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J. Biol. Chem., Vol. 277, Issue 43, 40196-40205, October 25, 2002

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The Stereoenantiomers of a Pinacidil Analog Open or Close Cloned ATP-sensitive K⁺ Channels^*

Ulf Lange, Cornelia Löffler-Walz, Heinrich C. Englert, Annette Hambrock, Ulrich Russ, and Ulrich Quast^§

From the Department of Pharmacology and Toxicology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany and  Aventis Pharma Deutschland GmbH, D-65926 Frankfurt, Germany

Received for publication, July 5, 2002, and in revised form, August 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP-dependent K⁺ channels (K_ATP channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening K_ATP channels. In the vasculature, openers of K_ATP channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be K_ATP channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, the R-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC₅₀ ~50 nM in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that the R-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas the S-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a K_ATP channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP-dependent K⁺ channels (K_ATP channels)¹ are gated by the intracellular ATP/ADP ratio with ATP inducing channel closure and MgADP channel opening. Functionally, these channels link membrane potential and excitability to the metabolic state of the cell (1, 2). Pharmacologically, K_ATP channels are closed by the hypoglycemic sulfonylureas such as glibenclamide and their benzoic acid analogs, the glinides; these drugs are used in the treatment of diabetes type 2. K_ATP channels are activated by the K⁺ channel openers, a chemically heterogeneous group of compounds including the cyanoguanidines like pinacidil and P1075 and the benzopyrans such as levcromakalim; these compounds relax smooth muscle and induce hypotension (2-5).

K_ATP channels are heteromeric complexes composed of pore-forming inwardly rectifying K⁺ channels (Kir6.x) and sulfonylurea receptors (SURs) (6, 7) (for reviews, see Refs. 8-10). Kir6.x is encoded by either one of two genes giving rise to two subtypes, Kir6.1 and -6.2, with the latter being present in most K_ATP channels. ATP binding to Kir6.2 induces channel closure, thus accounting for the inhibitory action of the nucleotide (11). SUR is a member of the ATP-binding cassette protein superfamily (7, 12). It carries binding sites for nucleotides, the nucleotide binding folds (13), and for the sulfonylureas and the openers (12, 14, 15). SUR also is encoded by either one of two genes. SUR1, expressed in the pancreatic -cell and predominantly in neurons, exhibits high affinity for the sulfonylureas and low affinity for the openers. SUR2 is expressed in the various muscle types and shows high affinity for the openers but lower affinity for the sulfonylureas (14, 15) (for reviews, see Refs. 5 and 16). Alternative splicing of the SUR2 mRNA leads to two isoforms, SUR2A and SUR2B, which differ only in the last carboxyl-terminal exon. SUR2A is expressed in skeletal and heart muscle; SUR2B is expressed in smooth muscle (17, 18).

The nucleotides not only gate the channel, they also profoundly affect the potency and efficacy of the synthetic K_ATP channel modulators by allosteric interactions. First, there is a positive allosteric interaction between nucleotide and opener binding, enabling SUR to bind openers with high affinity (14-16, 19, 20). Conversely, openers enhance the ATPase activity of SUR2A, thereby promoting channel activation (21). Second, there is a negative allosteric interaction between nucleotide and sulfonylurea binding (22, 23). Third, there is a negative allosteric coupling between opener and sulfonylurea binding, generally leading to mutually exclusive binding of the two ligands to SUR (14, 15, 19, 24).

In varying the cyanoguanidine structure, Khan and colleagues (25) discovered that introduction of a phenyl ring in the side chain (Fig. 1) gave compounds that potently reversed the vasodilation produced by various K_ATP channel openers but not that by other vasodilatory maneuvers; in addition, the inhibitory effect resided in one (i.e. the R-) enantiomer. It was concluded that the novel compounds acted as K_ATP channel blockers. The results of their study may be interpreted in two ways. First, the compounds may have acted as "neutral antagonists" (i.e. by displacing the opener from SUR without directly affecting channel activity); channel closure would then be induced by the high intracellular ATP concentrations in the vascular smooth muscle cell. Alternatively, the compounds may have acted as "inverse agonists" (i.e. they displaced the opener, and, similar to the sulfonylureas, their binding induced channel block also in the absence of ATP). In order to decide between these alternatives, the enantiomers of one such compound, PNU-94750 (Fig. 1), were examined in electrophysiological experiments using recombinant K_ATP channels. Since K_ATP channel openers and blockers (such as sulfonylureas) differ in their allosteric coupling to MgATP binding (see above), we also investigated the effect of MgATP on the binding of the enantiomers in radioligand binding assays. The study showed that the enantiomers of PNU-94750 differed in their coupling to MgATP binding and that the R-enantiomer (PNU-96293) was negatively coupled and inhibited K_ATP channels, whereas the S-enantiomer (PNU-96179) was positively coupled and was an opener.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structures of P1075, pinacidil, and PNU-94750. PNU-94750 is the racemic mixture of PNU-96179 (S-enantiomer) and PNU-96293 (R-enantiomer). The asterisk indicates the chiral center of the compound.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection-- SUR2B(Y1206S) was constructed from murine SUR2B (GenBank^TM D86038 (18)) as described (26). Human embryonic kidney (HEK) 293 cells were cultured in minimum essential medium containing glutamine and supplemented with 10% fetal bovine serum and 20 µg/ml gentamycin (14). Cells were transfected with the expression vector pcDNA3.1 (Invitrogen) containing the coding sequence of murine SUR2B, SUR2B(Y1206S), murine SUR2A (GenBank^TM D86037 (18)), or rat SUR1 (GenBank^TM X97279), and cell lines stably expressing these proteins were generated as described (14). Cotransfection of SUR with murine Kir6.2 (D50581 (17)) was done transiently at a molar plasmid ratio of 1:1 using LipofectAMINE and Opti-MEM (Invitrogen) as described previously (14). In cotransfections prepared for electrophysiological experiments, the pEGFP-C1 vector (CLONTECH, Palo Alto, CA), encoding for green fluorescent protein, was added for easy identification of transfected cells. 2-4 days after transfection, cells were used for binding studies and electrophysiological experiments.

Electrophysiological Experiments-- The patch-clamp technique was used in the inside-out, the cell-attached, and the whole-cell configuration as described by Hamill et al. (27). Patch pipettes were drawn from borosilicate glass capillaries (GC 150 or GC 150T; Harvard Apparatus, Edenbridge, UK) and heat-polished using a horizontal microelectrode puller (Zeitz, Augsburg, Germany). For experiments using inside-out patches, bath and pipette were filled with a high K⁺-Ringer solution containing 142 mM KCl, 2.8 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 11 mM D-(+)-glucose; 10 mM HEPES, titrated to pH 7.4 with NaOH at 22 °C. After filling with buffer, pipettes had a resistance of 1-1.5 megaohms. After excision of the patch, the pipette was moved in front of a pipe with a high K⁺-EGTA-Ringer solution containing 143 mM KCl, 0.85 mM MgCl₂, 1 mM CaCl₂, 5 mM EGTA, 11 mM D-(+)-glucose, 10 mM HEPES, titrated to pH 7.2 with NaOH at 22 °C. The PNU compounds and glibenclamide were dissolved in stock solutions as described under "Materials"; ATP was dissolved in buffer, and an appropriate amount of MgCl₂ was added to keep free Mg²⁺ constant at 0.7 mM. Substances were applied to the patch via the pipe, and patches were clamped to 50 mV. For evaluation of the inhibition by PNU-96293, traces were individually corrected for run down.

For experiments in the cell-attached configuration, bath and pipette were filled with the high K⁺-Ringer solution described above. After filling, the pipettes had a resistance of 1-1.5 megaohms. Patch experiments were performed at 37 °C, and patches were clamped at 50 mV.

Experiments using the whole-cell configuration were performed as described by Russ et al. (28). The bath solution was 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 at 37 °C. Patch pipettes were filled with 132 mM potassium glutamate, 10 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 1 mM EGTA, 1 mM Na₂ATP, titrated to pH 7.2 with NaOH, and had a resistance of 3-5 megaohms. Isolated cells were chosen. After establishing the whole cell configuration, cells were clamped to 60 mV, and every 12.5 s, the following square pulse protocol was applied. After 1 s at 60 mV, cells were clamped to the test potential of 110 mV for 0.5 s and then, after 1 s at 60 mV, to the next test potential of 90 mV, etc. (seven steps increasing by 20 mV from 110 to 10 mV). At the end of the protocol, capacitance and series resistance were measured, and the latter was compensated by 70%. To show the time-dependent current at a specified voltage, data were averaged over the last 125 ms spent at that voltage during the cycle and spliced together to give a continuous curve.

Data were recorded with an EPC 9 amplifier (HEKA, Lambrecht, Germany) using the "Pulse" software (HEKA). Signals were filtered at 200 Hz using the four-pole Bessel filter of the EPC9 amplifier and sampled with 1 kHz.

Membrane Preparation, Equilibrium Competition Experiments, and Dissociation Kinetics-- For cells stably expressing SUR alone, the antibiotic was withdrawn from the culture medium 1 week prior to membrane preparation. Membranes were prepared as described (14). Protein concentration was determined according to Lowry et al. (29) using bovine serum albumin as the standard.

In equilibrium binding assays, membranes (final protein concentration 0.2-0.5 mg/ml) were added to the incubation buffer containing 139 mM NaCl, 5 mM KCl, 5 mM HEPES, 2.2/1.2 mM MgCl₂, 1/0.003 mM Na₂ATP and supplemented with the radioligand ([³H]glibenclamide ~2.5 nM or [³H]P1075 ~2 nM) and the inhibitor of interest at 37 °C. In the Mg²⁺-free experiments, Mg²⁺ was omitted from the incubation solution, and EDTA (1 mM) was added. At equilibrium (15 min for [³H]glibenclamide and 30 min for [³H]P1075 binding), 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 rapid filtration under vacuum over Whatman GF/B filters (Whatman, Clifton, NJ). 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 Instrument Co., Meriden, CT). Nonspecific binding of [³H]P1075 was determined in the presence of 10 µM P1075, and that of [³H]glibenclamide was determined in the presence of 100 µM P1075 or 1 µM glibenclamide for SUR2B(Y1206S) and SUR1, respectively.

For measurement of the dissociation kinetics, membranes were incubated with 2 nM [³H]P1075 in the presence of 2.2 mM MgCl₂ and 1 mM Na₂ATP at 37 °C for 30 min. Dissociation was initiated by the addition of P1075 + PNU-96293 (10 µM each), and 300-µl aliquots were withdrawn at different times for filtration as described above.

Equilibrium Binding Experiments in Cells-- Experiments were conducted at 37 °C with an incubation time of 30 min as described previously (14, 28). Cells were suspended by rinsing with a HEPES-buffered physiological salt solution 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 and 5% CO₂; and titrated to pH 7.4 with NaOH at 37 °C. Binding experiments were started by the addition of cells (final concentration 1 × 10⁶ cells/ml, corresponding to 0.25 mg of protein/ml) to the buffer supplemented with ~2 nM [³H]P1075 and the inhibitor of interest. After 30 min, incubation was stopped as described above, and aliquots were filtered over Whatman GF/C filters.

Data Analysis-- Concentration dependencies were analyzed by fitting the logistic function

(Eq. 1)

to the data. Here, A denotes the extent of the effect (amplitude); n (= n_H) is the Hill coefficient, x is the concentration of the compound under study, and IC₅₀ the midpoint of the curve with px = log x and pIC₅₀ = log IC₅₀.

In binding experiments, the dependence of the midpoint of an inhibition curve (IC₅₀ value) on the concentration of the radioligand, L*, was calculated according to the equation by Cheng and Prusoff (30),

(Eq. 2)

where K_i is the inhibition constant and K_L is the equilibrium dissociation constant of the radioligand, L*. In general, this correction did not exceed a factor of 1.5. In cases when a homologous competition experiment (e.g. L*  L) was conducted in the presence of a fixed concentration of a further ligand, C, that also competed with the radioligand, Equation 2 was expanded to read as follows,

(Eq. 3)

where K_C is the equilibrium dissociation constant of competitor C. The homologous competition curves (in the absence and presence of competitor) may be transformed according to Scatchard (31),

(Eq. 4)

where B and F represent the bound and free concentration of the unlabeled ligand, L. B (in percent) is calculated from the radioactive ligand specifically bound, B*, as B = 100  B*.

Fits of the equations to the data were performed according to the method of least squares using the program SigmaPlot 6.1 (SPSS Science, Chicago, IL). Individual binding competition experiments were analyzed according to Equation 1. Errors in the parameters derived from the fit to a single curve were estimated using the univariate approximation (32). Amplitudes and pK values are normally distributed (33); here, K values with the 95% confidence interval in parentheses are given. Propagation of errors was taken into account according to Bevington (34).

Materials-- PNU-96293 and PNU-96179 were synthesized according to Humphrey et al. (35). [³H]P1075 (specific activity 4.5 TBq (118 Ci) mmol¹) was purchased from Amersham Biosciences, and [³H]glibenclamide (specific activity 1.85 TBq (50 Ci) mmol¹) was from PerkinElmer Life Sciences. The reagents and media used for cell culture and transfection were from Invitrogen (Karlsruhe, Germany). Na₂ATP was from Roche Diagnostics, and glibenclamide was from Sigma. P1075 was a kind gift from Leo Pharmaceuticals (Ballerup, Denmark). K_ATP channel modulators were dissolved in dimethyl sulfoxide/ethanol (1:1) 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 experiments 0.1% or below).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PNU-96293 as an Inhibitor of Kir6.2/SUR2B Channels-- Khan and colleagues (25) have shown that PNU-96293, the R-enantiomer, reversed the vasorelaxation induced by other K⁺ channel openers. We therefore examined the effect of this compound on the recombinant K_ATP channel in (nonvascular) smooth muscle, Kir6.2/SUR2B. After application of PNU-96293 (10, 30, and 100 µM) to an inside-out patch in the presence of 1 mM MgATP, no current developed (data not shown). In agreement with Khan et al. (25), this indicated that the compound was devoid of opener activity. When the channel was opened by reducing the ATP concentration to 3 µM, the compound inhibited the channel concentration-dependently (Fig. 2A). At 100 µM, the highest concentration tested, inhibition was ~50%. In comparison, glibenclamide, at the saturating concentration of 1 µM, inhibited the current by 80%. The inhibition curve of the PNU enantiomer extrapolated to a maximum inhibition of 46% with an IC₅₀ value of 16 µM (Fig. 2B, Table I). In the absence of ATP, the current was inhibited maximally by 30% with an IC₅₀ value of 12 µM (Table I).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   PNU-96293 inhibits Kir6.2/SUR2 channels in inside-out patches. A, original recording of Kir6.2/SUR2B channels in the presence of 3 µM ATP ([Mg2+]free = 0.7 mM) at 50 mV. MgATP (1 mM) was applied repetitively for 15 s, blocking the channel and inducing channel refreshment upon washout with 3 µM MgATP. The current developing during MgATP washout was inhibited by glibenclamide (1 µM) and PNU-96293 (10, 30, and 100 µM). B, concentration-dependent inhibition of Kir6.2/SUR2B () and Kir6.2/SUR2A () currents by PNU-96293 in the presence of 3 µM ATP; numbers in parentheses give the number of patches. Inhibition by 1 µM glibenclamide is shown for comparison. The parameters determined from the fit of Equation 1 with Hill coefficient 1 to the data are listed in Table I.

The effect of PNU-96293 was also studied in the whole-cell configuration at 37 °C (Fig. 3). Dialysis of the cell with 1 mM MgATP generated a current that was inhibited by the PNU enantiomer in the nanomolar concentration range (IC₅₀ = 78 nM) with a maximum inhibition of 74%; glibenclamide (1 µM) induced almost total inhibition (Table I). Analogous experiments using the Kir6.2/SUR2B(Y1206S) channel gave very similar results (Table I), showing that the mutation did not alter the sensitivity of the channel to PNU-96293.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   PNU-96293 inhibits Kir6.2/SUR2B channels in whole-cell experiments. A, original recording. The cell was clamped at 60 mV, and voltage was stepped from 110 to 10 mV in 20-mV steps as described under "Experimental Procedures." During dialysis of the cell with MgATP (1 mM) at 37 °C, a current developed that was inhibited by PNU-96293 in a concentration-dependent manner. After washout, glibenclamide (1 µM) produced total inhibition. B, concentration-dependent inhibition at 60 mV. The fit of Equation 1 with Hill coefficient 1 to the data (mean values from the number of patches given in parentheses) gave the parameters listed in Table I.

PNU-96179 as an Opener of the Kir6.2/SUR2B Channel-- Fig. 4A presents an original recording from an inside-out patch containing Kir6.2/SUR2B channels. The channel was kept closed by MgATP (1 mM); the addition of PNU-96179 (100 µM) generated a current that was completely inhibited by glibenclamide (3 µM) in a reversible manner. Further experiments showed that the current induced by PNU-96179 (100 µM) was about 10-20% of that produced by P1075 at the maximally effective concentration of 0.1 µM; lower concentrations of the PNU compound showed smaller and inconsistent effects.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   PNU-96179 opens Kir6.2/SUR2B channels. A, original recording from an inside-out membrane patch. After excision of the patch in the Mg2+-containing solution, strong run-down was observed. ATP (1 mM) induced channel closure; the further addition of PNU-96179 (100 µM) induced a current that was reversibly blocked by glibenclamide (3 µM). Note the "refreshment" of the channels upon washout of MgATP. B, cell-attached configuration. PNU-96179 and P1075 were applied as indicated by the bars. In both configurations, recordings were made in high [K+] buffer (140 mM) at a holding potential of 50 mV; temperature was ~22 °C in the inside-out and 37 °C in the cell-attached configuration.

The ability of PNU-96179 to open the Kir6.2/SUR2B channel was also examined in the whole-cell configuration; however, it proved difficult to keep the channel closed during prolonged dialysis of the cell with 3-10 mM MgATP. Experiments were therefore performed in the cell-attached configuration. Fig. 4B shows that PNU-96179 (100 µM) activated the channels to 13% (mean 15.6 ± 1.6; n = 6) of the level produced by P1075 (0.1 µM); at 10 µM, the PNU compound was ineffective. Collectively, the data show that PNU-96179 is an opener of the Kir6.2/SUR2B channel, albeit with low potency and efficacy. The opening properties were not investigated further, since channel opening is an effect expected for a cyanoguanidine.

Interaction of PNU Enantiomers with SUR2B: [³H]P1075 Binding Experiments-- Fig. 5 illustrates the inhibition of [³H]P1075 binding to SUR2B by the two enantiomers in membranes. In the presence of 1 mM MgATP, the compounds inhibited binding of the radioligand up to 100% with Hill coefficients of 1 and K_i values of 2.3 and 0.66 µM for PNU-96179 and PNU-96293, respectively (Table II). Hence, stereoselectivity of binding, measured as the eudismic (= K_i) ratio, was weak (3.5). Similar experiments were performed also at an ATP concentration of 3 µM (i.e. the lowest concentration at which high affinity binding of the radioligand (which requires MgATP) is detected) (14, 15). Under these conditions, the inhibition curve for PNU-96179 was shifted to the right by a factor of 2.7 and that of PNU-96293 to the left by a factor of 10, giving now a eudismic ratio of 100. Hence, increasing ATP increased the potency of PNU-96179 and strongly decreased that of PNU-96293, indicating positive allosteric coupling between PNU-96179 and MgATP binding, whereas for PNU-96293, the coupling to MgATP was negative.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Inhibition of [3H]P1075 binding to SUR2B by PNU-96179 and PNU-96293. Experiments (n = 3) were performed in the presence of MgATP (1 mM () and 3 µM ()) and evaluated individually. The mean parameters are listed in Table II. The arrows indicate the shift of the curves induced by decreasing MgATP concentration. [Mg2+]free was 1 mM, and [3H]P1075 was ~2 nM; specific binding (BS) (100%) corresponded to 217 ± 16 and 42 ± 4 fmol/mg protein at 1 mM and 3 µM ATP, respectively.

In order to facilitate comparison with the electrophysiological experiments, binding experiments were also performed in intact HEK cells cotransfected with Kir6.2 plus SUR2B. Under these conditions, the enantiomers inhibited binding of the radioligand with K_i values of 3.6 and 0.2 µM, respectively. Comparison with the K_i values determined in membranes with SUR2B alone at 1 mM ATP showed that coexpression with Kir6.2 and/or the whole cell environment increased the K_i value of PNU-96179 slightly by a factor of 1.6 ± 0.2 but decreased that of PNU-96293 by a factor of 3.4 ± 0.4 (Table II). The gain in potency of PNU-96293 obtained by these changes resembled that seen earlier with glibenclamide (26).

Since the PNU compounds and P1075 are structurally related (Fig. 1), we examined whether these ligands competed for the same site of SUR2B. To this end, homologous [³H]P1075-P1075 competition experiments were performed in the absence and presence of PNU-96179 (10 µM) or PNU-96293 (3 µM). The idea behind this was that the presence of the PNU compounds, if they competed with P1075 for the same binding site at SUR2B, should shift the P1075 binding curve to the right as predicted by Equation 3. Fig. 6 shows the inhibition curves in the Scatchard representation (see "Experimental Procedures"). Both PNU compounds reduced the slope but left the abscissa intercept unchanged. The observed reductions in P1075 affinity produced by 10 µM PNU-96179 (3.3×) and 3 µM PNU-96293 (4.4×) are in quantitative agreement with those calculated assuming competition with P1075 (Equation 3) and using the K_i values determined in Fig. 5.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Competition between the binding of PNU enantiomers and P1075 to SUR2B. [3H]P1075/P1075 inhibition curves were performed in the absence () and presence () of PNU-96179 (10 µM) and PNU-96293 (3 µM). Data are shown in the Scatchard representation (Equation 4) to highlight competitiveness. MgATP was 1 mM and specific binding (BS) in the absence of PNU compounds 202 ± 10 fmol/mg protein. PNU-96179 (10 µM) reduced specific binding to 32 ± 1% and PNU-96293 (3 µM) to 28 ± 1%.

If the PNU enantiomers bind to the same site as P1075, they should not affect the dissociation kinetics of P1075 from SUR2B. In control experiments, the addition of unlabeled P1075 in large excess (10 µM) to the SUR2B-[³H]P1075 complex induced dissociation of the complex with a half-time of 11 min. PNU-96293 (10 µM) in the presence of P1075 (10 µM) did not alter the half-time of dissociation (data not shown).

Interaction of PNU Enantiomers with SUR2B: [³H]Glibenclamide Binding Experiments-- Glibenclamide and opener binding sites at SUR2B are linked by strict negative allosteric coupling (14, 24); in addition, glibenclamide binding and MgATP binding are negatively allosterically coupled (36). The coupling between binding of the PNU enantiomers and ATP was therefore examined also using [³H]glibenclamide as the radioligand. Since high affinity glibenclamide binding is increased in the absence of MgATP, this offers the advantage of studying the coupling over a wider range of MgATP concentrations. However, the affinity of SUR2B for glibenclamide (K_D = 22 nM (36)) is too low to obtain a good signal; use was therefore made of the SUR2B(Y1206S) mutant, which has a 5.5 times higher affinity for glibenclamide (26). We have shown previously that neither opener binding nor the allosteric coupling between the binding of ATP, opener, and glibenclamide are perturbed by this mutation (26, 36).

PNU-96179 inhibited [³H]glibenclamide binding only partially, regardless of the presence or absence of MgATP (Fig. 7A and Table II). In the presence of 1 mM MgATP, the K_i value was 2.5 µM (i.e. identical with that measured using [³H]P1075 as the radioligand) (Table II). In the absence of MgATP (1 mM EDTA, neither Mg²⁺ nor ATP added), the K_i value was 83 µM (i.e. 33 times higher). This indicated positive allosteric coupling between PNU-96179 and MgATP binding, as observed also with [³H]P1075 as the radioligand. Surprisingly, PNU-96293 increased binding of [³H]glibenclamide in the presence of MgATP by 41 ± 3% with an EC₅₀ value of 1.7 µM; in the absence of MgATP, partial inhibition was seen with K_i = 18 nM (Fig. 7B). The opposite sign of the PNU-96293 effect depending on the presence of MgATP made a straightforward interpretation difficult. However, the 100-fold greater potency of the compound in the absence of MgATP suggested strong negative allosteric coupling between binding of this enantiomer and MgATP binding, again in agreement with the results obtained in the [³H]P1075 experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Modulation of [3H]glibenclamide binding to SUR2B(Y1206S) by the PNU enantiomers in the presence () and absence () of MgATP. Individual experiments (n = 3 each) were normalized to 100% specific binding (BS) in the absence of PNU compounds. Specific binding in the absence of MgATP (1 mM EDTA, no extra Mg2+ or ATP) was 352 ± 24 fmol/mg protein at 2.5 nM [3H]glibenclamide; the presence of MgATP reduced specific binding to 21 ± 1%. Fitting parameters are listed in Table II.

Experiments with Other SUR Subtypes/Recombinant K_ATP Channels-- In inside-out patches at 3 µM MgATP, PNU-96293 inhibited the Kir6.2/SUR2A current with IC₅₀ = 21 µM and a maximum inhibition of 54% (Fig. 2B, Table I) (i.e. with parameters similar to those found with the Kir6.2/SUR2B channel). In the whole cell mode, a large scatter of the data was noted. Data were therefore divided into two groups according to their glibenclamide sensitivity. Analyzing the group with high sensitivity to glibenclamide (at 1 µM glibenclamide: 79 ± 6% block; n = 3), the inhibition curve extrapolated to 68 ± 4% block at saturation with an IC₅₀ value of 14 (8, 23) nM (Table I). The inhibition curve of the cells with poor sensitivity to glibenclamide (block at 1 µM: 47 ± 15%; n = 4) gave a maximum extent of inhibition of 39 ± 11% and an IC₅₀ value 0.5 (0.2, 1.3) µM. This showed that the channels with poor sensitivity to glibenclamide were also quite insensitive to PNU-96293. In [³H]P1075 binding studies with SUR2A, binding of the PNU-96179 was positively allosterically coupled to MgATP binding, whereas PNU-96293 showed negative coupling (Table II). These results were qualitatively similar to the observations made with SUR2B; however, the ATP-induced shifts were smaller (Table II).

In inside-out patches in the absence of ATP, PNU-96293 (100 µM) inhibited the Kir6.2/SUR1 current by 40 ± 5% (n = 13); similar inhibition was obtained with tolbutamide at 100 µM (Table I). In binding experiments, PNU-96179 proved very weak in inhibiting [³H]glibenclamide binding to SUR1: at 1 mM, inhibition amounted to ~55% independent of the presence of MgATP. Extrapolating the curves to 100% inhibition at saturation gave IC₅₀ values of ~550 µM (i.e. an ~100-fold lower potency than found for the SUR2 subtypes) (Table II). PNU-96293 exhibited a behavior qualitatively similar to that observed at SUR2B(Y1206S) (Table II). In the presence of MgATP, the compound increased [³H]glibenclamide binding to 121% with EC₅₀ = 31 µM; in the absence of MgATP, partial inhibition was observed (maximum 22% and IC₅₀ = 1.4 µM). Hence, the R-enantiomer was again more potent in the absence of MgATP, indicating negative allosteric coupling to MgATP binding as observed also in the binding experiments with SUR2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we report that the enantiomers of a racemic compound derived from the K_ATP channel opening cyanoguanidines interact specifically with the channel, the R-enantiomer (PNU-96293) inducing channel inhibition and the S-enantiomer (PNU-96179) opening.

Inhibition of Recombinant K_ATP Channels by PNU-96293-- In whole cell experiments and in the presence of ~1 mM MgATP, PNU-96293 inhibited the Kir6.2/SUR2 channels with IC₅₀ values in the low nanomolar range and a slight selectivity for the Kir6.2/SUR2A channel. This is a potency similar to that of glibenclamide under the same conditions (26). The efficacy of PNU-96293, however, was lower than that of glibenclamide at both Kir6.2/SUR2 channels (Table I). This may be related to differences in the negative allosteric coupling to MgATP binding at SUR. Here we have shown that MgATP decreases the affinity of PNU-96293 for SUR2B; in the case of glibenclamide binding, however, the number of binding sites is reduced (23, 26).

Another point deserves comment. For the Kir6.2/SUR2B channel, the binding and the electrophysiological experiments were performed under similar conditions (whole cells, high MgATP, 37 °C); however, the channel inhibition curve (IC₅₀ = 78 nM) was shifted leftward from the binding curve (K_i = K_D = 200 nM) by a factor of ~3. A leftward shift of roughly this magnitude is expected if the channel has four equal and independent sites for the ligand and if occupation of a single site is sufficient for channel inhibition (37). Alternatively, if binding of four molecules is required for channel block, the shift is by the same factor ((2^1/4  1)¹ = 5.3 (28)) but in the opposite direction.

Using the inside-out configuration, the potency and efficacy of PNU-96293 in blocking the Kir6.2/SUR2 channels were reduced. The loss in potency was dramatic (IC₅₀ values in the 20 µM range); it was also unexpected for two reasons. First, the contrary is observed with sulfonylureas (e.g. glibenclamide) and analogs (e.g. repaglinide); in these cases, the sensitivity for channel block is increased in the inside-out patch (glibenclamide (26, 38) and repaglinide (39)). Second, the IC₅₀ values for channel inhibition are 190-250 times higher than the K_i values of the respective binding curves. Despite some differences in the experimental conditions (binding experiments were done at 37 °C and to SUR alone), these observations suggest that the transduction of PNU binding into to channel inhibition is more complex than in the case of the sulfonylureas. One may speculate that the more physiological environment of the (dialyzed) whole cell offers a component (e.g. another protein, a metabolic product) not present in the inside-out patch. Further work is needed to resolve this question. The other point is the lower efficacy of block observed in the inside-out configuration. This is less surprising, since it has generally been observed with all K_ATP channel inhibitors acting via SUR (e.g. see Refs. 38, 40, and 41). The reason for this behavior remains unknown.

Binding Sites of the Enantiomers-- Information on the binding site(s) of SUR for the PNU enantiomers was obtained from the binding experiments. First we recall that SUR accommodates the binding sites for the openers, the sulfonylureas, and nucleotides, and that these sites are linked by allosteric interactions (Fig. 8A; see also the Introduction). The partial inhibition of or even the increase in [³H]glibenclamide binding to SUR2B(Y1206S) and SUR1 excluded the possibility that the enantiomers bound to the glibenclamide site of SUR. Instead, these results showed that ternary complexes of SUR with glibenclamide and a PNU enantiomer existed at equilibrium and that the site(s) of the PNU enantiomers and the glibenclamide site were linked by allosteric interactions. Similarly, the modulation of PNU binding by MgATP showed allosteric linkage between the PNU site(s) and nucleotide binding to the nucleotide binding folds of SUR. The equilibrium binding experiments with [³H]P1075 in Fig. 6 and the kinetic experiments, however, were in quantitative agreement with a model in which the PNU enantiomers and P1075 competed for the same binding site of SUR2B. In view of the structural similarity between these three ligands (Fig. 1), competition for the same binding site seems plausible; however, more direct biochemical evidence (e.g. by photoaffinity labeling) is desirable. A first consequence of the competitive mechanism was that the K_i values determined in [³H]P1075 inhibition assays (Table II, Fig. 5) can be equated with K_D values for binding of these compounds to SUR2B. A second consequence was that the two enantiomers bind to the same site of SUR2B, which is of importance in view of their opposite coupling to MgATP binding and their opposite effects on channel activity.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Allosteric interactions between binding sites of SUR. A, SUR has binding sites for nucleotides (N), openers (O), and sulfonylureas (SU). These sites are linked by positive and negative allosteric interactions as indicated. For reasons of simplicity, the two nucleotide binding sites of SUR were condensed into one. B, in the presence of MgATP (N), PNU-96293 increases [3H]glibenclamide (G) binding. Top, SUR with [3H]glibenclamide and MgATP occupying their sites linked by negative allosteric interaction. Bottom, by strong negative coupling to the MgATP site, binding of the blocker PNU-96293 to the opener site induces dissociation of MgATP from the MgATP site, leading to a SUR-[3H]glibenclamide-PNU complex with increased occupancy of the [3H]glibenclamide site. The latter suggests that the negative allosteric interaction between [3H]glibenclamide and PNU is weaker than that between [3H]glibenclamide and MgATP. The strength of the different allosteric interactions is indicated by the thickness of the arrows. C, in the absence of MgATP, PNU-96293 partially displaces glibenclamide by the negative allosteric coupling between the respective sites.

Allosteric Coupling of PNU-96293 and MgATP Binding-- MgATP inhibited binding of PNU-96293 to SUR via a negative allosteric interaction, regardless of the radioligand used. Two points deserve comment. First, MgATP induced a large reduction in the affinity of PNU-96293 binding to SUR2B(Y1206S) (rightward shift ~100-fold), whereas for glibenclamide the number of binding sites was reduced (23, 36). Regarding binding to SUR1, MgATP induced a rightward shift in both cases, and the shift for PNU (22, Table II) was larger than that for glibenclamide (~7 (38)). Assuming that the negative allosteric coupling between PNU-96293 and MgATP binding is stronger than that between glibenclamide and MgATP, one can explain that in the presence of MgATP, PNU-96293 actually increased the binding of [³H]glibenclamide to SUR2B(Y1206S) (Fig. 7B) and to SUR1 (Table II). Including MgATP, one is faced with a system of three binding sites, linked by negative allosteric interactions of differing strength (Fig. 8B). As PNU-96293 binds to the SUR-[³H]glibenclamide-MgATP complex, the strong negative coupling between the PNU enantiomer and MgATP induces dissociation of MgATP from SUR, and one is left with a SUR-[³H]glibenclamide-PNU complex. Since more [³H]glibenclamide is bound, one must conclude that the negative allosteric interaction between glibenclamide and PNU-96293 binding is weaker than that between glibenclamide and MgATP binding (Fig. 8B). In the absence of MgATP (Fig. 8C), only the weak negative allosteric coupling between PNU-96293 and glibenclamide comes into play, and partial inhibition of glibenclamide binding results.

Second, the rightward shift in the K_i values of PNU-96293 upon increasing ATP depended on the SUR subtype (SUR2B > SUR2A; SUR2B(Y1206S) > SUR1; Table II); this suggested differences in the strength of the negative allosteric coupling between PNU-96293 and MgATP binding with the SUR subtype. Variable MgATP-induced rightward shifts were observed previously for the binding of glibenclamide and of the sulfonylthiourea, HMR 1883, to SUR subtypes; in this case, the maximum shift (measured as the ratio of the K_i values) was ~8 for glibenclamide binding to SUR1 (38). In addition, the glinide, AZ-DF265 (42), also displayed a rightward shift of 2.8-fold in its interaction with SUR2B.² Collectively, these data show that compounds of different chemical structures, which inhibit K_ATP channels by binding to the SUR subunit, are negatively allosterically coupled to MgATP binding to SUR.

Allosteric Coupling of PNU-96179 and MgATP Binding-- Increased MgATP induced a leftward shift in binding of the S-enantiomer to SUR2, indicating positive allosteric coupling between the respective binding sites. With [³H]P1075 binding to SUR2B and an increase in MgATP from 0.003 to 1 mM, the leftward shift measured for PNU-96179 was ~3-fold (Table II). This is in the range typical for openers; for P1075 it was 1.7-fold (14), and for the benzopyran 217-774 (43) it was 3.8-fold.² Much higher shifts were found with [³H]glibenclamide as the radioligand, probably because one can start from MgATP = 0. In this case, and using the mutant SUR2B(Y1206S), the shift for PNU-96179 amounted to a ratio of 33; for typical openers, it ranged from 100 to 300 (44). These data show that openers of different chemical structures are positively allosterically coupled to MgATP binding. In view of the fact that openers increase the ATPase activity of SUR (21), this is not surprising.

Eudismic Ratio and Affinity of the PNU Enantiomers-- At 1 mM MgATP, the PNU enantiomers bound to the different SUR subtypes in the low (SUR2) or high (SUR1) micromolar range (i.e. binding was relatively weak). The eudismic (= K_D) ratio of the enantiomers was also weak, ranging from ~1 (SUR2B(Y1206S)) to 17 (SUR1). Hence, the largest ratio was found for the SUR with the weakest affinity for the PNU compounds (i.e. SUR1). This was also true for the K_i values at low or 0 MgATP (Table I). These observations are not in accordance with Pfeiffer's rule, which states that the weaker the racemate, the smaller the potency difference between the enantiomers (45). One also notes that for all SUR subtypes, PNU-96293 was more potent than PNU-96179, which agrees with the electrophysiological experiments.

Significance of Results-- The major result of this study was that the R-enantiomer of the racemic cyanoguanidine, PNU-94750, exhibited the higher potency (25), was negatively coupled to MgATP binding, and inhibited K_ATP channels; for the S-enantiomer, the converse was true in all points. Stereoselective gating of K_ATP channels has been observed before, both for openers (e.g. for cromakalim (e.g. see Ref. 46) and for cyanoguanidines like pinacidil (47)) and for blockers (e.g. the benzoic acid sulfonylurea analogue AZ-DF 265 (42, 48)). In all of these cases, the distomer affected the channel in the same way as the eutomer but with reduced potency. Within the cyanoguanidine class, the PNU eutomer exhibited the R configuration as in the case of pinacidil. However, the S-enantiomer can also be the eutomer, indicating that SUR has considerable flexibility in accommodating the ligand (47) (for a review, see Ref. 4). More importantly, the PNU eutomer was a K_ATP channel blocker, and the two enantiomers had opposite molecular effects.

These observations are reminiscent of some dihydropyridines where, depending on membrane voltage and channel kinetics, one enantiomer can open and the other close the L-type Ca²⁺ channel (49, 50). These opposite actions are apparently mediated by binding to the same binding site of the channel (51). The enantiomers shift the channel inactivation curve by different degrees to more negative potential, which, in a certain voltage window, results in an increase or decrease in the number of activable channels (50). Regarding the PNU enantiomers, the available evidence suggests that they also bind to the same binding site of their receptor (SUR); however, they show opposite effects in their allosteric coupling to ATP binding (and possibly ATPase activity) and in their modulation of K_ATP channel activity. Within the framework of the biochemical mechanism of K_ATP channel openers (21, 52) and blockers (13), one may hypothesize that it is not the binding of a ligand to the opener site of SUR per se but the modulation by this ligand of ATP binding and ATPase activity of SUR that determines whether the compound acts as an opener or a blocker of the channel.

	ACKNOWLEDGEMENTS

We thank Drs. Y. Kurachi and Y. Horio (Department of Pharmacology II, Osaka University) for the generous gift of the murine clones of SUR2A, SUR2B, and Kir6.2; Dr. C. Derst (Department of Physiology, University of Marburg, Marburg) for the rat clone of SUR1; and Leo Pharmaceuticals (Ballerup, Denmark) for the kind gift of P1075. We also acknowledge C. Müller for excellent technical assistance with cell culture and transfections.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Qu 100/2-4 (to A. H. and U. Q.) and Qu100/3-1 (to U. Q.); by the fortüne program of the Medical Faculty of the University of Tübingen (to U. L.); and by the Federal Ministry of Education, Science, Research, and Technology Grant Fö 01KS9602 and the Interdisciplinary Center of Clinical Research (IZKF) Tübingen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 49-7071-29-76774; Fax: 49-7071-29-4942; E-mail: ulrich.quast@uni-tuebingen.de.

Published, JBC Papers in Press, August 8, 2002, DOI 10.1074/jbc.M206685200

² U. Quast, unpublished results.

	ABBREVIATIONS

The abbreviations used are: K_ATP channel, ATP-sensitive K⁺ channel; P1075, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine; Kir, inwardly rectifying K⁺ channel; SUR, sulfonylurea receptor; HEK, human embryonic kidney; PNU-96293, (R)-N-cyano-N'-(1-phenylpropyl)-N"-3-pyridylguanidine; PNU-96179, (S)-N-cyano-N'-(1-phenylpropyl)- N"-3-pyridylguanidine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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