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Keywords:

  • ABCC8;
  • ATPase activity;
  • KATP channel;
  • nucleotide-binding domain;
  • sulfonylurea receptor

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The ATP-sensitive potassium (KATP) channel couples glucose metabolism to insulin secretion in pancreatic β-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 Pi·s−1·(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 µm), 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.

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.

ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability and transmembrane ion fluxes. In pancreatic β-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 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 β-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]. 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[αP] and 8-azido-[32P]ATP[γP][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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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.

image

Figure 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 Å.

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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 monomers 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 Å and the inner diameter was 40–75 Å (Fig. 1C). A similar 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 (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 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 fitting the data to the Michaelis–Menten equation, was similar to that of ATP.

Table 1.   ATPase activities and kinetic constants.
ConstructTurnover rate (nmol Pi·s−1· nmol−1 protein)Vmax (nmol Pi·min−1· mg−1)Km (mm)n
SUR10.03 ± 0.0059.0 ± 1.70.10 ± 0.036
MBP–NBD10.03 ± 0.00323.8 ± 2.400.6 ± 0.0914
MBP–NBD20.13 ± 0.01103.81 ± 8.700.65 ± 0.1312
MBP–NBD1 + 20.08 ± 0.0161.22 ± 6.780.56 ± 0.1110
image

Figure 2.  ATPase activity of SUR1. (A) ATPase activity in the presence (filled circles, n = 6) or absence of Mg2+ (crosses, n = 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 = 1). (B) GTPase activity of SUR1 (n = 4). The line is fitted to the Michaelis–Menten equation, with an estimated Vmax and Km of 10 nmol Pi·min−1·mg−1 and 0.86 mm, respectively. (C) ATPase activity of SUR1 containing the mutations K719A (triangles, n = 2) or K1385M (diamonds, n = 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 = 4) and by BeF (filled circles, n = 3). Data are expressed as a fraction of the turnover rate in the absence of inhibitor.

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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 = 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.

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 µm; 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).

Table 2.   Inhibition constants for ADP and BeF. ND, not detected. n indicates the number of different protein preparations.
ConstructIC50(ADP) (mm)Ki(ADP) (mm)NIC50(ADP) (mm)Ki(BeF) (mm)n
SUR1NDND40.072 ± 0.020.016 ± 0.0113
MBP–NBD15.7 ± 1.52.12 ± 0.5430.087 ± 0.020.033 ± 0.0083
MBP–NBD22.2 ± 0.70.84 ± 0.2530.048 ± 0.010.019 ± 0.0024
MBP–NBD1 + 21.6 ± 0.30.60 ± 0.130.050 ± 0.010.020 ± 0.0034

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]).

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).

image

Figure 3.  ATPase activity of NBD1. (A) ATPase activity of NBD1 in the presence (filled circles, n = 14) or absence (open circles, n = 2) of Mg2+, at 37 °C. MBP control (open triangles, n = 2). (B) GTP (filled circles, n = 3) and ADP (filled triangles, n = 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 Pi·min−1·mg−1 and 2.6 mm, respectively. (C) Inhibition of ATPase activity at 1 mm MgATP by ADP (open circles, n = 3) or BeF (filled triangles, n = 3). Data are expressed as a fraction of the turnover rate in the absence of inhibitor.

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Table 3.   ADPase activities and kinetic constants.
ConstructTurnover rate (nmol Pi·s−1· nmol−1 protein)Vmax (nmol Pi· min−1·mg−1)Km (mm)n
MBP–NBD10.005 ± 0.0013.83 ± 1.260.17 ± 0.083
MBP–NBD20.014 ± 0.00311.26 ± 2.400.07 ± 0.023
MBP–NBD1 + 20.012 ± 0.0029.80 ± 2.690.06 ± 0.013

BeF blocked ATP hydrolysis at NBD1 with a Ki of 33 µm(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 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+.

image

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

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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 µm) 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.

image

Figure 5.  ATPase activity of NBD1 + NBD2. (A) ATPase activity of the NBD1 + NBD2 mixture in the presence (filled circles, n = 10) or absence (open circles, n = 2) of Mg2+, at 37 °C. (B) GTP (circles, n = 3) and ADP (triangles, n = 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 Pi·min−1·mg−1 and 1.7 mm, respectively. (C) Inhibition of ATPase activity at 1 mm MgATP by ADP (open circles, n = 3) or BeF (filled triangles, n = 4). Data are expressed as a fraction of the turnover rate in the absence of inhibitor.

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GTP was hydrolyzed by the NBD1 + NBD2 mixture with Km > 1 mm (Fig. 5B). As observed for the 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 µm (Table 2, Fig. 5C), which is not significantly different from that of either NBD1 (30 µm) or NBD2 (19 µm) 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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 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 Pi·min−1·mg−1) is at the lower end of the range found for MRP1: from 5 to 10 nmol  Pi· min−1·mg−1[37] to 470 nmol Pi·min−1·mg−1[38]. It is less than that reported for CFTR (60 nmol  Pi· min−1·mg−1 protein [39]), or P-glcyoprotein (320–3900 nmol  Pi· min−1·mg−1[40,41]), but higher than that found for ABCR (1.3 mol Pi·min−1·mg−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.

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 µm) did not vary significantly between the isolated NBDs (NBD1, NBD2 or the NBD1–NBD2 mixture). Previous studies yielded somewhat lower values of 290 µm and 350 µm for NBD1 and NBD2, respectively [19]. For comparison, values for the NBDS of SUR2A were 220 µm for NBD1 [19] and ranged from 370 µm[19] to 4.4 mm[45] for NBD2. The rates of ATP hydrolysis that we observed are about two-fold (NBD1) and up to five-fold (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 nucleotide 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 EM, the proteins formed ring-like structures with an outer diameter of ∼ 120–140 Å. This is similar to the outer diameter of the purified octameric KATP complex (180 Å) [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 Å. 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 Å 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, in agreement with the electrophysiological data. The same mutations shifted the IC50 for ATP block of the KATP channel to a value (13–16 µm) [6]) intermediate between that seen for wild-type channels in the presence (∼ 30 µm) [6] and absence (6 µm) [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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 full-length 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 lb·in−2, and centrifuged at 200 g for 10 min using a Beckman Allegra 6KR centrifuge with S/N02E3297 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 µm 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 µg·L−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-β-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 lb·in−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 mg·L−1 for NBD1–MBP and 0.8 mg·L−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).

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 α-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 µg·µL−1, and 300 µL 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 mg·mL−1, and 250 µL 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 mg·mL−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 µm 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 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 µm for BeF inhibition and 3 µm 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 (µg/µg) 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 µm.

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.

The Michaelis–Menten equation was fitted to concentration–activity relationships and used to obtain the Km. All activities were expressed as Vmax (nmol Pi released· min−1·mg−1 protein) and as maximal turnover rate (nmol Pi released·s−1·nmol−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 = 181 kDa; MBP–NBD1 = 87 kDa; MBP–NBD2 = 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:

  • image

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 = 0 for complete inhibition). Ki values were then calculated from the IC50 using the equation for competitive inhibition of Cheng & Prusoff [31]:

  • image

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

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank the Wellcome Trust (F. M. Ashcroft and C. Vénien-Bryan), the EU (F. M. Ashcroft, EuroDia-LSHM-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.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • 1
    Ashcroft FM (2005) ATP-sensitive potassium channelopathies: focus on insulin secretion. J Clin Invest 115, 20472058.
  • 2
    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 characterization of the purified KATP channel complex Kir6-2-Sur1 EMBO J 24, 41664175.
  • 3
    Tucker SJ, Gribble FM, Zhao C, Trapp S & Ashcroft FM (1997) Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 387, 179183.
  • 4
    Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement JPT, Gonzalez G, Aguilar-Bryan L, Permutt MA & Bryan J (1996) Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 272, 17851787.
  • 5
    Gribble FM, Tucker SJ & Ashcroft FM (1997) The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide. EMBO J 16, 11451152.
  • 6
    Gribble FM, Tucker SJ, Haug T & Ashcroft FM (1998) MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit. Proc Natl Acad Sci USA 95, 71857190.
  • 7
    Bryan J, Munoz A, Zhang X, Dufer M, Drews G, Krippeit-Drews P & Aguilar-Bryan L (2007) ABCC8 and ABCC9: ABC transporters that regulate K+ channels. Pflugers Arch 453, 703718.
  • 8
    Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, Thomas PJ & Hunt JF (2002) ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol Cell 10, 139149.
  • 9
    Locher KP, Lee AT & Rees DC (2002) The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296, 10911098.
  • 10
    Dawson RJ & Locher KP (2006) Structure of a bacterial multidrug ABC transporter. Nature 7108, 156157.
  • 11
    Hollenstein K, Frei DC & Locher KP (2007) Structure of an ABC transporter in complex with its binding protein. Nature 7132, 213216.
  • 12
    Campbell JD, Sansom MS & Ashcroft FM (2003) Potassium channel regulation. EMBO Rep 4, 10381042.
  • 13
    Campbell JD, Proks P, Lippiat JD, Sansom MS & Ashcroft FM (2004) Identification of a functionally important negatively charged residue within the second catalytic site of the SUR1 nucleotide-binding domains. Diabetes 53 (Suppl. 3), S123S127.
  • 14
    Vergani P, Lockless SW, Nairn AC & Gadsby DC (2005) CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433, 876880.
  • 15
    Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JPT, Boyd AE 3rd, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J & Nelson DA (1995) Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268, 423426.
  • 16
    Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, Matsuzawa Y & Kurachi Y (1996) A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel. J Biol Chem 271, 2432124324.
  • 17
    Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J & Seino S (1996) A family of sulphonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16, 10111017.
  • 18
    Tammaro P & Ashcroft FM (2006) Functional effects of naturally occurring KNCJ11 mutations causing neonatal diabetes on cloned cardiac KATP channels. J Physiol 571, 314.
  • 19
    Masia R, Enkvetchakul D & Nichols CG (2005) Differential nucleotide regulation of KATP channels by SUR1 and SUR2A. J Mol Cell Cardiol 39, 491501.
  • 20
    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, 233245.
  • 21
    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-sensitive K+ channels. J Biol Chem 277, 1420614210.
  • 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, 2875728763.
  • 23
    Mikhailov MV & Ashcroft SJ (2000) Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell KATP channels. J Biol Chem 275, 33603364.
  • 24
    Hough E, Mair L, Mackenzie W & Sivaprasadarao A (2002) Expression, purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor. Biochem Biophys Res Commun 294, 191197.
  • 25
    Mikhailov MV, Mikhailova EA & Ashcroft SJ (2000) Investigation of the molecular assembly of beta-cell K (ATP) channels. FEBS Lett 482, 5964.
  • 26
    Shevchenko A & Shevchenko A (2001) Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal Biochem 296, 279283.
  • 27
    Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M & Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116, 190199.
  • 28
    Frank J (1990) Classification of macromolecular assemblies studied as ‘single particles’. Q Rev Biophys 23, 281329.
  • 29
    Chifflet S, Torriglia A, Chiesa R & Tolosa S (1988) A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases. Anal Biochem 168, 14.
  • 30
    Clausen JD, McIntosh DB, Woolley DG, Anthonisen AN, Vilsen B & Andersen JP (2006) Asparagine 706 and glutamate 183 at the catalytic site of sarcoplasmic reticulum Ca2+-ATPase play critical but distinct roles in E2 states. J Biol Chem 281, 94719481.
  • 31
    Cheng Y & Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22(23), 30993108.
  • 32
    Trapp S, Tucker SJ & Ashcroft FM (1997) Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits. Proc Natl Acad Sci USA 94, 88728877.
  • 33
    Sankaran B, Bhagat S & Senior AE (1997) Inhibition of P-glycoprotein ATPase activity by beryllium fluoride. Biochemistry 36, 68476853.
  • 34
    Werber MM, Peyser YM & Muhlrad A (1992) Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes. Biochemistry 31, 71907197.
  • 35
    Kidd JF, Ramjeesingh M, Stratford F, Huan LJ & Bear CE (2004) A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity. J Biol Chem 279, 4166441669.
  • 36
    Ramaen O, Sizun C, Pamlard O, Jacquet E & Lallemand 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, 481490.
  • 37
    Chang XB, Hou YX & Riordan JR (1998) Stimulation of ATPase activity of purified multidrug resistance-associated protein by nucleoside diphosphates. J Biol Chem 273, 2384423848.
  • 38
    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, 6982.
  • 39
    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, 3905139057.
  • 40
    Urbatsch IL, al-Shawi MK & Senior AE (1994) Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. Biochemistry 33, 70697076.
  • 41
    Callaghan R, Berridge G, Ferry DR & Higgins CF (1997) The functional purification of P-glycoprotein is dependent on maintenance of a lipid–protein interface. Biochim Biophys Acta 1328, 109124.
  • 42
    Sun H, Molday RS & Nathans J (1999) Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease. J Biol Chem 274, 82698281.
  • 43
    Schwappach B, Zerangue N, Jan YN & Jan LY (2000) Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter. Neuron 26, 155167.
  • 44
    Ramjeesingh M, Li C, Garami E, Huan L-J, Galley K, Wang Y & Bear CE (1999) Walker mutations reveal loose relationship between catalytic and channel-gating activites of purified CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Biochemsitry 38, 14631468.
  • 45
    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, 19431952.
  • 46
    Annereau JP, Ko YH & Pdersen PL (2003) Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-dinding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate. Biochem J 371, 451462.
  • 47
    Randak C & Welsh MJ (2003) An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR. Cell 115, 837850.
  • 48
    Ueda K, Inagaki H & Sieno S (1997) MgADP antagonism to Mg2+-independent ATP binding of sulfonylurea receptor SUR1. J Biol Chem 272, 2298322986.
  • 49
    Stratford FL, Ramjeesingh M, Cheung JC, Huan LJ & Bear CE (2007) The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer. Biochem J 401, 581586.
  • 50
    Randak CO & Welsh MJ (2005) Adenylate kinase activity in ABC transporters. J Biol Chem 280, 3438534388.
  • 51
    Randak C, Neth P, Auerswald EA, Eckerskorn C, Assfalg-Machleidt I & Machleidt W (1997) A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase. FEBS Lett 410(2–3), 180186.
  • 52
    Ueda K, Inagaki N & Seino S (1997) MgADP antagonism to Mg2+-independent ATP binding of the sulfonylurea receptor SUR1. J Biol Chem 272, 2298322986.
  • 53
    Nishida M & MacKinnon R (2002) Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 Å resolution. Cell 111, 957965.
  • 54
    Pegan S, Arrabit C, Zhou W, Kwiatkowski W, Collins A, Slesinger PA & Choe S (2005) Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification. Nat Neurosci 8, 279287.
  • 55
    Biswas-Fiss EE (2006) Interaction of the nucleotide binding domains and regulation of the ATPase activity of the human retina specific ABC transporter, ABCR. Biochemistry 45, 38133823.