J Biol Chem, Vol. 275, Issue 2, 717-720, January 14, 2000

ACCELERATED PUBLICATION
Pharmaco-topology of Sulfonylurea Receptors
SEPARATE DOMAINS OF THE REGULATORY SUBUNITS OF K_ATP CHANNEL ISOFORMS ARE REQUIRED FOR SELECTIVE INTERACTION WITH K⁺ CHANNEL OPENERS^*

Andrey P. Babenko, Gabriela Gonzalez, and Joseph Bryan

From the Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

	ABSTRACT

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The differential responsiveness of (SUR1/K_IR6.2)₄ pancreatic -cell versus (SUR2A/K_IR6.2)₄ sarcolemmal or (SUR2B/K_IR6.0)₄ smooth muscle cell K_ATP channels to K⁺ channel openers (KCOs) is the basis for the selective prevention of hyperinsulinemia, myocardial infarction, and acute hypertension. KCO-stimulation of K_ATP channels is a unique example of functional coupling between a transport ATPase and a K⁺ inward rectifier. KCO binding to SUR is Mg-ATP-dependent and antagonizes the inhibition of (K_IR6.0)₄ pore opening by nucleotides. Patch-clamping of matched chimeric human SUR1-SUR2A/K_IR6.2 channels was used to identify the SUR regions that specify the selective response of sarcolemmal versus -cell channels to cromakalim or pinacidil versus diazoxide. The SUR2 segment containing the 12th through 17th predicted transmembrane domains, TMD12-17, confers sensitivity to the benzopyran, cromakalim, and the pyridine, pinacidil, whereas an SUR1 segment which includes TMD6-11 and the first nucleotide-binding fold, NBF1, controls responsiveness to the benzothiadiazine, diazoxide. These data are incorporated into a functional topology model for the regulatory SUR subunits of K_ATP channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

K⁺ channel openers, KCOs,¹ are a structurally diverse group of compounds with no obvious common pharmacophore linking their ability to antagonize the inhibition of ATP-sensitive K⁺ (K_ATP) channels by intracellular nucleotides (1). After the identification of K_ATP channels in cardiomyocytes and pancreatic -cells using the patch-clamp technique (2, 3), Trube et al. (4) showed the benzothiadiazine KCO, diazoxide, stimulates -cell K_ATP channels, and Escande et al. (5) reported the benzopyran KCO, cromakalim, increased the activity of sarcolemmal channels in inside-out membrane patches indicating a direct interaction with these channels. Cloning of -cell and sarcolemmal K_ATP channel subunits led to the understanding that they are (SUR1/K_IR6.2)₄ and (SUR2A/K_IR6.2)₄ complexes, respectively, and that their differential responses to KCOs and to sulfonylureas are determined by the SUR isoform (Refs. 6-8, and reviewed in Ref. 9). Recent studies have shown that pharmacologically significant binding of the pyridine KCO, [³H]P1075, an analog of the potent "cardiovascular" KCO, pinacidil (10), to SURs requires hydrolyzable nucleotide triphosphates, Mg²⁺ or Mn²⁺, and intact nucleotide-binding folds (both NBF1 and NBF2) (11-13). Diazoxide does not stimulate homomeric K_IR6.2 channels (14) and [³H]P1075 does not interact with K_IR6.0 (13). The effect of diazoxide on native and reconstituted sarcolemmal channels in the presence of quasi-cytosolic [Mg-ATP] is negligible in comparison with stimulation by both cromakalim and pinacidil (1, 9, 15). SUR2A/K_IR6.2 channels can be stimulated by diazoxide at supra-physiologic [ADP]_i and submillimolar [ATP]_i in the presence of Mg²⁺ (16). This suggests diazoxide can bind to SUR2s in agreement with the stimulation of SUR2B/K_IR6.2 channels (17) and with the displacement of [³H]P1075 from both SUR2 isoforms, differing in their final 42 amino acids, by diazoxide (13). By exchanging segments between SUR1 and SUR2A, D'Hahan et al. (18) have shown the importance of the C-terminal set of transmembrane domains of SUR2 for stimulation of K_ATP channels by SR47063, a cromakalim analog. Uhde et al. (19) were able to identify two smaller sequences within this region critical for [³H]P1075 binding and stimulation.

We have used matched human SUR1-SUR2A chimeras, previously employed to define the segments critical for SUR isoform-specific ATP-inhibitory gating (20) and high-affinity tolbutamide inhibition (21), to validate the requirement of TMD12-17 of SUR2 for stimulation by cromakalim or pinacidil and to demonstrate that TMD6-11 and NBF1 control the responsiveness to diazoxide in the presence of Mg-ATP.

	EXPERIMENTAL PROCEDURES

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The human SUR1, SUR2A, and K_IR6.2 cDNAs have been described previously (22), as have the cDNAs encoding chimeric SUR I through VI, IX through XIV (20), and XVII (21) (see Fig. 1, left panel). These chimeras were constructed initially to allow swapping of seven major domains of SUR using existing restriction sites in human SUR1 and SUR2A and, when missing, by engineering matching restriction sites. The new chimeras, XIX through XXIV, were constructed by swapping the indicated segments (Fig. 1). The co-transfection with SUR and K_IR6.2 and the cultivation of COSm6 cells was done as described (15). Recording of currents from inside-out patches was done at 22-24 °C as described previously (15) using an Axopatch 200B amplifier (Axon Instruments, Inc., Foster City, CA) at a holding potential of 40 mV. Pipettes were filled up with the K⁺-rich external solution containing (in mM) 145 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, pH 7.4 (KOH). The Mg²⁺-free internal solution was (in mM) 140 KCl, 5 EDTA, 5 HEPES, 10 KOH, pH 7.2 (KOH), whereas the Mg²⁺-containing "intracellular" solution was (in mM) 140 KCl, 1 MgCl₂, 5 EGTA, 5 HEPES, 10 KOH, pH 7.2 (KOH). Pinacidil was from Lilly Research Laboratories, and ATP (ultra-pure, di-sodium salt) and other compounds were from Sigma. The free Mg²⁺ concentration in all Mg²⁺-containing internal solutions was maintained at a quasi-cytosolic level of ~0.7 mM, as described previously (15). Tolbutamide (200 mM stock solution in 0.1 N KOH) was added to the nucleotide-free internal solution to a final concentration of 200 µM. Diazoxide, cromakalim, or pinacidil (100 mM stock solutions in dimethyl sulfoxide, Me₂SO) were added to the 0.1 mM ATP- and Mg²⁺-containing internal solution. The final concentrations were 300 µM diazoxide (0.3 volume % Me₂SO), 200 µM cromakalim, or 100 µM pinacidil. Bathing solutions were applied using a programmable rapid solution changer (RSC-200, Biologic Inc, Claix, France). The relative NPo, used as a measure of channel activity in the presence of a test compound, was estimated as described previously (15) after applying corrections for run-down and for reactivation by Mg-ATP. Addition of 0.3 volume % Me₂SO to the ~0.1 mM Mg-ATP containing internal solution induced a small, 1.25 ± 0.26- and 1.27 ± 0.28-fold, increase in the NPo values of -cell and cardiac K_ATP channels, respectively (mean ± S.D.). The latter estimate was used to define the lower limit for a significant increase in the averaged NPo values, expressed as mean ± S.D., in the presence of either KCO (see Fig. 1). The differences in NPo values with p < 0.05 (unpaired Student's t test) were considered significant.

	RESULTS

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In agreement with our original demonstration (8), the SUR1/K_IR6.2 and SUR2A/K_IR6.2 channels display differences in sulfonylurea sensitivity and in responsiveness to diazoxide and cromakalim (Fig. 1). The SUR1/K_IR6.2 channel currents, activated upon excision of an inside-out patch into nucleotide-free internal solution, are inhibited by ~60% by 200 µM tolbutamide, a concentration which saturates the high-affinity binding site (21), whereas the SUR2A/K_IR6.2 channels are inhibited by ~15% (15) as a result of low-affinity interaction(s) (21). With ~0.1 mM Mg-ATP in the internal solution to ensure maximal binding of KCOs (11-13) and pre-inhibit channel activity, a supra-pharmacological concentration of diazoxide, 300 µM, sufficient to half-maximally saturate SUR1 and SUR2 (13), significantly attenuated nucleotide-inhibition of SUR1/K_IR6.2, but not SUR2A/K_IR6.2, channels. A concentration of cromakalim, sufficient to saturate SUR2A, almost completely antagonized the inhibition of SUR2A/K_IR6.2 channels by 0.1 mM ATP but did not stimulate SUR1/K_IR6.2 channels. In the absence of either, or both, nucleotides and Mg²⁺ (~10⁹ M free Mg²⁺ in the Mg²⁺-free internal solution), stimulation by diazoxide was undetectable and stimulation by cromakalim or pinacidil was reduced ~10-fold (not shown). The remaining nonphysiologic effect of cromakalim in the absence of Mg-ATP was not examined further.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Delineation of SUR segments that specify differences in the pharmacologic profiles of SUR1/KIR6.2 versus SUR2A/KIR6.2 channels. The left panel illustrates the SUR chimeras used (see Refs. 20 and 21 for descriptions) with the topology of SUR (29) at the top and numbering of amino acids at the bottom. Segments from SUR1 and SUR2A are shown in white and gray, respectively. The middle panel shows records of current through SUR/KIR6.2 channels. The thin horizontal line shows the level of currents when KATP channels are closed; downward deflections correspond to inward currents. A 15 s bar is shown near each trace. The current bars are (in pA): 10, chimera XIX, and XXI through XXIII; 20, SUR2A, chimeras II through VI, IX through XI, and XX; 50, chimeras I, XIV, XVII, and XXIV; 100, SUR1, and chimera XIII; and 500, chimera XII. The different horizontal bars indicate applications of ATP or drugs, as indicated. The record of macrocurrent through chimera XII/KIR6.2 channels shows the slower wash-out for the cromakalim versus diazoxide stimulation; the vertical arrow above the trace indicates the application of 1 mM ATP. All of the chimeric channels were more inhibited by 0.1 mM ATP in the presence than absence of Mg2+ (20), and all were more inhibited than homomeric KIRs (14, 27, 28), implying that interactions mediating the SUR-induced increase in apparent ATP-sensitivity of KIR and Mg-ATP stimulation were preserved in all of the chimeras. The right panel gives a comparison of tolbutamide inhibition and stimulation by KCOs. For each patch, the NPo value in the presence of tolbutamide was normalized to that in nucleotide-free internal solution, and the NPo value in the presence of the KCO was normalized to that at 0.1 mM ATP with Mg2+. The relative NPo values 1 are plotted on an expanded scale. The two thin vertical lines indicate the interval where NPo changes were insignificant (see "Experimental Procedures"). All data points with a mean value (± S.D.) outside this interval represent significant effects (p < 0.05) with the exception of diazoxide stimulation of chimeras XI/KIR6.2 and XXII/KIR6.2 channels where p < 0.1.

Comparison of the stimulation of chimeric SUR/K_IR6.2 channels by diazoxide and cromakalim demonstrated all four possible types of channels including those differentially responsive to the two compounds, those that failed to respond to either drug and those that responded to both drugs. Analysis of six pairs of matched chimeric SURs (Fig. 1, underlined) suggested that stimulation by cromakalim was determined by the presence of TMD12-17 from SUR2 in the chimeric receptor (chimeras V through XII), whereas matched channels lacking this segment were not stimulated by cromakalim. Stimulation by diazoxide was correlated with the presence of either NBF1 and TMD6-11 from SUR1 (chimeras I through III and XII through XIV, and XI, respectively). Chimeras II and XII, containing both NBF1 and TMD6-11 of SUR1 conferred the maximal response to diazoxide, whereas chimera XI, containing TMD6-11 of SUR1 and NBF1 of SUR2, specified a lower efficacy response. Analysis of three additional pairs of chimeras with reciprocally exchanged single segments reinforces the conclusion that TMD12-17 of SUR2 confers a selective interaction with either cromakalim or pinacidil, whereas TMD6-11 and NBF1 of SUR1 contribute additively to diazoxide responsiveness. Substitution of TMD6-11 and NBF1 of SUR1 into SUR2A (chimera XXIV) was essential for maximal response to diazoxide.

The separable nature of the segments that specify the selective responses of K_ATP channel isoforms to the two KCOs is demonstrated by the generation of channels that are stimulated by both the "-cell specific" and "cardiovascular" KCOs (chimeras XII, XIX through XXI, and XXIV) versus those that are not stimulated by either KCO (chimera IV and chimera XVII). The inhibition (~60%) of these KCO-insensitive currents by 200 µM tolbutamide ensured that TMD12-17 was functionally coupled to K_IR6.2 (21). A direct comparison of currents through the channels responding to both types KCOs suggests a slower off-rate for the benzothiadiazine or pyridine versus benzopyran KCOs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results show that two separate regions in SURs are necessary to determine the selective effects of diazoxide versus cromakalim or pinacidil. We propose that segment(s) of SUR2, from the glutamate-rich motif following NBF1 through the intracellular segment preceding NBF2 (red tones in Fig. 2), confer sensitivity to the benzopyran and pyridine derivatives, whereas TMD6-11 and NBF1 of SUR1 specify a -cell channel-like response to the pyrimidine KCO, diazoxide. Our demonstration that TMD12-17 of SUR2 is required for cromakalim stimulation of human K_ATP channels complements the observation of D'Hahan et al. (18) that transfer of TMD12-17 from rat SUR2A into hamster SUR1 is sufficient for SR47063-responsiveness of chimeric SUR/K_IR6.2 channels and complements the report by Uhde et al. (19) that smaller segments, Thr¹⁰⁵⁹-Leu¹⁰⁸⁷ and Arg¹²¹⁸-Asn¹³²⁰, of rat SUR2 confer specific binding of [³H]P1075, displaceable by pinacidil and levcromakalim, when placed in a hamster SUR1 background. These results imply, but do not prove, that TMD12-17 is essential to form a KCO binding pocket. D'Hahan et al. (18) used a limited number of chimeras and were unable to delineate a region critical for the selective action of diazoxide, while Uhde et al. (19) assumed TMD12-17 was sufficient to determine the response to different KCOs and did not examine the effects of diazoxide. Using a matched set of SUR chimeras, we show the action of diazoxide requires more than TMD12-17. The demonstration that stimulation by diazoxide involves domains of SUR1 other than TMD12-17 suggests these domains either couple diazoxide binding at TMD12-17, or at another segment, to the K_IR6.2 gating mechanism or form an additional KCO-binding site. Consistent with a coupling mechanism, diazoxide can displace [³H]P1075 from SUR2B, and [³H]glibenclamide from SUR1, yielding similar apparent K_D values for both SUR1 and SUR2 isoforms (13) and can stimulate both K_ATP channel isoforms (16, 17). The results are compatible with a minimal model in which different classes of KCOs, including diazoxide, occupy the same site in TMD12-17 and that this site, in SUR1, is in close proximity to sulfonylurea binding pocket (19, 21, 23). The proximity of these sites implies the potential for negative allosteric interactions. We infer that multiple regions of SUR contribute to coupling KCO-occupied site(s) with the K_IR gating machinery. In SUR1/K_IR6.2 channels, efficient coupling of diazoxide binding requires TMD6-11 and NBF1, whereas the C terminus is important in SUR2B/K_IR6.2 channels. The involvement of NBF1 in stimulation by diazoxide suggests nucleotide binding and/or hydrolysis could contribute to the efficacy of this compound. This would be consistent with diazoxide stimulation of SUR2A/K_IR6.2 channels under conditions which may saturate ADP binding (16) and with isoform differences in the sequences of NBF1. NBF1 and NBF2 are known to serve different functions (24), and we have observed more efficient stimulation of -cell versus cardiac K_ATP channels by increasing the [ADP]/[ATP] ratio.² Based on four lines of evidence that 1) the analogous TMD12-17 segment of MRP, a structurally related transport ATPase, is important for conferring drug-resistance (25), 2) substrates are known to activate the ATPase activity of transport ATPases, 3) hydrolyzable Mg-ATP is required for KCO action, and 4) K_ATP channel stimulation is regulated by changes in the [ATP]/[ADP] ratio, we hypothesize that KCOs increase an ATPase activity of SUR and/or stabilize Mg-ADP on NBF2 of SUR (24). Within this framework, the requirement of Mg-ATP for KCO binding could reflect an ordered ATP hydrolytic mechanism and suggests KCOs may be transported. This suggests that drug binding to TMD12-17 and cooperative binding (24) of ATP and Mg-ADP to NBF1 and NBF2, respectively, may influence each other allosterically. This would explain the requirement for Mg-ATP and intact NBFs for KCO binding (13), modulation of this binding by Mg-ADP (26), and sulfonylurea-inhibition of the stimulatory action of nucleotides (21), and the release of pre-bound 8-N₃ATP from SUR1 by sulfonylureas (24) (as illustrated in Fig. 2).

View larger version (58K): [in this window] [in a new window]   Fig. 2.   A summary schematic showing the determinants and interactions which specify differential modulation of (SURx/KIR6.0)4 channels by nucleotides, KCOs, and sulfonylureas. Two SURx/KIR6.0 pairs are shown with SUR1 and SUR2A surrounding a central KIR6.2 pore. The -helix, beginning at Arg50, and -strands in the KIR N terminus are based on secondary structure predictions (30). Truncation of >44 N-terminal amino acids from KIR6.2 inhibits functional expression of KATP channels, indicating possible involvement of this region in co-assembly, while substitution of a Gln at Arg50 affects gating of SUR1/KIR6.2K185Q channels, implying there are interactions between the cytoplasmic sequences adjacent to M1 and M2 (31). We speculate that nucleotide-sensitive conformational changes in the cytoplasmic domains of KIR modulate nucleotide-independent motions of M2 helices that control gated access to the pore. The swapped segments of SURs are colored differently. TMD1-5 is placed near the N terminus of KIR6.2 because the N-terminal segments of SUR, including the long intracellular loop between TMD5 and 6 (32) and the N terminus of KIR6.2, are involved in controlling spontaneous bursting of KATP channels (20, 31). The C terminus of SUR is near the segment of KIR6.2 from Glu179 through Leu355 which has been assumed to contain a low-affinity nucleotide-binding site (14, 27, 28) and "linker domain" (33) since the last 42 amino acids of SUR1 specify, independently of the Po(max), a lower steady-state IC50(ATP) in -cell versus sarcolemmal KATP channels (20). The separable effects of SUR on the occupancy of the K+ driving force-independent closed state and on the IC50(ATP) suggest that ATP may bind to KIR6.2 in both closed and open conformations. Tolbutamide (Tlb), whose sulfonylurea moiety is identical to that in glibenclamide, is positioned near cytoplasmic loops of TMD12-17 of SUR1 based on identification of the segment(s) required for high-affinity tolbutamide inhibition and [3H]glibenclamide binding (19, 21, 23). The SUR segments which specify stimulation by diazoxide versus cromakalim or pinacidil are color-coded (blue versus red tones). The two darker segments in TMD12-17 of SUR2 are the predicted cytoplasmic regions which may form a KCO binding pocket based on [3H]P1075 binding (19) and thus match the color of pinacidil (Pin). Cromakalim (Crm), a less potent KCO, is shown displaced from the pocket. The equivalent segments in SUR1 are unavailable when the intervening sulfonylurea pocket is occupied (19); this provides a mechanistic explanation for the apparent negative allosterism between sulfonylurea- and diazoxide- (Dzx) binding. The "trees" identify the glycosylation sites (note differences between SUR isoforms). The glutamate-rich motif is indicated by EEE. The ER retention (34) and putative anterograde (35) signals, controlling surface expression of (SURx/KIR6.0)4 channels, are indicated by rrr and aaa, respectively. Interestingly, the rrr of SUR2 contains RKQ versus RKR in SUR1, and the numbers of channels with the regulatory subunits containing the rrr region of SUR1 appear to be higher in comparable patches (note different current scales for representative records in Fig. 1).

Fig. 2 presents a summary model integrating currently available data on the functional topology of SURs. In addition to sequences in TMD1-5 and the C terminus that specify differences in P_o(max) and IC_50(ATP) of K_ATP channel isoforms (20), results from four groups (18, 19, 21, 23) demonstrate that TMD12-17 is critical for the action of both sulfonylureas and KCOs, suggesting this region forms the binding pockets for these compounds. Subdivision of TMD12-17 of SUR1 implicates intracellular loops in high-affinity sulfonylurea binding (19, 21, 23). The corresponding regions in SUR2 separates sequences required for high-affinity binding of [³H]P1075. Contribution of separate domains to coupling the energy of binding to re-configurations of the channel gate show the molecular mechanism of action of these drugs cannot be understood simply in terms of drug binding sites. Recent studies (13, 14) have largely eliminated early models based on direct competition for a nucleotide binding site. The relative currents in the presence of ~0.1 mM Mg-ATP and 200 µM cromakalim (see also Ref. 18) are higher than expected if KCOs "uncouple" SUR from K_IR6.2 and thus eliminate the ability of SUR to increase the sensitivity of K_IR6.0 to ATP, judged by comparing the IC_50(ATP) of heteromeric versus homomeric channels (14, 27, 28). The data argue that KCOs have a net stimulatory effect, beyond the ~9-fold and ~33-fold decrease in IC_50(ATP) induced by SUR2A and SUR1, respectively (20, 27). We propose that there is no requirement for a decrease in the affinity of the putative ATP-inhibitory site on K_IR6.0 or need for dissociation of ATP for KCOs to open K_ATP channels. Our finding that the first half of the N terminus of K_IR6.2 "couples" high-affinity sulfonylurea- but not diazoxide-binding to SUR1 with K_IR gate (21) implies there are at least two parallel pathways through which SURs can converse with the K_IR6.0 pore gate.

	ACKNOWLEDGEMENTS

We thank Li-Zhen Song for excellent technical assistance with cell culture and transfections.

	FOOTNOTES

^* The work was supported by grants from Juvenile Diabetes Foundation International and American Heart Association (to A. P. B.) and by National Institutes of Health grants (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 713-798-4996; Fax: 713-790-0545; E-mail: ababenko@bcm.tmc.edu.

² A. P. Babenko, unpublished data.

	ABBREVIATIONS

The abbreviations used are: KCO, K⁺ channel opener; SUR, sulfonylurea receptor; K_ATP, (SURx/K_IR6.0)₄ channels; NBF, nucleotide binding fold; IC_50(ATP), IC₅₀ value for ATP; P_o(max), the maximal mean open channel probability in the absence of nucleotides; TMD, transmembrane domain.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES