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 ATP channel openers [1,3]. SUR has multiple transmembrane Keywords ATP-binding cassette transporter; K ATP channel; sulfonylurea receptor; SUR2A; SUR2B Correspondence 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 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 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 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 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. In the presence of Mg 2+ , 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 K ATP 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. 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. 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). 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, k cat 190 MBP–SUR2 NBDs 68 kDa 66 kDa 1234 250 150 100 50 37 25 20 15 10 75 82 40 16 7 31 12 179 kDa B A SUR2A 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- 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). 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 2655 was intermediate between that of the individual NBDs, and the K m 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. 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 K m remained unchanged, at  1mm ATP. 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 rates of 6.1 · 10 )3 s )1 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 not shown). SUR2B showed a 10-fold lower K m than SUR2A, suggesting that it binds ATP more tightly than SUR2A. The K m values of all four isolated NBDs were significantly larger than that of SUR2B. Inhibition of ATP hydrolysis by MgADP and beryllium fluoride MgADP inhibited ATP hydrolysis by NBD1, NBD2A and NBD2B with a K i 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). 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. BA [ATP] (mM) 0.01 0.1 1 10 0 5 10 15 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, • , 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 V max values of 37, 26, 8 and 31 nmol P i ÆminÆmg )1 , and K m 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 K m values of 995 and 878 l M, and V max values of 31 and 27 nmol P i Æmin )1 Æmg )1 protein, respectively. The dashed line is the average of the ATPase activities of NBD1 and NBD2B. 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. 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 SUR2A 6.1 ± 2.3 2.6 ± 0.8 373 ± 93 4 SUR2B 2.3 ± 0.3 0.8 ± 0.1 38 ± 11 3 ATPase activity of SUR2A and SUR2B H. de Wet et al. 2656 FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS SUR2BSUR2A [ATP] (mM) 0.01 0.1 1 10 0.0 1.0 2.0 3.0 0.5 1.5 2.5 3.5 0.001 C AB [ATP] (mM) 0.001 0.01 0.1 1 10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [ATP] (mM) 0.001 0.01 0.1 1 10 0.0 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 2+ . (B) ATPase activity of purified SUR2B in the presence ( • , n = 3) or absence (s, n =1)of10m M Mg 2+ . (C) ATPase activi- ties of SUR2A and SUR2B plotted on the same scale. The lines are fitted to the Michaelis–Menten equation using a K m of 460 l M,aV max of 2.52 nmol P i ÆminÆmg )1 and an offset of 0.1 nmol P i ÆminÆmg )1 for SUR2A, and a K m of 41 lM,aV 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 1.2 0.2 0.6 1.0 C SUR2A 0.2 0.6 1.0 1.4 1.8 Fractional activity SUR2B ATP ATP + ADP Fig. 4. Inhibition by MgADP. (A, B) Inhibi- tion of ATPase activity at 1 m M MgATP by ADP for (A) NBD1 ( , n = 4), NBD2A (s, n = 3), and NBD2B ( • , n = 3), and (B) for a mixture of NBD1 and either NBD2A (s, n = 3) or NBD2B ( • , n = 3). (C) ATPase activities of SUR2A and SUR2B at 1 m M MgATP with (white bars) or 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 than that reported previously, the V max being 31 nmol P i Æmin )1 per mg protein as compared with earlier values of 6–9 nmol P i Æmin )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 hydrolytic activity of our NBD2A (V max of 21 nmol - P i Æmin )1 per mg protein) was also greater than previ- ously reported (10 nmol P i 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 that previously reported for a similar construct (Gly1306–Thr1498) (in nmol P i Æmin )1 per mg protein, 11 [11], 18 [12], or 78 [14]). Importantly, the k cat 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. 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 Table 2. Inhibition by ADP and beryllium fluoride. n, number of preparations; ND, not determined. Construct ADP (l M) n Beryllium 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 ( • , 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 NBD2B ( • , n = 3). Data are expressed as a fraction of the turnover rate in the absence of inhibitor. Lines are fitted to Eqn (1), and K i values were calculated using Eqn (2). ATPase activity of SUR2A and SUR2B H. de Wet et al. 2658 FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS 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]. ATP hydrolysis by full-length SUR2A and SUR2B The ATPase activities of purified SUR2A (V max of 3 nmol P i Æmin )1 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 )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]. As previously reported for SUR1 [9], the K m 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 K m 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 K i values for ATP inhibition of 8-azido-[ 32 P]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 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 i 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. 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 K ATP channel. Current evidence suggests that the presence of MgADP at NBD2 results in K ATP channel opening, and that MgATP must be hydrolyzed to MgADP in order for channel activation to occur [7]. Consistent with the fact that the K i 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]. The IC 50 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 Mg 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 ⁄ - 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 K ATP 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. Experimental procedures 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 [ 3 H]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 l m 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. 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 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). 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 was measured at 0.2–0.5 mgÆmL )1 . Selwyn’s control test showed that P i 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 were 1 lm for beryllium fluoride (BeF 3 ) and BeF 4 2) ) 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 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 released per min per mg protein) and as maximal turnover rate (nmol P i released per s per nmol Protein) to allow direct comparison between proteins of different sizes. IC 50 values for MgADP and beryllium fluoride inhibition were calculated by fitting the Langmuir equation to the data: ATPase activity of SUR2A and SUR2B H. de Wet et al. 2660 FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS y ¼ B þ 1 1 þ [I] IC 50  ð1Þ where y is the ATP hydrolysis rate, IC 50 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). K i values were calculated from IC 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). References 1 Nichols CG (2006) K ATP channels as molecular sensors of cellular metabolism. Nature 440, 470–476. 2 Seino S & Miki T (2003) Physiological and pathophysi- ological roles of ATP-sensitive K + channels. Prog Bio- phys Mol Biol 81, 133–176. 3 Tucker SJ, Gribble FM, Zhao C, Trapp S & Ashcroft FM (1997) Truncation of Kir6.2 produces ATP-sensi- tive K + channels in the absence of the sulphonylurea receptor. Nature 387, 179–183. 4 Oldham ML, Davidson AL & Chen J (2008) Structural insights into ABC transporter mechanism. Curr Opin Struct Biol 18, 726–733. 5 Reimann F, Gribble FM & Ashcroft FM (2000) Differ- ential Response of K ATP channels containing SUR2A or SUR2B subunits to nucleotides and Pinacidil. Mol Pharm 58, 1318–1325. 6 Tammaro P & Ashcroft F (2007) The Kir6.2-F333I mutation differentially modulates K ATP channels com- posed of SUR1 or SUR2 subunits. J Physiol 581, 1259–1269. 7 Zingman LV, Hodgson DM, Bienengraeber M, Karger AB, Kathmann EC, Alekseev AE & Terzic A (2002) Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sen- sitive K + channels. J Biol Chem 277, 14206–14210. 8 Park S, Lim BB, Perez-Terzic C, Mer G & Terzic A (2008) Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines K ATP channel SUR2A catalytic activity. J Proteome Res 7, 1721–1728. 9 de Wet H, Mikhailov MV, Fotinou C, Dreger M, Craig TJ, Venien-Bryan C & Ashcroft FM (2007) Studies of the ATPase activity of the ABC protein SUR1. FEBS J 274, 3532–3544. 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. 11 Masia R, Enkvetchakul D & Nichols CG (2005) Differ- ential nucleotide regulation of K ATP 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 K ATP channel complex. FASEB J 14, 1943–1952. 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. 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. 15 Dietrich D, Schmuths H, De Lousa Marcos C, Baldwin JM, Baldwin SA, Baker A, Theodoulou FL & Holds- worth MJ (2009) Mutations in the Arabidopsis peroxi- somal ABC transporter COMATOSE allow differentiation between multiple functions in plants: insights from an allelic series. Mol Biol Cell 20, 530– 543. 16 De Lousa Marcos C, Dietrich D, Johnson B, Baldwin SA, Holdsworth MJ, Theodoulou FL & Baker A (2009) The NBDs that wouldn’t die: a cautionary tale of the use of isolated nucleotide binding domains of ABC transporters. Commun Integr Biol 2, 97–99. 17 Rosenberg MF, Kamis AB, Aleksandrov LA, Ford RC & Riordan JR (2004) Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR). J Biol Chem 279, 39051–39057. 18 Chang XB, Hou YX & Riordan JR (1998) Stimulation of ATPase activity of purified multidrug resistance-asso- ciated protein by nucleoside diphosphates. J Biol Chem 273, 23844–23848. 19 Mao Q, Leslie EM, Deeley RG & Cole SP (1999) ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells. Biochim Biophys Acta 1461, 69–82. 20 Sun H, Molday RS & Nathans J (1999) Retinal stimu- lates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease. J Biol Chem 274, 8269–8281. 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 2661 21 Mikhailov MV, Campbell JD, de Wet H, Shimomura K, Zadek B, Collins RF, Sansom MS, Ford RC & Ashcroft FM (2005) 3-D structural and functional char- acterization of the purified K ATP channel complex Kir6.2-SUR1. EMBO J 24, 4166–4175. 22 Matsuo M, Tanabe K, Kioka N, Amachi T & Ueda K (2000) Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B. J Biol Chem 275 , 28757– 28763. 23 Reimann F, Gribble FM & Ashcroft FM (2000) Differ- ential response of K ATP channels containing SUR2A or SUR2B subunits to nucleotides and pinacidil. Mol Pharmacol 58, 1318–1325. 24 de Wet H, Proks P, Lafond M, Aittoniemi J, Sansom MS, Flanagan SE, Pearson ER, Hattersley AT & Ashcroft FM (2008) A mutation (R826W) in nucleo- tide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes. EMBO Rep 9, 648–654. 25 Chen Y & Prusoff WH (1973) Relationship between the inhibition constant (K i ) and the concentration of inhibi- tor which causes 50 per cent inhibition (IC 50 )ofan enzymatic reaction. Biochem Pharmacol 22, 3099–3108. 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. Please note: As a service to our authors and readers, 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. ATPase activity of SUR2A and SUR2B H. de Wet et al. 2662 FEBS Journal 277 (2010) 2654–2662 ª 2010 The Authors Journal compilation ª 2010 FEBS . The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids Heidi de Wet,. 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