Introduction

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ATP-sensitive K⁺ channels (K_ATP channels) are a family of K⁺ channels with weak inward rectification that are gated by intracellular nucleotides and couple cell metabolism to membrane potential. Pharmacologically, they are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by the K_ATP channel openers, which act preferentially in the vasculature producing hypotension (Ashcroft and Ashcroft, 1990; Quast, 1992; Edwards and Weston, 1993; Quayle et al., 1997). Most studies (for review, see Quayle et al., 1997) agree that the opener-activated channel in the vasculature has a low unitary conductance (15-30 pS, Kajioka et al., 1991; Beech et al., 1993b; Kamouchi and Kitamura, 1994). It is relatively insensitive to inhibition by ATP (Beech et al., 1993a; Xu and Lee, 1994) and is activated by nucleoside diphosphates in the presence of Mg²⁺ (Beech et al., 1993a); therefore, it has also been termed nucleoside diphosphate-dependent K⁺ channel (K_NDP; Beech et al., 1993a). If no opener is present, the K_NDP channel is inhibited by GBC with IC₅₀  20 to 40 nM (Beech et al., 1993a; Xu and Lee, 1994; Quast, 1996); in the presence of opener, higher concentrations of GBC are required (e.g., Beech et al., 1993b; Quast, 1996). Using a [³H]GBC binding assay, we have identified a GBC binding site in rat aorta with K_D = 20 nM, that matches exactly the pharmacological profile of the vascular K_NDP channel (Quast et al., 1993; Löffler and Quast, 1997).

The K_ATP channel in several tissues has been shown to be a heteromultimeric complex of sulfonylurea receptor (SUR) and inwardly rectifying K⁺ channel (Kir6.x) subunits (reviews: Ashcroft and Gribble, 1998; Babenko et al., 1998). For the pancreatic -cell K_ATP channel, which is composed of SUR1 and Kir6.2, an octameric structure with 4:4 stoichiometry (SUR1/Kir6.2)₄ has been proven (Clement et al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1997). The Kir6.x subunits presumably form the pore of the channel and determine the sensitivity of the channel to inhibition by ATP. Electrophysiological experiments (Ashcroft and Gribble, 1998; Babenko et al., 1998) and radioligand binding assays (Hambrock et al., 1998, 1999; Schwanstecher et al., 1998) have shown that SUR is endowed with the binding sites for the openers and the sulfonylureas that mediate the pharmacological effects of these compounds. From the recombinant K_ATP channels known to date, the construct SUR2B/Kir6.1 matches best the properties of the K_NDP channel (Yamada et al., 1997). The recombinant channel has a unitary conductance of 33 pS in high-K⁺ solution and is activated by low (µM), but inhibited by high (mM) concentrations of ATP. It is inhibited by 10 µM GBC (Yamada et al., 1997; Satoh et al., 1998); its exact GBC sensitivity, however, is not yet known.

If one wants to ascertain that the SUR2B/Kir6.1 channel represents indeed the vascular K_NDP channel, the precise determination of its GBC sensitivity is of importance. Binding experiments using the radiolabeled opener, [³H]P1075 [[³H]N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine; Bray and Quast, 1992; Manley et al., 1993] showed that in membranes from cells expressing SUR2B alone, GBC inhibited opener binding with K_i values ranging from 0.3 (Schwanstecher et al., 1998) to 2.4 µM (Hambrock et al., 1998). This is difficult to reconcile with the fact that GBC blocks the native K_NDP channels with IC₅₀ values between 20 and 40 nM (see above). To resolve this question we have performed binding studies in human embryonic kidney (HEK) cells transfected with SUR2B or SUR2B + Kir6.1 using [³H]GBC as the radiolabel; the results were compared with binding studies using [³H]P1075. In addition, the GBC sensitivity of the current through SUR2B/Kir6.1 channels was measured. It is known that the binding affinity of GBC to the vascular K_ATP channel (Löffler-Walz and Quast, 1998) and the potency of GBC in blocking the cardiac K_ATP channel (Brady et al., 1996; Yokoshiki et al., 1997) depend on the presence of an intact actin cytoskeleton. Therefore, all experiments were performed in whole cells and at 37°C. The results show that coexpression with Kir6.1 increases the affinity of SUR2B for GBC and that the GBC sensitivity of the recombinant channel is similar to that reported for the native K_NDP channel. When this manuscript was being submitted, a paper by Dörschner et al. (1999) appeared that addresses similar questions. The results of their study, which was conducted under quite different conditions, differ in central aspects from ours (see Discussion).

	Experimental Procedures

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Cell Culture and Transfection. 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 containing glutamine and supplemented with 10% fetal bovine serum and 20 µg/ml gentamycin. Cells were transfected with the pcDNA 3.1 vector (Invitrogen, San Diego, CA) containing the coding sequence of murine SUR2B and murine Kir6.1 (GenBank accession numbers D86038 and D88159, respectively; Isomoto et al., 1996; Yamada et al., 1997). Cell lines stably transfected with SUR2B were isolated as described previously (Hambrock et al., 1998). Transient transfections were performed using lipofectAMINE and OPTIMEM (Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions. HEK cells were cotransfected with SUR2B + Kir6.1 at a molar plasmid ratio of 1:1 or 1:4. In general, pEGFP-C1 vector (Clontech, Palo Alto, CA), encoding for green fluorescent protein (GFP), was added for easy identification of transfected cells; this did not affect the results of the binding studies. Cells were allowed to express transfected DNA for 48 h and were then used for binding studies and electrophysiological experiments.

Equilibrium Competition Experiments. Transiently transfected HEK 293 cells were harvested 48 h after transfection at 60 to 80% confluence, nontransfected cells or permanently transfected cells at a similar density. Cells were suspended by rinsing with a HEPES-buffered physiological salt solution (PSS) containing 139 mM NaCl; 5 mM KCl; 1.2 mM MgCl₂; 1.25 mM CaCl₂; 11 mM D(+)-glucose; 5 mM HEPES; gassed with 95% O₂/5% CO₂; and titrated to pH 7.4 with NaOH at 37°C. After two centrifugations at 500g for 5 min, cells were resuspended in PSS. Incubation was started by the addition of cells (final concentrations 2 × 10⁶ and 3 × 10⁶ cells/ml for SUR2B and SUR2B/Kir6.1, respectively, corresponding to 0.5 and 0.7 mg protein/ml) to PSS containing 2.0 to 4.1 nM [³H]GBC or 1.0 to 1.5 nM [³H]P1075 and the inhibitor of interest in a total volume of 1 ml at pH 7.4 and 37°C. After 30 min, incubation was stopped by diluting 0.3-ml aliquots in triplicate into 8 ml of ice-cold quench solution (50 mM Tris-(hydroxymethyl)-aminomethane, 154 mM NaCl, pH 7.4) and by rapidly filtrating under a vacuum over Whatman GF/C 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). In case of [³H]P1075 experiments, nonspecific binding was determined in the presence of 10 µM unlabeled P1075; in the [³H]GBC studies, nonspecific binding could not be determined because at the highest concentration of GBC soluble (200 µM), low-affinity binding was not yet saturated.

Patch-Clamp Experiments. The patch-clamp technique was used in the whole-cell configuration as described by Hamill et al. (1981) with an extracellular buffer containing 142 mM NaCl; 2.8 mM KCl; 1 mM MgCl₂; 1 mM CaCl₂; 11 mM D(+)-glucose; 10 mM HEPES; titrated to pH 7.4 with NaOH. Temperature in the bath was 37°C. Patch pipettes were drawn from filament borosilicate glass capillaries (GC 150F-15; Clark Electromedical Instruments, Pangbourne, UK) and heat polished using a horizontal microelectrode puller (Zeitz, Augsburg, Germany). After filling with 132 mM K-glutamate; 10 mM NaCl; 2 mM MgCl₂; 10 mM HEPES; 1 mM ethylene glycol-bis-(2-aminoethyl ether)-N, N,N',N'-tetraacetic acid; 1 mM Li₂GDP; 0.3 mM Na₂ATP; titrated to pH 7.2 with NaOH, pipettes had a resistance of 3 to 5 M. Data were recorded with an EPC 9 (HEKA, Lambrecht, Germany) amplifier using Pulse software (HEKA). Cell capacitance (6-22 pF) and series resistance (5-14 M) were measured before the start of each pulse train with the EPC9 amplifier-system. Series resistance was compensated by 70% with the compensation circuit of the EPC9 patch-clamp amplifier. The voltage potentials were corrected for a liquid junction potential of +10 mV. Two to three days after transfection, isolated cells showing GFP fluorescence were clamped to 60 mV and every 12 s seven square pulses ranging from 110 to 10 mV (0.5 s each) were applied. Signals were sampled with 1 kHz and filtered at 200 Hz using the four-pole Bessel filter of the EPC9 amplifier. For evaluation of the GBC-block, the current at 60 mV was used.

Data Analysis and Modeling. Equilibrium inhibition curves were analyzed according to the logistic equation for up to three components:

(1)

Here A_i denotes the amplitude of component i, n_i (= n_{H, i}) the Hill coefficient, and IC_50,i the midpoint of component i with pIC_50,i = logIC_50,i; x is the concentration of the compound under study with px = logx. Because IC₅₀ values are log-normally distributed (Christopoulos, 1998), IC₅₀ values followed by the 95% confidence interval in parentheses are given in the text. IC₅₀ values were converted into K_i by correcting for the presence of the radioligand, L, according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973):

(2)

where K_D is the equilibrium dissociation constant of the radioligand. In case of homologous competition experiments, K_i is identical with the K_D value.

Materials. [³H]P1075 [specific activity = 4.5 TBq (121 Ci)/mmol] was purchased from Amersham Buchler (Braunschweig, Germany) and [³H]GBC [specific activity = 1.85 TBq (50 Ci)/mmol] from DuPont-NEN (Bad Homburg, Germany). The reagents and media used for cell culture and transfection were obtained from Life Technologies. Na₂ATP and Li₂GDP were purchased from Boehringer Mannheim (Mannheim, Germany) and GBC from Sigma (Deisenhofen, Germany). P1075 was a gift from Leo Pharmaceuticals (Ballerup, Denmark). K_ATP channel modulators were dissolved in dimethyl sulfoxide/ethanol (50:50, v/v) and further diluted with the same solvent or with incubation buffer. In binding studies, the final solvent concentration in the assays was always below 0.3%, in electrophysiological studies 0.1.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

[³H]P1075 Binding Experiments. Specific binding of [³H]P1075 was only found in HEK cells transfected with SUR2B or SUR2B/Kir6.1; in nontransfected cells and in cells expressing Kir6.1 alone, no specific binding was detected. Figure 1 shows the inhibition of specific [³H]P1075 binding by P1075 and GBC in HEK cells transfected with SUR2B and with SUR2B + Kir6.1. The inhibition curves of P1075 were closely together with K_D values of 4.3 (SUR2B) and 5.8 nM (cotransfection, see Table 1). For GBC, however, there was a significant difference; in cells transfected with SUR2B, GBC inhibited [³H]P1075 binding with a K_i value of 1 µM; coexpression with Kir6.1 reduced the K_i value to 280 nM. In these experiments, cotransfection was performed at a molar plasmid ratio of 1:1 and it was uncertain whether all SUR2B was complexed with Kir6.1; therefore, cells were also transfected with a SUR2B to Kir6.1 plasmid ratio of 1:4. Figure 1 shows that this did not change the K_i value of GBC more, indicating that at a molar ratio of 1:1, most SUR2B existed as the complex with Kir6.1. For additional experiments, cotransfections were therefore performed at the ratio of 1:1. In the course of these experiments we noted that total [³H]P1075 binding was considerably reduced when SUR2B was coexpressed with Kir6.1 at a ratio of 1:1 and even more at a ratio of 1:4 [see total binding (B_TOT) values in the legend to Fig. 1]. This may in part reflect differences between permanently (SUR2B) and transiently (cotransfection) transfected cells; in addition, the protein synthesizing machinery of the cell may become limiting so that less SUR2B is expressed at higher Kir/SUR ratios.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Inhibition of specific [3H]P1075 binding by P1075 and GBC in HEK cells transfected with SUR2B and SUR2B + Kir6.1 at 37°C. SUR2B: P1075, ; GBC, ; cotransfection with SUR2B + Kir6.1 at a ratio of 1:1: P1075, ; GBC, ; cotransfection with a ratio 1:4: GBC, . Data are means ± S.E. from three to five experiments, and fits of the logistic equation with one component to the data are shown. IC50 values were corrected for the presence of the radiolabel according to eq. 2 and are listed in Table 1; Hill coefficients were close to 1. [3H]P1075 concentration was 1.0 (SUR2B) or 1.5 nM (cotransfection); BTOT (fmol/mg) was 248 ± 24 (SUR2B), 61 ± 6 (cotransfection 1:1), and 15 ± 8 (cotransfection 1:4); nonspecific binding was 8 ± 1, 15 ± 3, and 35 ± 15% of BTOT, respectively.

[³H]GBC Binding Experiments. [³H]GBC binding was found in control and transfected HEK cells and it was first examined whether openers interfered with this binding. P1075 (10 µM) did not displace any radioactivity in control cells and cells transfected with Kir6.1. However, in cells transfected with SUR2B or SUR2B + Kir6.1, the opener reduced total [³H]GBC binding by 15 ± 2% (n = 13, SUR2B) and 15 ± 1% (n = 10, cotransfection); levcromakalim and diazoxide produced similar inhibitions (13%). The effect of P1075 was examined in detail. Figure 2 shows inhibition curves with P1075, giving K_i values of 6.6 and 3.2 nM in cells expressing SUR2B and SUR2B/Kir6.1, respectively. These values are in good agreement with those obtained in the homologous competition studies using [³H]P1075 (Fig. 1). This suggested that the P1075-sensitive fraction of total [³H]GBC binding in transfected cells represents GBC binding to SUR2B (or to the SUR2B-Kir6.1 complex).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Inhibition of total [3H]GBC binding to HEK cells by P1075. Control cells, ; cells transfected with SUR2B, ; and with SUR2B + Kir6.1, . The fit of the logistic equation with one component and Hill coefficient 1 to the data gave for SUR2B- and SUR2B + Kir6.1-transfected cells amplitudes of 14 ± 1 and 15 ± 1% BTOT. The Ki values derived from the fits are listed in Table 1. [3H]GBC concentrations and BTOT values were 3.4 nM/163 ± 3 fmol mg1 (SUR2B) and 4.1 nM/160 ± 9 fmol mg1 (cotransfection); data are means from three to four experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Homologous competition of total [3H]GBC binding with GBC in HEK cells. Control cells, ; SUR2B-, , and SUR2B + Kir6.1-transfected,  HEK cells. [3H]GBC was 3 nM and samples were incubated for 30 min at 37°C. Data are means ± S.E. from four to six experiments. Control cells: the logistic equation with two components (i.e., A1 = 0) was fitted to the data giving for the amplitudes (A2/A3, % BTOT) 39 ± 2/37 ± 1%; the KD values were (KD2/KD3, confidence interval in parentheses) 305 (220,420) nM/31 (15,63) µM. The data from SUR2B- and from cotransfected cells were analyzed according to eq. 1 setting A1 = 15% and inserting for pIC50,2 and pIC50,3 the values determined for control cells (see text). From the fit the following parameter values were obtained (SUR2B/cotransfection): KD1: 32 (16,65)/6.3 (3.5,11.5); A2 (%): 17 ± 1/14 ± 1; A3 (%): 40 ± 3/42 ± 2; Hill coefficients were set to 1. BTOT (fmol/mg) was 109 ± 9, 134 ± 6, and 164 ± 13 for control, SUR2B-, and cotransfected cells, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Inhibition of total [3H]GBC binding to SUR2B-transfected HEK cells by GBC in the absence () and presence () of P1075 (10 µM). Experiments ± P1075 were done side by side; means of five experiments are shown. The curve relating to the data in the absence of P1075 was calculated using the parameters determined in Fig. 3; the lower curve (+ P1075) was calculated setting A1 = 0. The curves do not represent an attempt to fit the equations to the data; they merely illustrate the hypothesis that P1075 exclusively interferes with high-affinity GBC binding.

Electrophysiological Experiments. After establishing the whole cell configuration, currents were small and the calculated resting potential of the HEK cells was positive to 50 mV. In HEK cells transiently transfected with SUR2B + Kir6.1 or with SUR2B + Kir6.1 + GFP, dialysis of the cell with GDP (1 mM) in the presence of ATP (0.3 mM) activated a hyperpolarizing current (I_{K, NDP}) within 3 to 5 min (Fig. 5A). I_{K, NDP} reversed around the Nernst potential for potassium ( 100 mV, Fig. 5B) and was blocked to 95% by 0.3 µM GBC in the experiment shown in Fig. 5A. The concentration dependence of this block was measured applying GBC cumulatively to the bath and correcting for the slow fading of the current in the absence of GBC (3%/min). The resulting curve, shown in Fig. 5C, gave an IC₅₀ value of 43 nM and a Hill coefficient close to 1 (1.1 ± 0.2).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Activation of the SUR2B/Kir6.1 current and inhibition with GBC. Recordings are from a green fluorescent HEK cell transfected with SUR2B + Kir6.1 + GFP; experiments were performed at 37°C and under a physiological K+ gradient. A, current traces. After establishing the whole cell configuration, currents were initially small (right). Dialysis of the cell with GDP (1 mM) in the presence of ATP (0.3 mM) activated a hyperpolarizing current, IK, NDP (middle). GBC (0.3 µM) blocked this current by 95% (right). B, current-voltage relationships obtained from the traces in (A). Initial current, . The calculated resting potential of the HEK cells was positive to 50 mV. Current after cell dialysis with GDP and ATP, IK, NDP, . The current reversed around the Nernst potential for potassium ( 100 mV). Residual current after block by 0.3 µM GBC, . C, concentration-dependent inhibition of IK, NDP by GBC. From the fit of eq. 1 with one component to the data, an IC50 value of 43 (33,55) nM and a Hill coefficient of 1.1 ± 0.2 were obtained. Each data point represents the mean of five to eight experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

[³H]GBC versus [³H]P1075 Competition Experiments in Cells and Membranes. A major goal of this study was to determine the affinity of SUR2B for GBC. To this end we performed homologous competition studies of [³H]GBC binding to whole cells yielding a K_D value of 32 nM; in contrast, experiments using the opener [³H]P1075 gave about 30 to 45 times higher K_i values. This is in contrast to results published recently by Dörschner et al. (1999), who reported identical values using the two radiolabels (300 nM). However, in agreement with the observations presented here, we had found earlier in rat aorta that binding studies using [³H]GBC gave a K_D value for GBC of 20 nM (Löffler and Quast, 1997) whereas the K_i value, determined in heterologous [³H]P1075-GBC inhibition experiments, was 20 times higher (400 nM; Bray and Quast, 1992). Potential explanations for the different findings of the two groups will be discussed below; here we concentrate first on the question of why homologous and heterologous competition experiments can give different results.

Affinity of GBC for SUR2B and the SUR2B-Kir6.1 Complex. In this study, the K_D value for GBC binding to SUR2B has been determined to 30 nM. This represents an appreciable affinity; however, this value is about 60 times higher than that for binding to SUR1, the high-affinity SUR typical of the K_ATP channels in pancreatic -cells and in neurons (0.55 nM; Schwanstecher et al., 1998). On the other hand, early work has suggested that the SUR2 subtypes had a low affinity for GBC with K_D values in the µM range (review: Babenko et al., 1998); in their recent publication, Dörschner et al. (1999) reported a value of 0.3 µM.

Stoichiometry of GBC Binding and Channel Closure. GBC inhibited the current through the SUR2B/Kir6.1 channel with IC₅₀ = 43 (33,55) nM and Hill coefficient = 1.1 ± 0.2. This is in excellent agreement with the value of Dörschner et al. (1999) for the recombinant channels formed from SUR2B or 2A with Kir6.2 (42 nM), and it agrees very well with the IC₅₀ value of GBC inhibiting the vascular K_NDP channel in the absence of opener [rabbit portal vein: 25 nM, Beech et al., 1993a; canine coronary artery: 20 nM, Xu and Lee, 1994; rat aorta: 20-40 nM (using ⁸⁶Rb⁺ efflux), Quast, 1996]. Collectively, this is strong pharmacological evidence for the view first expressed by Yamada et al. (1997) that the SUR2B/Kir6.1 channel is indeed the opener-sensitive K_NDP channel in the vasculature.

1