Recognition of Sulfonylurea Receptor (ABCC8/9) Ligands by the Multidrug Resistance Transporter P-glycoprotein (ABCB1)

ATP-sensitive K+ (KATP) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block KATP channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.


INTRODUCTION
ABC proteins form a large superfamily of mostly membrane proteins. They present as common characteristics a nucleotide binding domain able to hydrolyze ATP which includes the sequence motifs Walker A and B and the signature C (1). As membrane active transporters, they are expressed in virtually all branches of the living reign, and they exhibit a very broad diversity of transported substrates, handled in either direction of influx or efflux (2). In higher mammals, they are involved in various pathophysiological situations and genetic diseases (3).
The K ATP channel results from the constitutive association of 4 pore-forming Kir6.x subunits and 4 regulatory SUR subunits [(4); (5)]. In various combinations of the SUR isoforms, SUR1, SUR2A, and SUR2B, and the Kir6 isoforms, Kir6.1 and Kir6.2, these channels are present in most excitable cells -including neuronal, cardiac muscle, smooth muscle, and endocrine cells - (6) where they serve to couple membrane electrical properties to intracellular metabolism.
SUR, a member of the ABCC/MRP subfamily of ABC proteins, is the site of action of numerous drugs that cause either closing (K ATP channel blockers) or opening (K ATP channel openers) of the Kir6.x potassium pore (7). Blockers include antidiabetic drugs, sulfonylureas like glibenclamide, as well as non-sulfonylureas like meglitinide. Openers present interesting therapeutic opportunities, in particular for their antihypertensive and cardioprotective properties: a few, like diazoxide, pinacidil, nicorandil, and minoxidil are in clinical use as treatment against hypertension, angina pectoris, and alopecia, respectively (8).
Among ABC proteins of higher organisms, SUR stands out because its only known function is that of a channel regulator while most others are active (or passive in the case of CFTR) transporters. Yet, SUR possesses strong sequence homologies with other eukaryotic ABC proteins, like the multidrugresistance transporters MRP1 and P-gp (Pglycoprotein), suggesting that these proteins could share some structural and pharmacological properties (9). Indeed, SUR is the target of a panel of structurally unrelated ligands, like P-gp and other multidrug transporters. Multispecific drug recognition by P-gp is still a misunderstood molecular property, in spite of a mass of published data including extensive mutational analyses (10), as well as a pharmacophore model of some of its known ligands (11). As binding of SUR ligands has also been linked to some particular residues [(12); (13)], it is of interest to compare the ligand recognition features of these two ABC proteins, with the aim of gaining new insights in both of them.
In addition, regulation of several ABC transporters by K ATP channel modulators has been reported: CFTR is inhibited by glibenclamide, and less potently by a few other K ATP channel blockers and openers (14). Glibenclamide also inhibits MRP1 (15) and P-gp (16), and it appears to be a substrate of the latter. These observations are of interest since they can complete the pharmacological toolbox of researchers -glibenclamide is now the most widelyused blocker of CFTR channels -and they can point to potential side-effects and altered biodisposition. This is specially important for P-gp, which is an essential gate-keeper at the blood-brain barrier as well as in cancer cells [(17); (18)].
In this work, we have therefore focused on P-gp and examined whether representative compounds among K ATP channel blockers and openers could interact with P-gp. As blockers, we used glibenclamide, as a reference, and its two moieties, tolbutamide and meglitinide; as openers, we used diazoxide, SR47063, a derivative of cromakalim, and P1075, a derivative of pinacidil. Our findings demonstrate that these molecules bind to and are possibly transported by P-gp. They imply a possible structural similarity between SUR and P-gp, which could extend to a mechanistic similarity, in line with our recent work [(19); (12)], suggesting that K ATP channel openers and blockers interact with SUR much like transport substrates would, although intrinsic active transport by SUR has not yet been demonstrated.
In an effort to rationalize the observed data, and in the absence of high resolution crystallographic data, we built new homology models of human P-gp and SUR, based on multiple alignments with the bacterial ABC transporters MsbA and Sav1866, whose structures have been solved by X-ray crystallography in different conformations [(20); (21)], and on mouse P-gp whose structure has been recently solved in one conformation (22). Highlighting the various residues known to be implicated in drug binding specificity helped to delineate different hotspots for drug binding in the P-gp and SUR structures. Their 3D arrangement revealed two main binding domains for the drugs verapamil/SR47063 and for vinblastine/glibenclamide in P-gp, and their counterpart for binding of SR47063 and of glibenclamide in SUR.
In this work, we used structural models derived from comparative modeling, because it offers the advantage over crystallographic data to interpret a huge amount of binding data and to address the multispecificity character of these ABC proteins. Homology modeling also allowed us to interpret the results in various protein conformations, in relation to the enzymatic turn-over.

P-glycoprotein-containing membrane vesicles preparation.
The MDR cell line used, DC-3F/ADX, was selected from spontaneously transformed Chinese hamster lung fibroblast DC-3F on the basis of resistance to actinomycin D. This resistance is due to overexpression of the pgp1 gene. The DC-3F/ADX cells and DC-3F cells, their drug-sensitive parental counterparts, were cultured, harvested by scraping and washed in PBS in the presence of antiproteases. The cells in suspension were then disrupted by sonication. The resulting centrifuged supernatant was layered on a sucrose cushion to isolate the total membrane vesicle fraction. Membrane protein concentrations were determined by Bradford's method. In vesicles prepared from DC-3F/ADX, P-glycoprotein accounts for about 12-15% of total membrane proteins, whereas P-gp cannot be detected in the control vesicles prepared from the DC-3F cells (23). Experiments were performed using DC-3F cells as a control and DC-3F/ADX cells as a P-gp test. Because SUR displays ATPase activity modulated by its ligands (19), our results could be tainted by the unlikely presence of different amounts of SUR in DC-3F and DC-3F/ADX cells. The effect would be however negligible because the ATPase activity of SUR is >100-fold less than P-gp [(24);(25); (26)].
Measurement of ATPase activity. The experiments are based on the enzymatic determination of ATPase activity of native membrane vesicles containing high amounts of Pglycoprotein, measured at 37°C by using a standard coupled-enzyme assay, comprising an ATPregenerating system and continuous spectrophotometric detection of NADH absorbance at 340 nm, which allows the monitoring of NADH consumption in the reaction medium, that is stoichiometric to ADP production.
The reaction medium contained 30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM KCl, 2 mM MgCl 2 , 1 mM dithiothreitol, 1 mM MgATP, supplemented with 0.1 mg/ml pyruvate kinase, 1 mM phosphoenol pyruvate, 0.1 mg/ml lactate dehydrogenase and 0.5 mM NADH. ATPase activity was determined with about 20 µg/ml of membrane proteins. The assay medium was supplemented with 10 mM sodium azide, 0.5 mM ouabain and 1 mM EGTA, to inhibit the H + -ATPases, Na + /K + -ATPase, and Ca 2+ -ATPases, respectively. The residual ATPase activity measured in the presence of the specific inhibitors of these enzymes and in the absence of any added drug was essentially due to the basal activity of Pglycoprotein. MgATP concentration was kept throughout the experiments at the sub-saturating level of 1 mM since we have previously reported that the Km value of P-gp for MgATP remains essentially unchanged and independent of transported substrates [(23); (27) Although activity vs concentration curves often show an inhibition at the highest concentrations, we fitted these curves using only one high affinity specific binding site because : (i) this inhibition may be due to a non-specific membrane perturbation by amphiphilic drugs, rather than to a low-affinity specific binding site; (ii) curve fitting using a two-site binding model would require twice as many parameters as a single-site model, producing more uncertainty and no more qualitative information about the mutual relationships exhibited by the compounds tested. [See for example the parameters determined in Fig.4 compared with those in supplemental Fig.S1A  Absorbance spectra for all tested compounds (data not shown) revealed that only SR47063, which has an absorbance peak at 328 nm, absorbed significantly at our assay wavelength of 340 nm. The resulting interference decreased the signal-tonoise ratio at the highest SR47063 concentrations used but would not alter the slope of the signal which is the parameter used to measure the ATPase rate.
Since some ABC proteins are monomeric (fullsize transporters) whereas others are dimeric (halfsize transporters), and since some of them exhibit an additional N-terminal TMD0 domain, we used the "Conserved Domain Database" (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd .shtml (32)) of NCBI to detect the respective consensus regions in the proteins. This roughly corresponds to one monomer of the dimeric proteins (+/-a dozen of residues), and amino acid sequences with a size of about 600-650 residues were selected for the phylogenetic study, thereby excluding the TMD0 sequences (Table S1).
Multiple amino acid sequence alignment was performed with DIALIGN (33).
A maximum likelihood tree was constructed using PhyML (34) and we made 100 bootstrap replicates [(35,36)] to assess confidence on the results. Indeed, this statistic technique is used to improve the robustness of a constructed tree and to inform about its reliability. As a general rule, if the boostrap value for a given branch is found higher than 90%, then the topology of that branch is considered as "correct". The phylogenetic tree was edited using TreeViewX (http://taxonomy.zoology.gla.ac.uk/sofware (37)): in this representation, each branch length reflects a statistical distance between the considered sequence and its "common ancestor".
Homology Modeling. The alignment of SUR, Pgp, Sav1866 and MsbA, shown in Figure S2, was edited using Jalview Software (38). The secondary structures, derived from the 3D model of P-gp, were added manually above the alignment.
-Template selection: PSI-BLAST (39) against Protein Data Bank (PDB) was used to select the templates for the homology modeling of P-gp and SUR1. This analysis confirmed that MsbA and Sav1866 have the closest sequences to these two mammalian ABC proteins. MsbA and Sav1866 share 28-31% identity with P-gp and 20-21% with SUR1 (see legend of Fig.S2). After the withdrawal of the first crystallographic MsbA structures, the new structures deposited were proposed to represent three distinct putative functional conformations of this representative ABC transporter (20), the open inward-facing, the closed inward-facing and the outward-facing conformations. They were thus used as templates to rebuild three structural models for the human P-gp (see Table 1); the outward-facing conformation also used Sav1866 as a template, allowing a two-template homology modeling. The recent crystallographic structure of mouse P-gp allowed us to rebuild another model for the human P-gp in the open inward-facing conformation based on a single pairwise alignment (87% sequence identity between human and mouse P-gp). For SUR1, only the outward-facing conformation was built using a two-template reconstruction from both St-MsbA and Sav1866 (Table 1), hence giving a higher confidence than a mono-template homology modeling.
-Model generation: For each modeling procedure, ten models were generated with the Modeller 9v6 software (40), with refinement settings 'slow' and the "automodel" procedure. The templates, which were either the N-and C-terminal halves of mouse P-gp structure or half-size bacterial transporters, do not contain the domain TMD0, the first intracellular loop and the linker region. Hence these regions were excluded from modeling.
-Model selection: A scoring program was applied to each generated model and the best scored model among the ten was selected. We have used a combination of the scoring programs available under Modeller, GA341 score and DOPE (Discrete Optimized Protein Energy) score.
-Rebuilding structural models in a lipid environment: The three modeled P-gp conformations were placed in a "native-like" environment in order to analyze the interactions of the protein with model phospholipids arranged in a double layer membrane. We chose POPC (1palmitoyl-2-oleoyl-phosphatidylcholine) because it is the most prominent lipid in mammalian cell membranes. The procedure included a first step of solvation, realized by the Solvate program http://www.mpibpc.mpg.de/home/grubmueller/ downloads/solvate/index.html, and then successive steps allowed by the VMD (Visual Molecular Dynamics) software (46), using the "solvation box" and the "membrane builder" procedures.
Ligand molecular modeling and alignment. The structures of glibenclamide and SR47063 were minimized and aligned under SYBYL (Tripos, St. Louis, MO) with the structures of vinblastine and verapamil, respectively, using procedures and data from Garrigues et al (11). Hydrophobicity and Hbond potential areas were calculated using MOLCAD routine of SYBYL. The compounds were superimposed manually and consensus groups selected. The compounds were then aligned using the multifit command of SYBYL.

Experimental assay validation.
Throughout this work, the ATPase activity of P-gp-enriched vesicles was used as a reporter of the function of native P-gp. This assay has been validated thoroughly in earlier works [(28); (11)]. To further ascertain its suitability in our experimental settings, tests were performed using the P-gp substrates verapamil, progesterone, and vinblastine (Fig.1A). Our results are fully consistent, qualitatively and quantitatively, with the expected effects of these substrates on P-gp ATPase activity (28). Verapamil and progesterone increased ATPase activity with K ½ of 1.3 and 17 µM, respectively, with no obvious cooperativity (Hill coefficients nearing 1). This activation declined at higher concentrations, a phenomenon that could be consistent with the presence of both high and lowaffinity sites as well as with a non-specific perturbation due to the insertion of an amphiphilic compound into the membrane bilayer. Vinblastine inhibited the basal ATPase activity of P-gp-enriched vesicles down to a level close to that of the control vesicles. As control vesicles were unaffected by these 3 compounds, it appears that the ~70% additional ATPase activity measured in P-gpenriched vesicles arose mainly from the presence of P-gp proteins.
K ATP channel blockers stimulate P-gp ATPase. Inhibitors of K ATP channels include sulfonylureas as well as non-sulfonylureas compounds, all known to act through direct binding to SUR (47). Evidence has been published that the prototypical sulfonylurea glibenclamide is a P-gp substrate able to block, at 100 µM, transport of colchicine by P-gp (16). The data from Fig.1B are in agreement with this finding as they show that glibenclamide augments the ATPase activity with a maximum attained near 100 µM. The dose-effect curve resembles that obtained in Fig.1A with verapamil.
Two other blockers were examined: meglitinide, which represents the non-sulfonylurea half of glibenclamide, and tolbutamide, the sulfonylurea half of glibenclamide. Both increased ATPase activity, although with lower affinities (Fig.1B). Although the maximum activation could not be reached as this would have required excessive concentrations and excessive levels of the vehicle DMSO, Hill equation fits to the data gave maximum activation levels of ~70% for the 2 compounds, a value similar to that achieved with the other ATPase activators, glibenclamide, verapamil and progesterone.
K ATP channel openers stimulate P-gp ATPase. K ATP channel openers form an extended family of seemingly unrelated molecules that bind to SUR at a site distinct from the sulfonylurea binding site and allosterically cause channel opening (7). We tested three representative openers: diazoxide; SR47063, a derivative of cromakalim; and P1075, a derivative of pinacidil. Diazoxide targets both SUR1 and SUR2 isoforms (48), while SR47063 and P1075 target only SUR2 isoforms through a common site separate from that of diazoxide.
All three compounds caused an increase in ATPase activity of P-gp-containing vesicles but not control vesicles (Fig.1C). P1075 caused the largest increase (V max = 101%) but had the lowest affinity (K ½ =1.5 mM). Diazoxide had also a low affinity of 0.6 mM. The affinity of SR47063 was highest at 36 µM but its effect was more modest (V max = 55%). These latter values likely underestimate the actual effects of SR47063 since the higher concentrations of SR47063 had an inhibitory effect on the contaminant sources of ATPase activity that appeared when the compound was tested in the control vesicles devoid of P-gp (Fig.1C). It should be noted that the effect of SR47063 was modest in the experiments using the full concentration range, but could be more sizeable in separate singleconcentration experiments as in the experiment of Fig.3

and 4.
Cumulative effects of glibenclamide and other K ATP channel modulators. Binding assays on SUR using radiolabelled glibenclamide have shown that most K ATP channel modulators displace glibenclamide binding (9). We therefore performed experiments designed to reveal interaction between glibenclamide and our other test compounds. Full concentration-effect curves for glibenclamide were repeated in the presence of mM concentrations of the K ATP channel blockers tolbutamide and meglitinide and the openers diazoxide and P1075. Fig.2 shows that the glibenclamide curve was merely shifted up by these compounds without increase in apparent affinity (i.e., K 1/2 value). Thus, effects on P-gp were cumulative without hints of the kind of interactions observed with SUR [(49); (50)]. Surprisingly, neither meglitinide nor tolbutamide competed with glibenclamide in spite of their very similar chemical structures.
Interactions between glibenclamide and established P-gp substrates. The compounds verapamil, progesterone, and vinblastine are typical P-gp substrates (Fig.1A) presumed to bind at distinct position within the P-gp binding pocket [(28); (51)]. As such, they constitute useful test drugs to characterize the binding of novel P-gp ligands such as those reported above. Each drug was therefore assayed in combination with selected K ATP channel modulators.
Experiments combining glibenclamide and verapamil, shown in Fig.3A and B, demonstrate that glibenclamide inhibited the verapamil-induced increase in ATPase activity, and vice versa. This inhibition was not accompanied by a significant increase in K 1/2 value of the control substance, implying a non-competitive interaction. Indeed, a "pure" competitive inhibition by 10 µM glibenclamide or by 1 µM verapamil, concentrations corresponding to their respective affinities, should have induced a 2-fold increase of K 1/2 value for verapamil and glibenclamide, respectively. The dose-dependency of the inhibition correlated well with the respective K 1/2 values of glibenclamide and verapamil observed for basal ATPase stimulation ( Fig.1A and B). Indeed, the K 1/2 value for verapamil activation of basal ATPase was close to 1 µM, and the same concentration was sufficient to reduce by half the glibenclamide-induced increase in ATPase activity (Fig.3B). Conversely, the K 1/2 value for glibenclamide activation of basal ATPase was near 10 µM and Fig.3A shows that, in the presence of that concentration of glibenclamide, the verapamilinduced increase in ATPase activity was roughly half what it was in control. This correlation suggests that a single glibenclamide binding site mediates both activation of basal ATPase and inhibition of verapamil-induced ATPase.
A similar observation was made when glibenclamide was tested against progesterone as shown in Fig.4A: glibenclamide inhibited the effects of progesterone with a half-maximal concentration close to 10 µM. Curve fitting of the ATPase activity vs. progesterone concentration data yielded K 1/2 values decreasing from 17 µM in absence of glibenclamide to a non-significantly different 12 µM in 10 µM glibenclamide. Lower K 1/2 values of 1.8 and 1 µM were obtained in 100 and 300 µM glibenclamide, respectively, but the shallow nature of the curves makes these values too uncertain to consider this decrease as significant and infer cooperativity rather than non-competitive inhibition.
The effects of vinblastine on the ATPase vs. glibenclamide concentration curve (Fig.5A) were also inhibitory, with a dose-dependent increase in K 1/2 (from 11 µM in control to 19 µM at 1 µM vinblastine) suggestive of a weak competitive interaction. Vinblastine inhibited the ATPase stimulation elicited by glibenclamide with full inhibition achieved at ~10 µM vinblastine, a concentration which, by itself, did not affect basal activity of P-gp (see Fig.1A). Half-maximal effect was observed at ~1 µM, which matches values previously determined from the inhibition by vinblastine of other drug-stimulated ATPase activities: verapamil (28), nicardipine (29), indinavir (52).
Interactions between K ATP channel openers and established P-gp substrates. The K ATP channel openers diazoxide and P1075, unlike glibenclamide, shifted upward the ATPase vs. verapamil concentration curve without modifying significantly the K 1/2 of verapamil (Fig.3C). We therefore conclude that the ATPase stimulation by these openers and verapamil were mostly additive, suggesting separate binding sites on P-gp. SR47063 increased the K 1/2 of verapamil from 1.1 to 1.9 µM, a slight change considering the data points scattering, and it caused a decrease in V max from 76% to 41%. Although this V max decrease could reflect saturation of the verapamil effect due to the large stimulation already elicited by SR47063 in those particular experiments, these results suggest a weak competitive inhibition between verapamil and SR47063. Experiments with progesterone ( Fig.4B) revealed that stimulation by diazoxide and progesterone were additive, whereas P1075 and SR47063 produced a decrease in the V max of progesterone from 66% to 40% with a reduction in K 1/2 with P1075 that appeared less significative because of experimental data uncertainties. This indicates a difference in the mechanism of action of diazoxide and P1075 or SR47063.
Finally, as with glibenclamide, 10 µM vinblastine fully inhibited the ATPase stimulation produced by SR47063 (Fig.5B), although the data with lower concentrations of vinblastine (not shown) did not permit to quantify changes in K 1/2 indicative of competitive or non-competitive interactions.
Analysis of molecular recognition elements in glibenclamide and SR47063 structures. Using SYBYL software, we identified hydrophilic (i.e., electron donor and acceptor groups) and hydrophobic (i.e., alkyl and aromatic groups) elements contained in the molecular structures of the two P-gp ligands, glibenclamide and SR47063. SR47063, which exhibits a competitive interaction with verapamil, has in common with verapamil an electron-donor group (CN) and an aromatic group (Fig.6A). Glibenclamide, which exhibits a competitive interaction with vinblastine, presents five parts in common with vinblastine when considering their 3D-alignment: two aromatic cycles, two hydrogen donor groups (NH) and one electron donor group (CO and SO 2 ) (Fig.6B). We have previously shown that multispecific ligand interaction with P-gp relies on the recognition of a 3D pattern of consensus hydrophilic and hydrophobic elements presented by these ligands, independently of their precise chemical structures (11). Furthermore, multidrug recognition capacity is amplified by the existence of different 3D patterns, each defining a pharmacophore on P-gp. Indeed, according to the multipharmacophoric model of the multidrug recognition domain of P-gp established on the basis of mutual interactions between several Pgp ligands, verapamil and vinblastine were identified as representatives of two distinct pharmacophores, pharmacophores I and II, respectively (11). The combined enzymological and structural evidence presented here suggests that SR47063 and glibenclamide belong to the pharmacophores I and II, respectively (Fig.6C). Indeed, the aromatic and the electron-donor groups of SR47063 and of glibenclamide correspond to the consensus elements defining these two pharmacophores.
These data about pharmacophoric-type recognition processes by P-gp have prompted us to investigate the structural features of the P-gp protein involved in the selective molecular recognition of its various ligands.
Phylogenetic study of membrane ABC proteins.
The phylogenetic tree of a set of membrane ABC proteins composed of the membrane human ABC transporters and two bacterial ABC transporters of known structure is shown in Fig.7. Its branches split along the five human subfamilies, A, B, C, D and G, previously defined on the basis of sequence similarities signing divergent evolution (3). Using either the N-or C-terminal sequence of the monomeric full-size ABC transporters in the alignment did not change the topology of the tree (not shown). Based on the high bootstrap values, the phylogenetic tree could be interpreted with a good confidence. Two distinct "clusters" are found. They represent ABCA-G and ABCB-C-D. It appears that P-gp (ABCB subfamily) and SUR1/2 (ABCC subfamily) belong to the same cluster. Moreover, inside this cluster, the ABCC branch is closer to the ABCB than the other branches in term of sequence evolution, as indicated by the length of the (horizontal) line defining these branches.
Interestingly, the two multispecific bacterial transporters MsbA and Sav1866 belong to the ABCB branch, in close proximity to P-gp (ABCB1), as well as ABCB4 (MDR3) and ABCB11 (BSEP). This confirms that MsbA and Sav1866 can be considered as suitable templates for modeling P-gp structure by homology.
Molecular modeling of P-gp.
-Selection and assessment of the final models. All the modeled structures generated, for Pgp as well as for SUR, were validated by GA341 scores >0.9. The selection of the "best" models (one for each conformation) was performed using the DOPE score, even though we are aware that it was initially designed for globular soluble proteins, and is applied to membrane proteins for lack of more suitable tools. The quantitative assessment of the selected models is presented in Table S2. Even for the two inward-facing conformations, constructed from the Ec-and Vc-MsbA low-resolution Cαpositions-only structures, all models show very satisfying quality, as evidenced by Ramachandran plots that show in all cases that >90% of the residues are in favorable or allowed regions.
-Structural model of P-gp. From the three conformations of the bacterial ABC transporters (2 nucleotide-free "apo conformations": "open inwardfacing" and "closed inward-facing"; 1 nucleotidebound: "outward-facing"), we generated three different P-gp structural models, that could reflect different enzymological states of the active transporter during its catalytic turn-over (see coordinates files in pdb format in supplemental data).
The open inward-facing conformation model (Fig.S3), derived from the template Ec-MsbA (20), displays the following features: (i) the two NBDs are far apart (distance between P-loop1 [Ser429] and P-loop2 [Ser1072] ≈ 48.4 Å, mean distance between P-loop and LSGGQ segment [Leu531/Leu1176] ≈ 65.4 Å); (ii) the transmembrane helices TM1-2 and 4-5 are not exactly symmetrical compared with their C-terminal counterparts, helices TM7-8 and 10-11, respectively; (iii) the TM4-5/10-11 are intertwined between the two halves of the protein; (iv) all the helices of the transmembrane domain are in continuity with their corresponding intracellular helices; (v) these helices, oblique with respect to the membrane plane, form an internal cavity ("chamber") lined by the TM4, 6, 7, 10 and 12 at the level of the cytosolic leaflet of the membrane.
During the course of this work, the publication of an X-ray structure for murine P-gp (87% of identity with human P-gp) in the open inward-facing conformation (22), with a better resolution than that of Ec-MsbA and that of the previously published structures obtained by electron microscopy [(53); (54)], although solved "by halves" and phased on MsbA, allowed us to generate another model of this conformation with a better reliability (Fig.8A). For this reason, we will only consider this model in the following. Compared with P-gp modeled from Ec-MsbA, the main difference is that the two NBDs are closer (distance between P-loop1 and P-loop2 ≈ 35.0 Å, mean distance between P-loop and LSGGQ segment ≈ 23.7 Å).
The closed inward-facing conformation model (Fig.8B), derived from the template Vc-MsbA (20), differs from the open inward-facing models by the following features: (i) the two NBDs are in close proximity (distance between P-loop1 and P-loop2 ≈ 8.0 Å; but, mean distance between P-loop and LSGGQ segment ≈ 36.5 Å); (ii) the transmembrane helices are perpendicular to the membrane plane, and no longer form a "chamber".
The outward-facing conformation models (Fig.8C&D), built from templates St-MsbA (20) and Sav1866 (21), differ from the inward-facing models: (i) the two NBDs are associated (distance between P-loop1 and P-loop2 ≈ 23.5 Å; mean distance between P-loop and LSGGQ segment ≈ 11.6 Å, indicating a rotation of the NBDs from their position in the closed inward-facing conformation); (ii) the transmembrane helices are oblique with respect to the membrane plane, with an orientation opposite to that in the open inward-facing conformation, and form an extracellularly-accessible pocket in the phospholipid membrane, that is best seen upon rotation of the model (Fig.8D). At variance with our model, the model of the same conformation elaborated by O'mara & Tieleman (55) presents a region TM1-EC1-TM2 which extends in the extracellular medium with a marked asymmetry between the 2 halves of the protein; The model of Ravna et al (56) also predicts a slight asymmetry between the two halves of the protein, which is not apparent in our model. For an unobstructed view of the protein structure, the models are presented devoid of their membrane embedding in Fig.S4.
-3D localization of P-gp residues involved in substrate recognition. The P-gp models permit spatial localization of 52 residues (Table 3) reported to be involved in substrate recognition, as previously collected from point mutagenesis and chemical labeling data both for human and for mice P-gp [(10);(57)] as well as from crystallographic data (22). In Fig.9A Fig.9B, the same residues are highlighted in the model of the closed inward-facing conformation. As a consequence of helices motion, they now even more clearly segregate between "lower" and "upper" hotspots reminiscent of the "hotspot model" proposed by Shilling et al. (10), especially when considering the top-view of the structure.
In the outward-facing conformation of Fig.9C, the important residues are also distributed between "lower" and "upper" hotspots. We can thus divide these hotspots into 4 classes: (i) "cytosolic Nterminal", located in TM1, (ii) "cytosolic Cterminal" located in TM10-11, (iii) "exoplasmic Nterminal", located in TM4-5-6, (iv) "exoplasmic Cterminal", located in TM7-11-12. These 4 hotspot classes include 49 out of the 52 selected residues. The 3 outsider residues (354, 937 and 938) are rather isolated and located at the membrane interface. From the inward-facing conformations to the outward-facing one, there is a significant reorientation of the residues of helices TM1-6-7-12 due to a rotation of these helices consistent with recent interpretation of crystallographic data (58).
Molecular modeling of SUR. SUR1 in the outward-facing conformation was modelled with the St-MsbA and Sav1866 templates (see coordinates files in pdb format in supplemental data). This model (Fig.10A) based on ~20% sequence identity was of good quality in terms of ProSA and QMEAN scores and Ramachandran analysis. (Table S2). It reveals the position in the transmembrane domains of the residues previously shown to be involved in the binding of K ATP channel openers (12), namely Thr1285 and Met1289, and blockers (13), namely Ser1237 (Fig.10B). Their orientation towards the interior of the protein appears consistent with their presumed role. As already apparent in the alignment of Fig.S2, these residues, located in the upper (TM12E) and the lower part of the transmembrane domain (Fig.10C), correspond to P-gp residues of the above-characterized hotspots.

DISCUSSION
Evidence has been reported that the sulfonylurea glibenclamide is a substrate of P-gp (16). We show here, using an ATPase measurement assay, that other ligands of the ABC protein SUR that act as K ATP channel blockers and openers are also able to interact with the ABC protein P-gp. P-gp is a multidrug transporter responsible for the MDR phenotype of some tumor cells, and as such it is able to recognize and transport a broad panel of structurally unrelated molecules. The multispecific recognition of various substrates by P-gp constitutes the first step in the enzymatic cycle leading to the ATP hydrolysis-coupled expelling of the drugs out of the cell. However, multidrug recognition by P-gp, as well as by SUR, remains mysterious at the molecular level even after publication of several structures of multidrug transporters. Here we show that useful insight can be gained by comparing the binding properties and structures of these two rather homologous ABC proteins. Our strategy was to gather new information on shared ligands, develop models based on the latest high-resolution ABC proteins structures, and analyze the large body of existing functional, biochemical, and mutagenetic data in order to identify the structural determinants of drug recognition by both P-gp and SUR. This permitted to evidence and characterize hotspots for drug binding consistent with a multipharmacophoric model with a similar architecture for P-gp and SUR. K ATP channels blockers and P-glycoprotein. As representative K ATP channel blockers, we tested glibenclamide, tolbutamide and meglitinide. The sulfonylurea glibenclamide was the most potent (K 1/2 ~ 11 µM) at stimulating ATPase activity of Pgp. Meglitinide which represents the non-sulfonylurea half of glibenclamide had also a clear effect (K 1/2 ~ 300 µM). Tolbutamide, the sulfonylurea half of glibenclamide, was the weakest agonist (K 1/2 ~ 3 mM). The Hill coefficients for all three molecules were close to 1, suggesting a single binding site linked to ATPase activation.
Although concentrations for half-maximal stimulation of P-gp ATPase were lower by 2 to 3 orders of magnitude than those necessary to bind to SUR and block K ATP channels, the rank order of potency of these compounds was the same for P-gp and SUR1 [(49); (47)]. There appeared to be no obvious interactions between these 3 compounds in modulating P-gp ATPase since when tolbutamide or meglitinide -at high concentrations -were combined with glibenclamide, the resulting increase in ATPase activity was merely the sum of the increases elicited by each drug with no hint of competition. This finding could be considered as surprising since it rules out any overlap between the binding sites of these 3 molecules in spite of the large degree of resemblance of their chemical structures, since the benzoic acid derivative meglitinide and the sulfonylurea tolbutamide roughly represent the 2 halves of the glibenclamide molecule. This observation is reminiscent of what has been previously reported on P-gp when studying steroids (27) and dihydropyridines (29). It reveals the versatility of the possible interactions of P-gp drug binding domain with the various hydrophilic/hydrophobic recognition elements of its ligands. To explain the much higher channel blocking affinity of glibenclamide on SUR1 and its very slow dissociation rate, it has been postulated that SUR1 possesses a benzoic acid site and a sulfonylurea site, both sites being separate but close enough to allow the binding of a single molecule of glibenclamide [(49); (13)]. Such a scheme cannot apply to P-gp as it predicts a competitive behavior between glibenclamide and tolbutamide or meglitinide that we have not observed. Instead, evidence points to separate non-interacting sites on P-gp for each of the 3 molecules. This conclusion implies that one should not discard the hypothesis that SUR could also harbors distinct, though mutually exclusive sites for these molecules (see below).
To examine the relations between the site of action of glibenclamide on P-gp and the sites of established ligands of P-gp, we performed experiments combining both glibenclamide and either verapamil, progesterone, or vinblastine. Results are recapitulated in Fig.6C. Verapamil reduced the glibenclamide-induced increase in ATPase activity in a dose-dependent manner without significantly shifting the glibenclamide K 1/2 . Antagonism of activation by glibenclamide and increase in basal ATPase activity were both halfmaximal at about 1 µM verapamil, indicating that the same verapamil binding site mediates the two phenomena. The opposite experiment -effect of glibenclamide on verapamil-induced activitysimilarly supports the existence of a single binding site for glibenclamide on P-gp. These mutually noncompetitive inhibitions show that glibenclamide and verapamil bind to different, though allosterically connected, sites. The same could be concluded from the observed non-competitive antagonism of the progesterone-induced ATPase activity by glibenclamide.
Vinblastine also antagonized the glibenclamidestimulated ATPase activity much like it did for the verapamil-stimulated ATPase [(28); (51)]. Full inhibition was obtained at a vinblastine concentration of 10 µM that had by itself little impact on P-gp basal ATPase. Half-inhibition of the glibenclamide-stimulated ATPase activity was reached at ~1 µM vinblastine whereas halfinhibition of the basal activity was at 30 µM. There was also a moderate (about 2 fold) increase by 1 µM vinblastine of the K 1/2 of glibenclamide, indicative of a competition between vinblastine and glibenclamide. Assuming a 1:1 stoichiometry for this mutual exclusion leads to a K i value for vinblastine antagonism of glibenclamide stimulation of about 1 µM, which reinforces the existence of a high-affinity vinblastine binding site on P-gp, consistent with previous observations on the inhibition by vinblastine of P-gp ATPase stimulated by various P-gp substrates [(28);(29); (52)]. The observed inhibition of the basal ATPase activity by higher vinblastine concentrations is thus probably of a similar nature as the inhibition observed for verapamil above 50 µM or progesterone above 100 µM. The competition between glibenclamide and vinblastine could arise from a partial site overlap, leading to mutually exclusive steric constraints, since they possess the common hydrophilic/hydrophobic recognition elements of pharmacophore II (11), and likely bind to the same structural determinants of P-gp (see below). The absence of competition between glibenclamide and verapamil, whereas verapamil binds close to vinblastine and displays with it mutual exclusion by steric constraints without common recognition element (11), can be explained by the fact that glibenclamide molecule is smaller than vinblastine (Fig.6C).
K ATP channels openers and P-glycoprotein. Three prototypical K ATP channel openers were tested: SR47063, an analogue of cromakalim, P1075, an analogue of pinacidil, and diazoxide. The former two are known ligands of the SUR2 isoform, probably sharing overlapping binding sites (12) while the latter targets another site on both SUR1 and SUR2 isoforms (48). SR47063, P1075, and diazoxide augmented P-gp ATPase activity with K 1/2 values of 36 µM, 1.5 mM and 0.6 mM, respectively, and Hill coefficients near 1, indicative of a single binding site on P-gp for each of them. In comparison, radioligand binding assays on the SUR2B isoform (26) have yielded dissociation constants of ~300 nM for levcromakalim, a molecule close in structure and activity to SR47063, ~10 nM for P1075 and ~15 µM for diazoxide. Values for the SUR2A isoforms were about fivefold greater, while for the SUR1 isoform a dissociation constant of ~150 µM was registered for diazoxide and in the 1 mM range for the other 2 openers. Whatever the SUR isoform considered, there appears to be therefore little correlation between SUR and P-gp in terms of affinities for openers and rank order of potencies.
Binding experiments on various SUR isoforms have shown an apparent competition between K ATP channel openers and blockers [for review see (7)]. Such competition was not seen with P-gp between the two openers tested, P1075 and diazoxide, and glibenclamide, since the effects of these openers on the glibenclamide-stimulated ATPase activity were strictly cumulative. We infer that sites for openers and blockers are located in different regions of P-gp, as they probably are for SUR (50).
Combined experiments with verapamil indicate no evidence of competition between verapamil and P1075 or diazoxide. This suggests no overlap between the sites for P1075 and diazoxide and verapamil. Further tests showed a non-competitive inhibition of progesterone-stimulated activity by P1075 or SR47063 but no interaction between progesterone and diazoxide. Therefore, diazoxide and P1075/SR47063 interact differently with P-gp than they do with SUR. The experiment concerning SR47063 points to a moderate competitive inhibition between verapamil and SR47063 and to a clear inhibition by vinblastine of the SR47063stimulated activity, although the type of inhibition between these two drugs could not be determined. The existence of common recognition elements in the molecular structures of SR47063 and verapamil indicates that SR47063 may belong to the pharmacophore I, according to Garrigues et al (11). This is reinforced by the close structural resemblance of SR47063 with rhodamine 123, a compound for which recognition by P-gp has been determined to be competitive with verapamil (SM & SO, unpublished results) and to correspond to pharmacophore I (NL & SO, unpublished results). Due to the incomplete overlap with verapamil, SR47063 could sterically interfere with vinblastine, but this is difficult to ascertain experimentally. Fig.6C summarizes these observations in relation to the two pharmacophores previously described on Pgp. The data suggest a striking functional homology between SUR and P-gp for recognition of the tested ligands, especially glibenclamide and SR47063.
Implications for the molecular mechanisms of multispecific ligand recognition. Our results demonstrate a correlation between SUR and P-gp in terms of shared ligands. Does this correlation arise from the promiscuous nature of these ligands, which have a tendency to lack specificity (glibenclamide, for instance, has been postulated to be a universal ABC protein ligand (16)), or from the promiscuous nature of the target sites, or from a true structural homology between the target proteins, SUR and Pgp ? Because the structural data on eukaryotic ABC proteins are limited to the recently published structure of mouse P-gp in one conformation (22), we performed an in-silico structural modeling of Pgp and SUR, based on the various crystallographic structures of the bacterial ABC transporters MsbA and Sav1866. To validate this choice of template homologues, we performed a phylogenetic analysis of a large set of membrane ABC proteins using a global analysis method based on a maximum likelihood algorithm, that is less dependent on varying sequence lengths and that provides a quantitative measure of protein homology (59). This analysis positioned MsbA and Sav1866 close to Pgp in the ABCB branch of the phylogenetic tree. In addition, the SUR-containing ABCC branch was the closest to the ABCB branch. This supports the choice of MsbA and Sav1866 as structural homologues. Furthermore, the various satisfactory quantitative assessments of the models obtained for P-gp as well as for SUR confirmed the pertinence of this choice. Indeed, the models correctly predict (i) the intramembrane position of the various residues known to be involved in substrate recognition and (ii) the orientation of their side chains towards the interior of the protein where they delimit a variable-size "chamber", depending on the conformation of the protein.
In the case of P-gp, we modeled three different conformations, open inward-facing, closed inwardfacing and outward-facing, which are supposed to represent successive states of the enzyme during its transport cycle (20). The comparison between the open conformation and the two others revealed that residues involved in substrate recognition are not spatially distributed evenly, but segregate between several hotspots mainly located either in the "lower" / "cytosolic" or "upper" / "exoplasmic" region of the phospholipid membrane. Analysis of the abundant mutagenetic and chemical labeling data for verapamil and vinblastine binding shows that the set of hotspots 4E/5E/6E/7E/10C/11C/12E could constitute anchoring points for verapamil and the ligands belonging to its competition class; similarly, the set of hotspots 1C/6E/11C/11E could constitute anchoring points for vinblastine and its competition class. Thus, these two sets of hotspots correspond to pharmacophores I and II, respectively (11), which provides a valuable link between the enzymological analysis and the structural analysis that reinforces the multisite model for the molecular mechanisms involved in multispecific drug recognition. This rough description of the drug recognition by P-gp cannot obviously replace a deep and careful analysis of ligand binding using in-silico docking techniques, which would require a massive effort out of the scope of this report. However, this allows to propose the following interpretations. (i) The two hotspots, 6E and 11C, shared by verapamil and vinblastine, could represent the common point between the two pharmacophores that we have previously reported (11). (ii) The higher number of hotspots forming the verapamil recognition pattern could indicate that Pgp is able to bind simultaneously two verapamil molecules, of limited size, as observed for QZ-59SSS (22), but only one vinblastine molecule, which is much larger. (iii) The residues that interact with the two peptides QZ-59RRR and QZ-59SSS in the crystallographic structure of P-gp are mostly located outside the hotspots; not surprisingly, since these peculiar ligands do not share the hydrophilic/hydrophobic recognition elements of either verapamil or vinblastine (not shown). (iv) By comparing the three P-gp conformations, it appears that some of the hotspots do not move within the membrane plane (TM 6, 7, 8, 12) while others do (TM 1, 4, 5, 10, 11); this is true for both sets of hotspots linked to verapamil and vinblastine, and is consistent with the required affinity alteration for the transported substrate during the enzymatic turn-over of a typical active transporter. (v) The location of the hotspots within the membrane bilayer, found both at the level of the cytosolic leaflet and the exoplasmic leaflet, is consistent with the two dominant functional models proposed in the literature for P-gp, i.e. a membrane floppase, and a "vacuum-cleaner" expelling the transported substrate directly out of the membrane.
In the case of SUR1, we built a structural model for the outward-facing conformation, which is supposed to be the physiologically relevant enzymatic state since it is the nucleotide-bound form. SR47063 binding to SUR1 involves a protein region that corresponds to the "upper sites" of P-gp, namely residues Thr1285 and Met1289 [(12); (9)] that are equivalent to P-gp residues Ser979 and Phe983. Glibenclamide binding to SUR1 involves Ser1237 (13), equivalent to P-gp Ser931 located at the membrane interface just beyond the "lower sites" which are compatible with the size of the average conformation of free glibenclamide (18 Å). Thus, the two ligands present binding sites located in corresponding regions of SUR and P-gp when aligned. As a whole, these two multispecific ABC membrane proteins appear to harbor a common architecture of binding sites for a pattern of structurally diverse ligands.
Because the openers can augment the ATPase activity of SUR2 (19), we have hypothesized that these ligands could act as "pseudo-transport" substrates of SUR2 (12). The finding that the openers SR47063 and P1075 stimulate the ATPase activity of P-gp, suggests not only structural but also functional homology between P-gp and SUR.
Beyond these similarities, SUR and P-gp display distinct characteristics for drug recognition and binding: (i) The variety of ligands of P-gp exceeds that of SUR [(60);(61);(62)]; (ii) P-gp binds larger ligands (up to 1250 Da (60)) than SUR (up to 500 Da for glibenclamide (62)); (iii) the smallest Pgp ligands (in the Mw range of SUR ligands) have poor affinities (beyond the µM range (11)) whereas the smallest SUR ligands can have high affinity; (iv) P-gp ligands often display non-competitive interactions except if they have sufficient size (>ca 700 Da) (11), whereas SUR ligands are generally always competitive one with each other, even when they bind distinct sites [(63); (47)]. All these features can be reconciled in a model in which the volume of the "cavity" forming the P-gp multidrug binding domain is much larger than that presented by SUR. Schematically, in P-gp, the multidrug binding domain forms a continuous multispecific pocket, whereas in SUR it appears rather restrained to (at least) two discrete multispecific sites, even though the two multidrug binding domains appear built around common hotspots.
The phylogenetic analysis of the membrane ABC proteins, supported by the high values of bootstrap at several branches, shows the relative proximity of the various subfamilies. This analysis supports a close structural and functional relationship between P-gp, representative of the ABCB branch, and SUR, representative of the ABCC branch. In contrast, this cannot be extrapolated readily to the D branch, and a fortiori to the more remote A and G branches.
Therapeutical implications. Several K ATP channel blockers and openers are in clinical use for a variety of conditions including diabetes and hypertension [(64); (8)]. Since P-gp is present and active at the intestinal and blood-brain barriers as well as of excretory systems [(17); (18)], knowing if and how a molecule is transported by P-gp is useful in anticipating pharmacokinetics and tissue distribution, and potential side-effects due to drugdrug interactions. In this work, we have measured the effects of blockers and openers on P-gp ATPase activity. The correlation between ATPase activity and transport is complex and not yet fully resolved [(65); (66)]. Molecules that augment P-gp ATPase activity are likely to be transported although ATPase activation does not necessarily imply a net transport (67). Of the molecules tested here, direct evidence of transport has only be supplied for glibenclamide (16). Such evidence would be difficult to obtain for the other molecules which are either not available in labeled form or have weaker affinities. If transport indeed occurs, one can infer that the biodisponibility of these K ATP channel modulators could be altered and that interferences with P-gp transport of coadministered drugs could lead to drug-drug interaction and eventual side effects. This is expected to be a significant factor for compounds that have comparable affinities for P-gp and their intended target, SUR. This is the case for SR47063, which activates muscle K ATP channels at µM concentrations (68) and interacts with P-gp at the same concentrations. In contrast, the other compounds tested have much greater affinities for K ATP channels than for P-gp, as exemplified by glibenclamide which blocks pancreatic K ATP channels at nanomolar concentrations and activates P-gp ATPase activity at micromolar concentrations. We may anticipate that the capacity of these compounds to act across barriers should be high.
Keywords: K ATP channels, potassium channel openers, potassium channel blockers, ABC transporters, sulfonylurea receptor, MDR1/P-glycoprotein, molecular modeling, hotspots. Effect of standard P-gp ligands on ATPase activity using P-gp-containing vesicles. The ATPase activity of P-gp-devoid (control, open symbols) and P-gp-enriched (filled symbols) native membrane vesicles was measured over a range of concentrations for 3 well-characterized P-gp substrates: verapamil (squares), progesterone (triangles), and vinblastine (circles). Dashed lines represent fits of the Hill equation to the initial ligand-induced changes in ATPase activity of P-gp-enriched vesicles. Concentrations for half-maximal effect (K ½ ), Hill coefficient (h), and V max were: 1.3 µM, 0.94, and 73% for verapamil; 31 µM, 1.35, and 53% for vinblastine; and 17 µM, 2.1, and 66% for progesterone. Basal activity of P-gp-containing vesicles (i.e., 100% after normalization) was 120-300 nmol/min per mg of membrane protein while it was ~30 nmol/min/mg in control vesicles. When not visible, error bars were smaller than the symbols. Panel B. Effect of SUR ligands that block K ATP channels on ATPase activity using P-gp-containing vesicles, showing that SUR blockers are P-gp substrates with the same order of potency. ATPase activity of control (open symbols) and P-gp-enriched (filled symbols) vesicles was measured over a range of concentrations for the K ATP channel blockers, glibenclamide (squares), tolbutamide (triangles), and meglitinide (circles). The chemical structures of these compounds are illustrated on the right. Dashed lines represent fits of the Hill equation to the initial ligand-induced increases in ATPase activity of P-gp-enriched vesicles. K ½ , h, and V max were: 11 µM, 1.2, and 73% for glibenclamide; 276 µM, 0.75, and 70% for meglitinide; 3 mM, 0.68, and 70% for tolbutamide. Basal activities of P-gp enriched vesicles used for the glibenclamide, tolbutamide, and meglitinide assays were on average 126, 96, and 114 nmol/min per mg of membrane protein, respectively. Panel C. Effect of SUR ligands that open K ATP channels on ATPase activity using P-gp-containing vesicles, showing that SUR openers are low-affinity P-gp substrates. ATPase activity of control (open symbols) and Pgp-enriched (filled symbols) vesicles was measured over a range of concentrations for the K ATP channel openers, SR47063 (squares), P1075 (triangles), and diazoxide (circles). The chemical structures of these compounds are illustrated on the right. Dashed lines represent fits of the Hill equation to the ligand-induced increases in ATPase activity of P-gp-enriched vesicles. K ½ , h, and V max were: 36 µM, 1.3, and 55% for SR47063; 610 µM, 1, and 63% for diazoxide; 1.5 mM, 1.2, 101% for P1075. Basal activities of P-gp enriched vesicles used for the SR47063, P1075, and diazoxide assays were on average 150, 138, and 114 nmol/min per mg of membrane protein, respectively.

Figure 2.
Effect of various SUR ligands on the glibenclamide-induced stimulated P-gp ATPase activity, showing a lack of "cross-talk" between glibenclamide and other K ATP blockers and openers. The effects of glibenclamide on P-gp ATPase activity were measured in the absence (squares) and presence of other K ATP channel modifiers, meglitinide (1 mM, down triangles), tolbutamide (3 mM, diamonds), P1075 (1 mM, up triangles), or diazoxide (1 mM, circles). Dashed lines represent fits of the Hill equation to the glibenclamideinduced increases in ATPase activity. K ½ , h, and V max were: 11 µM, 1.2, and 73% in control; 11 µM, 0.9, and 78% with meglitinide added; 6.7 µM, 1.1, and 94% with tolbutamide; 6.5 µM, 1.1, and 81% with P1075; 9.8 µM, 1.3, and 69% with diazoxide.    Schematic representation of the functional interactions between the binding sites on P-gp for K ATP channel blockers (GLB, glibenclamide; MGL, meglitinide; TLB, tolbutamide; in dark grey), openers (SR, SR47063; DZX, diazoxide; P1075; in light grey), and known P-gp substrates (VRP, verapamil; VBL, vinblastine; PRG, progesterone; in white). Solid arrow lines represent mutual exclusiveness indicated by the competitive inhibition observed between glibenclamide and vinblastine, and between SR47063 and verapamil. Connecting lines represent binding to separate sites: either mutual destabilization reflected by observed noncompetitive inhibition, or mutual stabilization reflected by observed positive allosteric effect,or a lack of recorded interactions reflected by additive effects. The interaction between SR47063 and vinblastine is shown exclusively by a simple proximity since our data are insufficient to assess the competitive or noncompetitive nature of the observed inhibition. The dotted boxes represent the two pharmacophores previously characterized (11).    Outward-facing conformation model of SUR1. Panel B. Top-view of 3D transmembrane location of residues involved in the binding of K ATP channel openers (Thr1285 and Met1289) and blockers (Ser1237). Panel C. Structural alignment of TM11 (P-gp)/ TM16 (SUR1) and TM12 (P-gp)/ TM17 (SUR1). SUR1 residues involved in openers and blockers binding are represented and labeled in green, P-gp TM12E hotspots in red, and P-gp TM11 residue in blue.  K 1/2 : the half-activating concentration of the tested compound on P-gp basal ATPase activity; (-) indicates that it is the half-inhibiting concentration since there is no apparent activation. Ki: the half-inhibiting concentration of the tested compound on P-gp stimulated ATPase activity, as determined from the concentration-dependence curves of the substrate indicated in brackets. nd: not determined. ME: mutually exclusive, that is the mutual relationship between two drug substrates revealed by a competitive inhibiting effect on P-gp ATPase activity; NME: non-mutually exclusive, that is the mutual relationship between two drug substrates revealed by either a non-competitive inhibiting effect or an additive effect or a positive allosteric effect on P-gp ATPase activity.
Drug studied at increasing concentrations