Introduction

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ATP-sensitive K⁺ channels (K_ATP channels), first discovered in the heart (Noma, 1983; Trube and Hescheler, 1984), link the membrane potential to the metabolic state of the cell as reflected by the levels of nucleoside triphosphates and diphosphates (Ashcroft and Ashcroft, 1990; Quast, 1996; Yokoshiki et al., 1998). K_ATP channels have been shown to be a heteromeric complex of pore-forming subunits, which belong to the class of inwardly rectifying K⁺ channels (Kir6.x), and of sulfonylurea binding subunits (SURs) (Inagaki et al., 1995; Sakura et al., 1995; reviews: Ashcroft and Gribble, 1998; Babenko et al., 1998). SURs are members of the ATP binding cassette proteins (ABC proteins) and contain binding sites for sulfonylureas and nucleotides (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996). Based on multisequence alignments and hydropathy analyses, the classical topology of ABC proteins (i.e., 12 transmembrane helices with two intracellular nucleotide binding folds) has been proposed for the SURs with an extension of five transmembrane helices at the N terminus (Tusnády et al., 1997). Electrophysiological studies on recombinant K_ATP channels have shown that the SURs confer on the channel complex the sensitivity to the sulfonylureas, the openers, and the activating nucleotides and that they account for the major pharmacological differences between the K_ATP channels in various tissues (review: Babenko et al., 1998). The channel of the pancreatic -cell contains SUR1 (Inagaki et al., 1995; Sakura et al., 1995); current evidence suggests that the SUR in heart and skeletal muscle is SUR2A (Inagaki et al., 1996) and that in smooth muscle it is SUR2B (Isomoto et al., 1996; Yamada et al., 1997). In the SURs cloned to date (human, rat, and murine) there are species differences in the amino acid sequence (Isomoto et al., 1996; Aguilar-Bryan et al., 1998). However, SUR2A and 2B of the same species are splice variants differing only within the last carboxyl terminal exon (Aguilar-Bryan et al., 1998); in the case of the murine SUR2 isoforms, this involves the last 42 amino acids (Isomoto et al., 1996).

Recent studies examining the binding of the tritiated K_ATP channel opener [³H]P1075 ([³H]-N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine; Bray and Quast, 1992) to recombinant SUR2A (rat SUR2A, Schwanstecher et al., 1998) and SUR2B (murine SUR2B, Hambrock et al., 1998; human SUR2B, Schwanstecher et al., 1998) have provided strong support for the contention that these SURs are indeed the receptors for openers and sulfonylureas of the native K_ATP channels in cardiac and vascular smooth muscle. Binding studies with [³H]P1075 to rat cardiocytes in culture (Lemoine et al., 1996) and to cardiac membranes from rat (Löffler-Walz and Quast, 1998) and dog (Atwal et al., 1998), as well as to rat aortic rings (Bray and Quast, 1992; Quast et al., 1993), have provided detailed information on the ligand binding properties of native cardiac and vascular K_ATP channels.

In this study we investigated [³H]P1075 binding to membranes from human embryonic kidney (HEK)293 cells transfected with murine SUR2A and 2B. In agreement with other studies (Hambrock et al., 1998; Schwanstecher et al., 1998) we found that opener binding to SUR requires the presence of MgATP; the EC₅₀ values were 5 µM (SUR2A) and 3 µM (SUR2B). HPLC analysis, however, showed, that most of the nucleotide (3 µM ATP) added to the solution had been hydrolyzed at the end of the incubation period. In addition, we show for the first time that MgADP, which opens the channel by interacting with SUR (Nichols et al., 1996; Satoh et al., 1998; reviews: Ashcroft and Gribble, 1998; Babenko et al., 1998), also affects opener binding to SUR2A and 2B, modulating it in opposite directions. Significant differences between the two SURs were also observed in the kinetics and thermodynamics of [³H]P1075 binding and the modulator binding profile.

	Experimental Procedures

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Cell Culture, Transfection, and Membrane Preparation. HEK 293 cells were cultured in plastic dishes with a diameter of 9.4 cm at 37°C in a humidified atmosphere with 95% air and 5% CO₂ in minimum essential medium (MEM) containing glutamine and supplemented with 10% fetal bovine serum and 20 µg ml¹ gentamycin. At 60 to 80% confluence (10-16 million cells per dish), cells were transfected with the pcDNA 3.1 vector (Invitrogen, San Diego, CA) containing the coding sequence of murine SUR2A or murine SUR2B (GenBank accession numbers D86037 and D86038, respectively; Isomoto et al., 1996). Transfections were performed using lipofectAMINE and OPTIMEM (Life Technologies, Eggenstein, Germany) according to the manufacturer's instructions with 4 µg DNA and 25 µl lipofectAMINE per culture dish. Cells were allowed to express transfected DNA for 48 h. Control experiments were performed by omitting either DNA or lipofectAMINE. Isolation of stably transfected cells was achieved as described previously (Hambrock et al., 1998).

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Kinetics of [³H]P1075 Binding. Membranes were thawed and homogenized with a polytron homogenizer for 2 × 5 s at 10⁴ rpm at 4°C. To measure the association kinetics, membranes [final protein concentration: 200 µg ml¹ (SUR2A) and 50 µg ml¹(SUR2B)] were added to the incubation buffer containing: 139 mM NaCl, 5 mM KCl, 5 mM HEPES, 3.8 mM MgCl₂, and 3 mM Na₂ATP, so that the concentration of free Mg²⁺ was 1 mM (see below) and were supplemented with [³H]P1075 (2-5 nM) at 37°C. Aliquots (300 µl) were withdrawn at different times for separation of bound and free ligand by dilution into 8 ml of ice-cold quench solution (50 mM Tris and 154 mM NaCl, pH 7.4) and rapid filtration under vacuum over Whatman GF/B filters. Filters were washed twice with 8 ml of ice-cold quench solution and counted for ³H in the presence of 6 ml of scintillant (Ultima Gold; Packard Instruments, Meriden, CT). Nonspecific binding was determined in the presence of 10 µM unlabeled P1075 and did not change with time. Because the label concentration (L) was in large excess over the concentrations of binding sites, the data were fitted to a single exponential as function of time (t),

(1)

where B_eq denotes the concentration of the receptor-label complex at equilibrium and k_app the apparent rate constant of association. For a simple bimolecular association reaction, k_app depends on the rate constants of association and dissociation (k₊, k) and on the concentration of L as written in eq. 1 (Tallarida, 1995). K_D = k/k₊ is the equilibrium dissociation constant.

(2)

Equilibrium Competition Experiments. Membranes (SUR2A: 150 µg protein ml¹; SUR2B: 60 µg protein ml¹) were added to the incubation buffer described above containing [³H]P1075 (1.5-3 nM) and the inhibitor of interest in a total volume of 1 ml at pH 7.4 and 37°C. After equilibrium had been reached (SUR2A: 13 min; SUR2B: 30 min), incubation was stopped by diluting 0.3-ml aliquots in triplicate into 8 ml of ice-cold quench solution and filtrating as indicated above. Concentration dependencies were analyzed by fitting the logistic form of the Hill equation,

(3)

Here, b denotes the starting level of the curve, a the level at saturation, so that a-b represents the extent of the effect (amplitude); n (=n_H) is the Hill coefficient, x the concentration of the compound under study and K the midpoint of the curve with px = logx and pK = logK. The dependence of the midpoint of an inhibition curve (IC₅₀ value) on the concentration of the radioligand, L, was calculated according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973),

(4)

where K_i is the inhibition constant and K_D the equilibrium dissociation constant of the radioligand.

Determination of Nucleotides. Adenine nucleotides were analyzed with HPLC using a Grom-Sil 120 ODS-3 CP column (5 µm, 125 × 4 mm i.d.; Sykam, München, Germany) and an UV detector (UVIS 200; Sykam) for absorbance recording at 254 nm. The column was eluted at 30°C with a flow rate of 1 ml min^-1 and a low pressure gradient. Eluent A consisted of 65 mM potassium phosphate, pH 4.6, and 5 mM tetrabutylammonium sulfate as ion-pair forming agent. Solvent B was solvent A + 40% (v/v) acetonitrile. The mobile phase was kept at 100% solvent A for 3 min after injection of the sample (50 µl). After 4 min, a linear gradient was started to increase solvent B to 23% at 10 min and to 60% at 17 min; thereafter, solvent B was kept to 60% for 3 min. To reequilibrate the system, solvent A had to be kept at 100% for 8 min before the next sample injection. The chromatogram was completed within 30 min. AMP, ADP, and ATP were identified by their retention times (AMP, 5.8 min; ADP, 10.25 min; and ATP, 13.15 min) and quantified by peak area measurement by means of online computing integrator (AXXIOM chromatographic system 747; Sykam). Samples were prepared as described for the equilibrium binding studies. Incubation was stopped by filtration using FP 030/30 filters (Schleicher & Schuell, Dassel, Germany) and the filtrate stored immediately at 80°C for HPLC analysis.

Data Analysis. Fits of the equations to the data were performed according to the method of least-squares using the FigP program (Biosoft, Cambridge, UK). Errors in the parameters derived from the fit to a single curve were estimated using the univariate approximation (Draper and Smith, 1981) and assuming that amplitudes and pK values are normally distributed. In the text, pK ± S.E.M. or K values with the 95% confidence interval in parentheses are given. Propagation of errors was taken into account according to Bevington (1969).

Materials. [³H]P1075 (specific activity 121 Ci mmol¹) was purchased from Amersham Buchler (Braunschweig, Germany). The reagents and media used for cell culture and transfection were purchased from Life Technologies. Na₂ATP was purchased from Boehringer Mannheim (Mannheim, Germany); EDTA and UTP were purchased from Fluka (Deisenhofen, Germany); and creatine phosphate, creatine kinase, phosphoenol pyruvate, and pyruvate kinase were purchased from Sigma (Deisenhofen, Germany). Acetonitril and tetrabutylammonium sulfate (HPLC grade) were obtained from Merck (Darmstadt, Germany). The following drugs were kind gifts of the pharmaceutical companies indicated in parentheses: aprikalim (Rhône-Poulenc Rorer, Paris, France), AZ-DF 265 (4-[[N-(-phenyl-2-piperidino-benzyl) carbamoyl]methyl] benzoic acid (Thomae, Biberach, Germany), diazoxide (Essex Pharma, München, Germany), levcromakalim (SmithKline-Beecham, Harlow, UK), nicorandil (Chugai, Tokyo, Japan), P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine; Leo Pharmaceuticals, Ballerup, Denmark). Minoxidil sulfate and the active enantiomer of pinacidil (()pinacidil) were synthesized by Dr. W. P. Manley (Novartis, Basel, Switzerland). Glibenclamide was from Sigma. K_ATP channel modulators were dissolved in ethanol and dimethyl sulfoxide (1:1) and further diluted with the same solvent or with incubation buffer; the final solvent concentration in the assays was always below 0.3%.

	Results

Top Summary Introduction Experimental Procedures Results Discussion References

Kinetic Experiments. The association and dissociation kinetics of [³H]P1075 binding at 37°C to membranes from HEK cells transfected with SUR2A and 2B are illustrated in Fig. 1. With SUR2A, the kinetics at 1.9 nM [³H]P1075 were fast and close to the resolution limit of the filtration assay (fastest sampling rate 30 s per point). The rate constant of dissociation, k, was determined to 0.61 ± 0.01 min¹, corresponding to a half-time (T_1/2) of 1.1 min; the apparent rate constant of association, k_app, at 1.9 nM [³H]P1075 was 0.67 ± 0.03 min¹. Assuming a one-step bimolecular binding mechanism, the rate constant of association, k₊, is calculated according to eq. 1 to (3.2 ± 1.6)*10⁷ M¹ min¹, where the large error in this value follows from the laws of error propagation (Bevington, 1969). From these kinetic values, K_D is calculated to 19 ± 10 nM, a value in excellent agreement with the K_i value of 17 nM determined in equilibrium competition experiments (see Fig. 7 and Table 2. Figure 1A shows that after 10 min, [³H]P1075 binding to SUR2A started to decline, and, after 30 min, tended to plateau at 80%. Attempts to stabilize binding by coupling of ATP-regenerating systems like creatine kinase (20 U ml¹) and creatine phosphate (10 mM) or pyruvate kinase (20 U ml¹) and phosphoenol pyruvate (10 mM) in the presence of 13 mM Mg²⁺ did not improve stability, whereas in the presence of other nucleotides like UTP or ATPS (3 mM, instead of ATP), stability decreased (data not shown). For equilibrium experiments, incubation was stopped after 13 min when binding was still at its maximum; in view of the rapid kinetics (T_1/2 = 1.1 min), equilibrium was reached.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Kinetics of [3H]P1075 binding to membranes from HEK cells transfected with SUR2A (A) and SUR2B (B) at 37°C. A, , association of [3H]P1075 (1.9 nM) with SUR2A and , dissociation of the complex, induced by addition of 10 µM unlabeled P1075 after 10 min. B, , association of [3H]P1075 (4.8 nM) with SUR2B and , dissociation of the complex, addition of 10 µM unlabeled P1075 after 30 min. Note different time scales in A and B. Fit of monoexponential kinetics to data (n = 3) gave for kapp 0.67 ± 0.03/0.221 ± 0.006 min1 and for k 0.61 ± 0.01/0.070 ± 0.002 min1 for SUR2A/2B, respectively. Data are normalized to percentage of specific binding (Bs) at maximum, which was 57 ± 4/230 ± 10 fmol mg protein1 for SUR2A/2B, respectively.

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ATP Saturation Curves. Figure 2A shows the dependence of [³H]P1075 binding to SUR2A on [ATP] at a total Mg²⁺ concentration of 2.2 mM. ATP supported binding with an EC₅₀ value of 5 µM and Hill coefficient of 1. This value was 20 times lower than that found for the ATP dependence of [³H]P1075 binding in rat cardiac membranes (EC₅₀ = 100 µM; Löffler-Walz and Quast, 1998); however, the latter experiments had to be performed in the presence of an ATP-regenerating system to assure continued presence of ATP in spite of a high nucleotidase activity of the preparation (Löffler-Walz and Quast, 1998; see also Dickinson et al., 1997). Initially, we used the system also employed with cardiac membranes (Löffler-Walz and Quast, 1998), which consisted of creatine phosphate (20 mM), creatine kinase (50 U ml¹), and Mg²⁺ (25 mM) in the presence of 20 mM HEPES to preserve pH (Stryer, 1995). Later experiments showed that with SUR₂ this could be reduced to creatine phosphate (3 mM), creatine kinase (5 U ml¹), Mg²⁺ (10 mM) and 10 mM HEPES, and these concentrations were used routinely. Adding these components to the incubation solution, the ATP-dependence of [³H]P1075 binding to SUR2A was shifted from 5 to 110 µM, a value similar to that observed in cardiac membranes. Control experiments showed that neither high Mg²⁺, nor creatine phosphate, nor the enzyme alone had any effect and that only the three components together produced the rightward shift of the ATP activation curve (not illustrated).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Dependence of [3H]P1075 binding to SUR2A and 2B on total ATP concentration, [ATP]o, and effect of an ATP-regenerating system. ATP was added as Na2ATP. A, SUR2A: in absence ()/presence () of an ATP-regenerating system, ATP activated binding to SUR2A with pEC50 values of 5.30 ± 0.01/3.96 ± 0.05 and Hill coefficients = 0.97 ± 0.03/1.03 ± 0.11, respectively. B, SUR2B: , absence of ATP-regenerating system: ATP activated binding with pEC50 = 5.47 ± 0.08 and nH = 1.04 ± 0.18; at ATP >1 mM, binding increased further to a final level of 166 ± 4%. , Addition of ATP as MgATP. , Presence of ATP-regenerating system. ATP activated binding with pEC50 = 5.37 ± 0.04; amplitude = 183 ± 10%; nH = 1.18 ± 0.11. Membranes (SUR2A/2B) were incubated with [3H]P1075 (2.5/1.5 nM) for 13/30 min at 37°C in presence of 2.2 mM Mg2+(n = 4 for each point). The ATP- regenerating system consisted of creatine phosphate (3 mM), creatine kinase (5 U ml1), Mg2+ (10 mM) in presence of 10 mM HEPES to buffer pH against activity of the ATP-regenerating system that consumes 1 proton/cycle (Stryer, 1995). Data were normalized with respect to (specific) binding at 1 mM ATP, which was 58 ± 3/150 ± 50 fmol mg protein1 for SUR2A/SUR2B, respectively.

MgADP Dependence. It was thought that the effects of the ATP-regenerating system reflected the depletion of ADP. This hypothesis was tested in experiments performed at 30 µM ATP, a concentration at which [³H]P1075 binding to both isoforms of SUR2 is essentially saturated under control conditions but where coupling of the ATP-regenerating system should produce a major effect (Fig. 2). Indeed, adding the ATP-regenerating system depressed binding to SUR2A to 45% but increased binding to SUR2B to 188% of control (Fig. 3). Addition of 1 mM ADP reversed the effects of the ATP-regenerating system and brought binding back to control values (Fig. 3). Analogous experiments were performed using the phosphoenolpyruvate (3 mM)/pyruvate kinase (5 U ml¹) system (Mg²⁺= 10 mM). Coupling of this system produced effects similar to those obtained with the creatine-based system; again these changes were reversed by addition of ADP (n = 3; not shown). These experiments clearly show that it is depletion of ADP by the ATP-regenerating systems that produced the observed effects.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Reversal of effects of ATP-regenerating system by ADP. ATP-regenerating system was: creatine phosphate (3 mM), creatine kinase (5 U ml1), and Mg2+ (10 mM). Binding after addition of 30 µM ATP under control conditions was 48 ± 3 fmol mg protein1 and 120 ± 15 fmol mg protein1 at 2.5 and 1.5 nM [3H]P1075 for SUR2A (solid bars) and SUR2B (crosshatched bars), respectively.

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View this table: [in this window] [in a new window]   TABLE 1 Determination of adenine nucleotides in incubation samples Samples were incubated for 13 min (SUR2A) or 30 min (SUR2B) min at 37°C in absence or presence of SUR2A (150 µg membrane protein ml1) or SUR2B (60 µg membrane protein ml1), the ATP-regenerating (reg.) system (creatine phosphate, 3 mM; creatine kinase, 5 U ml1; Mg2+, 10 mM; HEPES, 10 mM) and ADP (1 mM). Each sample was prepared at least three times and analyzed three to five times.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Dependence of [3H]P1075 binding to SUR2A () and 2B () on [ADP] in presence of 1 mM ATP and [Mg2+]free 1 mM (n = 3-5). Data are normalized to 100% at standard conditions (1 mM ATP, 1 mM [Mg2+]free, no ADP added). First two ADP concentrations are from Table 1, other concentrations represent sum of endogenously formed + added ADP. Fits were performed according to eq. 3 with Hill coefficient = 1, giving for SUR2A/2B pK values of 3.47 ± 0.12/4.89 ± 0.10; starting levels (b) were 95 ± 2/340 ± 35%, and amplitudes (b-a) 41 ± 3/299 ± 34%, respectively (see text for details).

Mg²⁺ Dependence. The dependence of [³H]P1075 binding to SUR2A and 2B on [Mg²⁺]_free at saturating [ATP] (3 mM) is illustrated in Fig. 5. Both curves were biphasic. In either case, the first component of the curves was activatory with EC₅₀ values of 0.8 µM (SUR2A) and 0.6 µM (SUR2B) and Hill coefficients (n_H) of 1. With SUR2A, the second component showed a further activation leading to more than a doubling of binding with EC₅₀ 70 µM and n_H = 1. For SUR2B, the second component was inhibitory and binding decreased by about one-half with IC₅₀ 170 µM and n_H = 1. Basal binding in the absence of Mg²⁺ was 10%, caused by contaminations of Na₂ATP with Mg²⁺ (Hambrock et al., 1998). In the case of SUR2B, the creatine-based ATP-regenerating system was coupled at saturating [Mg²⁺]_free (>3 mM). Under these conditions binding increased from 100 to 165 ± 2% (n = 4, data not illustrated). This showed that at saturating Mg²⁺ ADP is important and it suggested that the second component of the [Mg²⁺]_free dependence reflects changes in MgADP similar to those seen in Fig. 2. Similarly, one may speculate that the first component reflects changes in MgATP. Indeed, a replot of these data as function of [MgATP] gave a regular concentration dependence for the first component with EC₅₀ values of 42 ± 6 and 20 ± 3 µM for SUR2A and SUR2B, respectively; however, the second component of this plot was extremely steep and compressed into less than 1 order of magnitude (replot not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Dependence of [3H]P1075 binding to SUR2A () and 2B () on [Mg2+]free, in presence of 3 mM ATP and 1 mM EDTA (n = 4). Data are normalized to 100% at standard conditions (1 mM ATP, 1 mM [Mg2+]free) with absolute values given in Fig. 2. Logistic form of Hill equation with two components was fitted to data, giving for first component pEC50 values for SUR2A/2B of 6.08 ± 0.10/6.21 ± 0.06 and amplitudes (%) of 39 ± 3/224 ± 8; for second component, pEC50 values were 4.14 ± 0.06/3.77 ± 0.08 and amplitudes of 52 ± 3/-135 ± 7%.

Temperature Dependence. Initial experiments at 0°C showed very low binding of [³H]P1075 to SUR2A but good binding to SUR2B; at 37°C, however, binding to SUR2A was increased 10-fold and that to SUR2B was decreased by 30%. These observations prompted us to investigate the temperature dependence of binding in more detail. First, the association kinetics were measured at 24, 12, and 0°C to determine the appropriate incubation times (see legend to Fig. 6). Figure 5A shows that binding of [³H]P1075 to SUR2A at equilibrium decreased continuously in the temperature range from 37 to 0°C to reach a level of 11 ± 2% at 0°C. In contrast, [³H]P1075 binding to SUR2B exhibited a bell-shaped temperature dependence increasing by more than a factor of 2 at 24 and 12°C; at 0°C, binding was still 150% of that at 37°C.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Temperature dependence of [3H]P1075 binding to SUR2A (solid bars) and SUR2B (cross-hatched bars). Specific binding (Bs) measured in presence of 2.8 nM [3H]P1075 (SUR2A) or 4.5 nM [3H]P1075 (SUR2B). Measurement of association kinetics indicated that following incubation times (minutes) were sufficient to reach equilibrium (SUR2A/2B): 37°C: 13/30; 24°C: 45/90; 12°C: 60/180; 0°C: 240 min for SUR2B. At 0°C, binding to SUR2A was too small to perform kinetic and homologous competition experiments and samples were incubated for 300 min. BS is normalized with respect to values at 37°C, which were 55/243 fmol mg protein1 for SUR2A/2B, respectively. Data are from three to four experiments.

Pharmacological Properties. [³H]P1075 binding was inhibited by K_ATP channel openers and blockers with regular inhibition curves (Hill coefficient 1) reaching 100% with the exception of minoxidil sulfate where maximum inhibition was only 70%. Figure 7 illustrates the inhibition curves of the openers pinacidil, minoxidil sulfate, and diazoxide and of the inhibitor, glibenclamide, in SUR2A-containing membranes; the pK_i values of all compounds tested are listed in Table 2. The results for several channel modulators obtained in membranes with SUR2B have been published before (Hambrock et al., 1998); they are included in Table 2 together with additional values for pinacidil and nicorandil determined in this study. Also listed are the pK_i values of these compounds obtained against [³H]P1075 in rat cardiac membranes (Löffler-Walz and Quast, 1998) and in rat aortic strips (Quast et al., 1993). The results of the correlation analysis are presented in Table 3. Excellent correlations were obtained comparing the potencies at SUR2A and SUR2B with those in heart membranes and rat aortic strips, respectively; in addition, slopes were near unity and the correlation lines were close to the line of identity. The comparison of opener potencies toward SUR2A with those toward SUR2B gave similar results concerning correlation coefficient and slope but showed that openers were on average 3.5 times more potent at SUR2B.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Competition between [3H]P1075 and selected KATP channel modulators binding to SUR2A. , P1075, , minoxidil sulfate, , glibenclamide, and , diazoxide. Data are means ± S.E.M. from four experiments. From fit of logistic equation to pooled data IC50 values of 19 nM (P1075), 166 nM (minoxidil sulfate), 370 nM (glibenclamide), and 46 µM (nicorandil) were calculated; Hill coefficients were close to 1. Note that maximum inhibition by minoxidil sulfate was only 73 ± 2%. mean pKi values determined from fits to individual inhibition curves and corrected for presence of radiolabel are listed in Table 2. Inhibition of [3H]P1075 binding was studied in presence of [3H]P1075 (3 nM) and inhibitor of interest at 3 mM ATP and a free Mg2+ concentration of 1 mM; 100% (specific) binding corresponds to 63 ± 3 fmol mg protein1 and nonspecific binding amounted to 29 ± 2% of total binding.

View this table: [in this window] [in a new window]   TABLE 2 Inhibition of [3H]P1075 binding by KATP channel modulators Binding experiments with SUR2A were performed as shown in Fig. 7. Logistic equation (see Experimental Procedures) was fitted to individual competition curves (n = 4) yielding pIC50, amplitudes (100% with exception of minoxidil sulphate, see below) and Hill coefficients nH (1). pIC50 values were corrected for presence of the radiolabel according to equation of Cheng-Prusoff which, on logarithmic scale, corresponded to addition of 0.07. Data for SUR2B are from Hambrock et al. (1998) with exception of values for nicorandil and () pinacidil, which were determined in this study. pKi values in rat heart microsomes are from Löffler-Walz and Quast (1998) with a Cheng-Prusoff correction of 0.06, and, those for rat aortic strips from Quast et al., 1993. Temperature was 37°C in all cases. In all four preparations, minoxidil sulfate inhibited [3H]P1075 binding only by 68 to 75%.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

MgATP and [³H]P1075 Binding to SUR2A and SUR2B. This study showed that addition of ATP in the presence of mM Mg²⁺ enabled binding of the opener [³H]P1075 to SUR2A and SUR2B with EC₅₀ values of 5 and 3 µM, respectively (see also Hambrock et al., 1998; Schwanstecher et al., 1998). In addition, Schwanstecher et al. (1998) showed that nonhydrolyzable ATP-analogs do not support opener binding to SUR2B. The HPLC measurements performed in this study showed, however, that at the end of the incubation period, >90% of the 3 µM ATP originally present had been hydrolyzed and that this was due mostly to the ATPase activity of proteins other than SUR. Hence, the EC₅₀ values for ATP determined here and elsewhere cannot be taken at face value. On the other hand, the ATPase rate of SUR is unknown and the nucleotides bound to SUR need not to be at equilibrium with the changing nucleotide composition of the incubation solution, in particular if the hydrolytic activity of SUR is low.

Effect of MgADP. A major new result of this study is the observation that, in the obligatory presence of MgATP, MgADP is an important modulator of [³H]P1075 binding. At SUR2A, MgADP (<100 µM) shifted the MgATP dependence of binding toward the left by 20 times; higher concentrations increased binding. At SUR2B, MgADP inhibited opener binding by three- fourths. Several points deserve comment: First, the opposite direction of the MgADP effect at the two SUR2 isoforms (which only differ in their carboxyl terminus) suggests that the carboxyl terminus affects the MgADP binding site. Second, the inhibitory effect of MgADP on opener binding to SUR2B is intriguing because MgADP opens the vascular K_ATP channel (=nucleoside diphosphate-dependent K⁺ channel, K_NDP; Beech et al., 1993) and the SUR2B/Kir6.1 construct (Satoh et al., 1998). Third, SUR2B, in the presence of 1 mM ATP, senses changes in the ADP concentration from 10 to 100 µM, suggesting that the nucleotide binding site that mediates the MgADP effects has a >10-fold selectivity for MgADP over MgATP. In this context it is of interest that on the multidrug resistance-associated protein, another ABC protein, a binding site with high selectivity for nucleoside diphosphates has been described that is distinct from the catalytic (nucleoside triphosphate) site (Chang et al., 1998). Occupation of this site stimulates the ATPase activity of the protein severalfold.

Temperature Dependence. A surprising result of this study is the opposite temperature dependence of [³H]P1075 binding to SUR2A and 2B; falling temperatures decreased binding to SUR2A monotonously but induced a bell-shaped increase in binding to SUR2B. Necessarily, these changes reflect the thermodynamics of the interaction of the opener and of the nucleotides with SUR and the temperature dependence of ADP formation. Lowering temperature will lead to a decrease in the amount of ADP formed and this may contribute to the observed decrease in binding to SUR2A and to the increase with SUR2B. A detailed interpretation of the data requires the direct measurement of nucleotide binding to SUR which, in turn, requires very high expression of the proteins.

Pharmacological Properties. Openers representative of the different chemical families of this class of drugs as well as glibenclamide inhibited [³H]P1075 binding to murine SUR2A with Hill coefficient of 1 and to completion; the exception was minoxidil sulfate with only 73% inhibition. Similar observations had been made for minoxidil sulfate in membranes from HEK cells transfected with murine SUR2B (Hambrock et al., 1998), in cardiac membranes (Löffler-Walz and Quast, 1998), in A10 cells (a cell line derived from embryonic rat aorta; Russ et al., 1997), and in calf coronary myocytes (Lemoine et al., 1996). For human SUR2B expressed in COS cells, Schwanstecher et al. (1998) reported a biphasic inhibition curve with about equal amplitudes for the low- and the high-affinity component. These results have mostly been interpreted as reflecting heterogeneity of the otherwise homogeneous opener sites, although allosteric mechanisms were not ruled out (Hambrock et al., 1998; Löffler-Walz and Quast, 1998; Schwanstecher et al., 1998). Alternatively, minoxidil sulfate could transfer its sulfate group to a neighboring amino acid, thereby inhibiting further binding of the drug to the receptor site (W. P. Manley, personal communication); protein sulfation by minoxidil sulfate has been reported to occur easily (Meisheri et al., 1993).

Conclusion. This study has shown that MgADP stimulates opener binding to SUR2A but inhibits binding to SUR2B. One may speculate that the carboxyl termini of these SURs fold back to affect the interaction of MgADP with its binding site. Finally, the results suggest that the carboxyl terminus may form part of the binding pockets for openers and glibenclamide; alternatively, it may affect these binding pockets allosterically. Further work, including mutational analyses of the SURs is required to decide between these possibilities.