR2B.
0.001
0.01
0.1
[ATP] (mM)
1
10
A
B
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.01
0.1
1
10
0.01
0.1
1
10
[ADP] (mM)
[ADP] (mM)
Fig. 4. Inhibition by MgADP. (A, B) Inhibi-
tion of ATPase activity at 1 mM MgATP by
ADP for (A) NBD1 ( , n = 4), NBD2A
C
1.8
1.4
1.0
0.6
0.2
(s, n = 3), and NBD2B ( , n = 3), and (B)
•
for a mixture of NBD1 and either NBD2A
ATP
ATP + ADP
(s, n = 3) or NBD2B ( , n = 3). (C)
•
ATPase activities of SUR2A and SUR2B
at 1 mM MgATP with (white bars) or
without (gray bars) 3 mM 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 Ki
values were calculated using Eqn (2).
SUR2A
SUR2B
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ATPase activity of SUR2A and SUR2B
H. de Wet et al.
Table 2. Inhibition by ADP and beryllium fluoride. n, number of
hydrolytic activity of our NBD2A (Vmax of 21 nmol -
PiÆmin)1 per mg protein) was also greater than previ-
ously reported (10 nmol PiÆmin)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].
preparations; ND, not determined.
Beryllium
Construct
ADP (lM)
n
fluoride (lM)
n
NBD1
443 107
368 109
305 52
3
3
3
3
3
3
3
26.0 4.6
25.8 3.3
28.3 5.7
23.9 1.4
22.7 2.3
ND
4
4
4
4
4
NBD2A
NBD2B
NBD1 + NBD2A
NBD1 + NBD2B
SUR2A
370 138
352 106
No inhibition
No inhibition
SUR2B
ND
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 (kcat 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 PiÆmin)1 per mg protein, within the range of
Beryllium fluoride inhibited the ATPase activity of
NBD1, NBD2A and NBD2B with a Ki of ꢀ 25 lm
(Fig. 5A; Table 2). Mixing NBD1 with either NBD2A
or NBD2B did not alter the Ki (Fig. 5B; Table 2).
Discussion
that previously reported for
a similar construct
ATP hydrolysis by the NBDs
(Gly1306–Thr1498) (in nmol PiÆmin)1 per mg protein,
11 [11], 18 [12], or 78 [14]). Importantly, the kcat was
greater than that of NBD2B but similar to that of
NBD2A. This suggests that the final 42 amino acids of
SUR2B may reduce its hydrolytic activity, and that
the catalytic activity of SUR2A is not measurably
affected by its final 42 amino acids.
Previous studies of ATP hydrolysis by the NBDs of
SUR2A have yielded a Km of 220 lm for NBD1 [11]
and Km 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
than that reported previously, the Vmax being 31
nmol PiÆmin)1 per mg protein as compared with earlier
values of 6–9 nmol PiÆmin)1 per mg protein [8,11,12].
These differences may be attributable to the amino
acids used for the various constructs: Gly635–Gly889
in this study, as compared with Ser684–Ser884 [11,12]
and Asp666–Glu890 [8] in previous work. Alterna-
tively, it might result from the different techniques that
were used to estimate protein concentration, or from
differences in the assay conditions. Likewise, the
In contrast to what was found for SUR2A, mixing
NBD1 and NBD2 of SUR2B enhanced ATPase activ-
ity (above the average of the individual NBDs), indi-
cating that the NBDs must interact, and emphasizing
the functional importance of the last 42 amino acids of
SUR2. One possibility is that interaction of the hetero-
dimer produces a conformational change that physi-
cally reduces the inhibitory effect of the last 42 amino
acids of SUR2B on ATPase activity. Presumably, this
conformational change is prevented by the presence of
the TMDs, as the activity of full-length SUR2B is
A
B 1.2
1.2
0.8
Fig. 5. Inhibition by beryllium fluoride. (A)
Inhibition of ATPase activity at 1 mM MgATP
by beryllium fluoride for NBD1 ( , n = 4),
0.8
0.4
0.0
NBD2A (s, n = 3), and NBD2B ( , n = 3).
•
(B) Inhibition of ATPase activity at 1 mM
MgATP by beryllium fluoride for a mixture
of NBD1 and either NBD2A (s, n = 3) or
0.4
0.0
NBD2B ( , n = 3). Data are expressed as
•
a fraction of the turnover rate in the
absence of inhibitor. Lines are fitted to
0.01
0.1
1
10
0.01
0.1
1
10
Eqn (1), and Ki values were calculated
[Beryllium fluoride] (mM)
[Beryllium fluoride] (mM)
using Eqn (2).
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H. de Wet et al.
ATPase activity of SUR2A and SUR2B
fourfold less than that of SUR2A. There is an increas-
ing body of evidence that suggests that isolated NBDs,
which are presumably free from the conformational
constraints imposed by their TMDs, behave very dif-
ferently from their full-length cousins, and our data
give further support for this idea [15,16].
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 KATP 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-
[32P]ATP[aP] binding to NBD1 and NBD2 of full-
length SUR2A and SUR2B [22]: the Ki for MgADP
previously measured for NBD2B (70 lm) was lower
than that found for the isolated NBD mixture
(350 lm), but that for NBD2A was not significantly
different.
ATP hydrolysis by full-length SUR2A and SUR2B
The ATPase activities of purified SUR2A (Vmax of
3 nmol PiÆmin)1 per mg protein) and SUR2B (0.8
nmol PiÆmin)1 per mg protein) are significantly less
than that of SUR1 (9 nmol PiÆmin)1 per mg protein)
[9]. They are also less than those of the cystic fibrosis
transmembrane conductance regulator (60 nmol
PiÆmin)1 per mg protein [17]) and MRP1 (5–470 nmol
PiÆmin)1Æmg)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
PiÆmin)1 per mg protein [20]). The lower ATPase activ-
ities of the various SURs may be related to their role
as channel regulators, rather than transporters. It is
also possible that ATP hydrolysis is enhanced when
SUR2A and SUR2B are coexpressed with Kir6.2, as is
found for SUR1 [9,21].
Implications for channel gating
Unlike other ABC proteins, SUR2 serves as a channel
regulator, and ATP hydrolysis by SUR2 plays a key role
in the metabolic regulation of the KATP channel.
Current evidence suggests that the presence of MgADP
at NBD2 results in KATP channel opening, and that
MgATP must be hydrolyzed to MgADP in order for
channel activation to occur [7]. Consistent with the fact
that the Ki for MgADP inhibition of ATPase activity is
similar for NBD2A and NBD2B, Kir6.2 ⁄ SUR2A and
Kir6.2 ⁄ SUR2B are activated by MgADP to about the
same extent [5].
As previously reported for SUR1 [9], the Km values
for ATP hydrolysis by SUR2A and SUR2B were lower
than those measured for the isolated NBDs. This sug-
gests that the TMDs induce conformational changes in
the NBDs, or in their association, that influence nucle-
otide handling.
The IC50 for MgATP inhibition of Kir6.2 ⁄ SUR2A
currents is less than that for Kir6.2 ⁄ SUR2B [23]. In
contrast, ATP blocks via both channels to a similar
extent in the absence of Mg2+. This suggests that
MgATP activation of Kir6.2 ⁄ SUR2A is less than that
of Kir6.2 ⁄ SUR2B [23]. In support of this idea, if KATP
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].
The Km for MgATP was substantially lower for
SUR2B (38 lm) than for SUR2A (400 lm) or SUR1
(100 lm [9]), suggesting that SUR2B binds MgATP
more tightly. This is in agreement with a previous
report that the Ki values for ATP inhibition of
8-azido-[32P]ATP[aP] binding to NBD1 and NBD2 of
native SUR2B were lower than those for the NBDs
of SUR2A [22]. SUR2A and SUR2B differ only in
their last 42 amino acids, which do not form part of
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 Km 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 (Ki of 0.3–0.4 mm), as
reported for NBD2 of SUR2A [14]. In contrast,
MgADP did not block ATP hydrolysis by full-length
We speculate that the lower rate of ATP hydrolysis
by SUR2B is associated with prolonged occupancy of
FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS
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ATPase activity of SUR2A and SUR2B
H. de Wet et al.
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 ꢀ 3 mgÆL)1 for
all NBDs, and comprised > 95% of total purified protein.
Proteins were separated on 4–12% gradient Bis ⁄ Tris gels,
and visualized by Coomassie staining (Invitrogen, Paisley,
UK).
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 ⁄-
SUR2B channels and then current slowly increases, as
though channels slowly accumulate in the MgADP-
bound activated state [5]. MgATP was also more effec-
tive at slowing the off-rate of KATP channel openers
on Kir6.2 ⁄ SUR2B than Kir6.2 ⁄ SUR2A, which might
also reflect longer occupancy of site 2 by MgADP [5].
We therefore conclude that the lower ATP hydroly-
sis rate of SUR2B is associated with longer occupancy
of the MgADP-bound activated state and thus
increased channel activation.
Nucleotide hydrolysis
ATPase activities were measured as described for SUR1
and SUR1 NBDs [9]. The ATPase activity of SUR2B was
measured using a protein concentration of > 1 mgÆmL)1 to
ensure a robust signal above background; that of SUR2A
Experimental procedures
was measured at 0.2–0.5 mgÆmL)1
.
Selwyn’s control test showed that Pi release for MBP–
NBDs was linear over the time course of the assay, and
that the relationship between protein concentration and
activity was linear (Fig. S1). The protein concentrations
Protein expression and purification
A FLAG tag was inserted into rat SUR2 between Ala1026
and Asp1027. Full-length SUR2A and SUR2B were
expressed in insect cells (Sf9), using a baculovirus expres-
sion system (Invitrogen, Paisley, UK), and purified essen-
tially as described for SUR1 [9]. Briefly, protein expression
was verified by [3H]glibenclamide binding to infected Sf9
cells 48 h after infection. Cells were lysed under high pres-
sure, and membranes were purified by a sucrose gradient
(10% ⁄ 46%, w ⁄ v) centrifugation step of 100 000 g for 1 h.
Membranes were then solubilized in 150 mm NaCl and
50 mm Tris ⁄ HCl (pH 8.8), supplemented with 0.5% (w ⁄ v)
DDM, for 20 min at room temperature. Solubilized mem-
branes were bound to anti-FLAG M2 affinity resin,
washed, and eluted with 100 lm 3-FLAG peptide at 4 ꢀC
(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.
)
were 1 lm for beryllium fluoride (BeF3 and BeF42)) inhibi-
tion and 3–10 lm for MgADP inhibition.
In some experiments, equal amounts of NBD1 and
NBD2 (w ⁄ w) were mixed and allowed to interact on ice for
45 min prior to the hydrolysis assay.
To control for contaminating Pi 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 Pi 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
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
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 Km. All activities
were expressed as Vmax (nmol Pi released per min per
mg protein) and as maximal turnover rate (nmol Pi released
per s per nmol Protein) to allow direct comparison between
proteins of different sizes.
IC50 values for MgADP and beryllium fluoride inhibition
were calculated by fitting the Langmuir equation to the
data:
2660
FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. de Wet et al.
ATPase activity of SUR2A and SUR2B
1
10 Ramaen O, Sizun C, Pamlard O, Jacquet E & Lalle-
mand JY (2005) Attempts to characterize the NBD
heterodimer of MRP1: transient complex formation
involves Gly771 of the ABC signature sequence but
does not enhance the intrinsic ATPase activity. Biochem
J 391, 481–490.
ꢀ
ꢁ
y ¼ B þ
ð1Þ
[I]
IC50
1 þ
where y is the ATP hydrolysis rate, IC50 is the concentra-
tion of inhibitor I at half-maximal inhibition, and B is the
ATPase activity remaining at maximal inhibition (where
B = 0 for complete inhibition). Ki values were calculated
from IC50 values by using the equation for competitive
inhibition of Chen and Prusoff (1973) [25]:
11 Masia R, Enkvetchakul D & Nichols CG (2005) Differ-
ential nucleotide regulation of KATP channels by SUR1
and SUR2A. J Mol Cell Cardiol 39, 491–501.
12 Bienengraeber M, Alekseev AE, Abraham MR,
Carrasco AJ, Moreau C, Vivaudou M, Dzeja PP &
Terzic A (2000) ATPase activity of the sulfonylurea
receptor: a catalytic function for the KATP channel
complex. FASEB J 14, 1943–1952.
IC50
ꢀ
ꢁ
Ki ¼
ð2Þ
½ATPꢁ
1 þ
KmðATPÞ
13 Bienengraeber M, Olson TM, Selivanov VA, Kathmann
EC, O’Cochlain F, Gao F, Karger AB, Ballew JD,
Hodgson DM, Zingman LV et al. (2004) ABCC9 muta-
tions identified in human dilated cardiomyopathy dis-
rupt catalytic KATP channel gating. Nat Genet 36,
382–387.
Acknowledgement
This work was supported by the Wellcome Trust, the
Royal Society and the European Union (EDICT:
201924).
14 Zingman LV, Alekseev AE, Bienengraeber M, Hodgson
D, Karger AB, Dzeja PP & Terzic A (2001) Signaling
in channel ⁄ enzyme multimers: ATPase transitions in
SUR module gate ATP-sensitive K+ conductance.
Neuron 31, 233–245.
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Supporting information
The following supplementary material is available:
Fig. S1. Selwyn’s test.
This supplementary material can be found in the
online version of this article.
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this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
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