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Br J Pharmacol. Oct 2002; 137(4): 429–440.
Published online Oct 2, 2002. doi:  10.1038/sj.bjp.0704905
PMCID: PMC1573525

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit

Abstract

  1. ATP-sensitive potassium channels (KATP channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg2+, the stilbene disulphonate, DIDS, irreversibly inhibits KATP channels by binding to the Kir subunit. Here, the effects of Mg2+ on the interaction of DIDS with recombinant KATP channels were studied in electrophysiological and [3H]-glibenclamide binding experiments.
  2. In inside-out macropatches, Mg2+ (0.7 mM) increased the sensitivity of KATP channels towards DIDS up to 70 fold (IC50=2.7 μM for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations [gt-or-equal, slanted]10 μM was irreversible.
  3. Mg2+ sensitized the truncated Kir6.2Δ26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg2+ did not require the presence of nucleotides.
  4. [3H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg2+ and by coexpression with Kir6.2.
  5. In the presence of Mg2+, DIDS inhibited binding of [3H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC50=7.9 μM by a non-competitive mechanism. Inhibition was fully reversible.
  6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg2+ binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg2+ effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the KATP channel.
Keywords: KATP channels, magnesium ion, sulphonylurea receptor subtypes, DIDS, stilbene disulphonates, patch-clamp, inwardly rectifying K+ channels, [3H]-glibenclamide

Introduction

ATP-sensitive K+ (KATP) channels are closed by ATP and opened by MgADP (and other nucleoside diphosphates); hence, these channels link the metabolic state of the cell to membrane potential and cellular excitability. In addition to their regulation by intracellular nucleotides, KATP channels are the target of important drugs, the sulphonylureas exemplified by glibenclamide and the potassium channel openers such as levcromakalim and pinacidil (Ashcroft & Ashcroft, 1990). KATP channels are composed of two types of subunits, the weakly inwardly rectifying K+ channels of the family Kir6.x and the sulphonylurea receptors (SURs), arranged in a hetero-octameric complex (Kir6.x/SURx)4 (Aguilar-Bryan et al., 1995; Sakura et al., 1995; Clement et al., 1997; Shyng & Nichols, 1997). The inhibition of KATP channels by ATP is mediated by the Kir subunit (Tucker et al., 1997) whereas the activation by MgADP is mediated by SUR (Nichols et al., 1996; Gribble et al., 1997).

SURs are members of the ATP-binding cassette proteins. They have two nucleotide binding folds (NBFs) with NBF1 binding ATP and NBF2 mediating the activation of the channel by ADP in Mg2+-dependent manner (Nichols et al., 1996; Gribble et al., 1997); in addition, SURs carry the binding sites for the sulphonylureas and the openers (Aguilar-Bryan et al., 1995; Sakura et al., 1995; Hambrock et al., 1998; Schwanstecher et al., 1998). Kir6.x and SURx each are encoded by two genes; this and alternative splicing of the SUR gene products lead to subtypes of SUR and Kir6 (reviewed in Aguilar-Bryan & Bryan, 1999). Different combinations of Kir6.x and SURx are the basis for the tissue-specific differences of KATP channels with Kir6.2/SUR1 being the channel in the pancreatic β-cell and neurons, Kir6.2/SUR2A in cardiomyocytes and skeletal muscle cells and Kir6.x/SUR2B in smooth muscle myocytes.

Stilbenes like DIDS (4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid), at high concentrations ([gt-or-equal, slanted]100 μM), inhibit anion transporters and channels (Cabantchik et al., 1978) and activate KvLQT1/Isk channels (Busch et al., 1994; Abitbol et al., 1999). In the low μM range, DIDS acts as an antagonist at P2-purinoceptors (Ralevic & Burnstock, 1998). Noting structural similarities of DIDS with sulphonylureas, Furukawa et al. (1993) investigated the effect of DIDS on KATP channels in guinea-pig ventricular myocytes. They found that DIDS, applied to the intracellular side of the membrane and in the absence of Mg2+, inhibited the channel with IC50=71 μM in an irreversible manner (Furukawa et al., 1993). The reactive SCN groups of DIDS are known to covalently modify cysteine and lysine residues of proteins (Gatto et al., 1997). Again in the absence of Mg2+ and using the truncated KATP channel Kir6.2Δ36 (which exhibits channel activity in the absence of SUR (Tucker et al., 1997)), Proks et al. (2001) showed that DIDS interacted with the Kir6.2 subunit. They also showed that ATP protected against (irreversible) DIDS block suggesting interaction of the ATP and the DIDS sites of Kir6.2. Interestingly, coexpression of Kir6.2 with SUR2A reduced both the rate and extent of channel block as compared to Kir6.2/SUR1, indicating that the sensitivity of the channel towards inhibition by DIDS was modulated by the SUR subunit (Proks et al., 2001).

We have found that purinergic antagonists such as phloxin B and DIDS inhibit binding of the opener [3H]-P1075 to SUR2B; in the presence of 3 mM MgATP, the IC50 value of DIDS was 1.6 μM (Russ et al., 2000), i.e. a potency more than 40 fold higher than that observed for channel block in the absence of Mg2+. This raised the question whether the presence of Mg2+ (or MgATP) affected the block of KATP channels by DIDS and whether the binding of DIDS to SUR was in any way transduced into modulation of channel activity. Here we present electrophysiological and radioligand binding studies designed to answer these questions. Since the effects of Mg2+ were most pronounced at the Kir6.2/SUR2B channel, experiments were focused on this channel.

Methods

Recombinant KATP channels, mutations, cell culture and transfection

Human embryonic kidney (HEK) 293 cells were cultured as described previously in minimum essential medium containing glutamine, supplemented with 10% foetal bovine serum and 20 μg ml−1 gentamycin (Hambrock et al., 1998). Cells were transfected with the pcDNA 3.1 vector (Invitrogen, Karlsruhe, Germany) containing the coding sequence of rat SUR1 (GenBank accession number X97279), murine SUR2A, SUR2B (GenBank accession numbers: D86037 and D86038; Isomoto et al., 1996) or SUR2B(Y1206S). Cell lines stably expressing these proteins were isolated as described previously (Hambrock et al., 1998).

The mutants SUR2B(Y1206S) (Hambrock et al., 2001), Kir6.2(G334D) (Drain et al., 1998) and a truncated form of Kir6.2, which lacks the C-terminal 26 amino acids (Kir6.2Δ26) were prepared as described by Hambrock et al. (2001).

For patch clamp and some binding experiments, cells were transiently transfected with Kir6.2 (GenBank accession number D50581; Inagaki et al., 1996) and SURx at a molar plasmid ratio of 1 : 1 using LipofectAMINE and OptiMEM (Invitrogen) as described previously (Hambrock et al., 1998). In cotransfections used for electrophysiological experiments, the pEGFP-C1 vector (Clontech, Palo Alto, CA, U.S.A.), encoding for green fluorescent protein, was added for identification of transfected cells. Cells were allowed to express transfected DNA for 48 h before use in electrophysiological experiments (Russ et al., 1999).

Patch-clamp experiments

The patch-clamp technique was used in the inside-out configuration as described by Hamill et al. (1981). Transfected HEK cells showing green fluorescence were chosen. Patch pipettes were drawn from borosilicate glass capillaries (GC 150T, Harvard Apparatus, Edenbridge, U.K.) and heat polished using a horizontal microelectrode puller (Zeitz, Augsburg, Germany). Bath and pipette were filled with a high K+-Ringer solution containing (in mM): KCl, 142; NaCl, 2.8; MgCl2, 1; CaCl2, 1; D(+)-glucose, 11; 4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid (HEPES), 10 titrated to pH 7.4 with NaOH. After filling with buffer, pipettes had a resistance of 1–1.5 MΩ. After excision of the patch, the pipette was moved in front of a pipe with a high K+-EGTA-Ringer solution containing (in mM): KCl, 143; MgCl, 0.85; CaCl2, 1; ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5; D(+)-glucose, 11; HEPES, 10 titrated to pH 7.2 with NaOH. If ATP was added, the concentration of free Mg2+ was kept constant at 0.7 mM. The Mg2+-free solution consisted of (in mM): KCl, 145; ethylenediaminetetraacetic acid (EDTA), 5; HEPES, 10 and D(+)-glucose, 11. For the bath solution at pH 6, 2-(N-morpholino)-ethanesulphonic acid (MES, 10 mM) was used as the buffering ion. All responses were normalized to the current prior to DIDS application. Patches were clamped to −50 mV. All experiments were done at a room temperature of 22°C.

Data were recorded with a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA, U.S.A.) using a MacLab interface and the Chart software (AD Instruments, Castle Hill, Australia). Signals were filtered at 200 Hz, digitized online at 100 Hz, and stored on a Power Macintosh 8200/120 computer for later analysis.

Membrane preparation and [3H]-glibenclamide binding experiments

For cells stably expressing SUR2B(Y1206S) alone, the antibiotic geneticin was withdrawn from the culture medium 1 week prior to membrane preparation; cells transiently expressing Kir6.2/SUR2B(Y1206S) were harvested 2–3 days after transfection. Membranes were prepared as described (Hambrock et al., 1998). In brief, cells were centrifuged for 5 min at 500×g and 4°C and lysed by addition of ice-cold hypotonic buffer containing (in mM): HEPES, 10; EGTA, 1 at pH 7.4. The lysate was centrifuged at 105×g and 4°C for 60 min and the resulting membrane pellet was resuspended in a buffer containing (in mM) HEPES, 5; KCl, 5; NaCl, 139; at pH 7.4 and 4°C at a protein concentration of ~1.5–3.0 mg protein ml−1 and frozen at −80°C. Protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin as the standard.

The interaction of DIDS with SUR was studied in [3H]-glibenclamide competition or saturation experiments. Membranes (final protein concentration 100–150 μg protein ml−1) were added to the incubation buffer (NaCl 139 mM, KCl 5 mM, HEPES 10 mM) supplemented with [3H]-glibenclamide (0.5–2.5 nM for competition experiments), Mg2+ (0/0.7/2.2 mM), EDTA (1/0/0 mM) and ATP (0/0/1 mM) at 22°C for 45 min or at 37°C for 15 min. Incubation was stopped by diluting, in triplicate 0.3 ml aliquots into 8 ml of quench solution (50 mM tris-(hydroxymethyl)-aminomethane, 154 mM NaCl, pH 7.4) at 0°C and filtration over Whatman GF/B filters. Nonspecific binding was determined in the presence of 100 μM unlabelled P1075 and ranged from 22–32% of total binding. Non-transfected HEK 293 cells possess endogenous low affinity glibenclamide sites which are not sensitive to P1075; however, P1075 completely displaces specific glibenclamide binding (=glibenclamide binding to mutant SUR2B) (Hambrock et al., 2001).

Data analysis

Data are shown as mean±s.e.mean. Concentration dependencies were analysed by fitting the logistic form of the Hill equation,

equation image

to the data. Here A denotes the extent of the effect (amplitude); n (=nH) is the Hill coefficient, x the concentration of the compound under study and K (=IC50) the midpoint of the curve with px=−logx and pK=−logIC50. For the analysis of biphasic curves, the superposition of two logistic terms was used and Hill coefficients were set to 1 if not stated otherwise.

In case of competitive inhibition, the IC50 value of the binding inhibition curve was corrected for the presence of the competing radioligand, L, according to Cheng & Prusoff (1973), to give the inhibition constant, Ki:

equation image

where KL is the equilibrium dissociation constant of the radioligand, L. The Cheng-Prusoff correction was generally less than 2.

Saturation experiments were analysed according to the equation,

equation image

Total binding (BTOT) is the sum of specific and nonspecific binding. Here, BMAX (fmol mg−1 protein) denotes the concentration of specific binding sites in the preparation, KD is the equilibrium dissociation constant, L the concentration of the radioligand and a the proportionality constant describing nonspecific binding as a linear function of L. Nonspecific binding was determined as described above. Specific binding was also plotted in the Scatchard presentation (Scatchard, 1949).

Fitting of equations to data was performed according to the method of least squares using the SigmaPlot6.1 programme (SPSS Science, Chicago, IL, U.S.A.). Errors in the parameters derived from fit to a single curve were estimated using the univariate approximation (Draper & Smith, 1981) and assuming that amplitudes and pIC50 values are normally distributed (Christopoulos, 1998). These were then averaged and pIC50 values±s.e.mean were converted to IC50 values with the 95% confidence interval in parentheses. Significance of differences between parameters obeying the normal distribution was determined using the two-tailed unpaired Student's t-test.

Chemicals

4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), glibenclamide, MgATP and β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) were from SIGMA (Deisenhofen, Germany). The pinacidil analogue, P1075 was a gift from Leo Pharmaceutical (Ballerup, Denmark). DIDS was dissolved in bath solution or incubation buffer and protected from light with aluminium foil and used within 3 h. Glibenclamide and P1075 were dissolved in dimethyl sulphoxide/ethanol (1 : 1) and further diluted with incubation buffer; the final solvent concentration was always below 0.3%. The reagents and media used for cell culture and transfection were from Life Technologies (Eggenstein, Germany).

Results

Interaction with recombinant KATP channels

Figure 1 shows the effects of DIDS on the Kir6.2/SUR2B channel in recordings from inside-out patches. Upon patch excision into a Mg2+ (0.7 mM)-containing nucleotide-free solution, the current underwent rapid run-down (Figure 1A,B). Addition of ATP (1 mM) induced complete channel block and, upon washout, a large ‘refreshment' of the current was observed. This cycle was repeated and application of DIDS (10 μM) inhibited the current by ~90% within 90 s (Figure 1A). Upon washout of the agent with 1 mM ATP, the inhibition proved essentially irreversible. At 100 μM DIDS, inhibition was almost complete within 10 s (Figure 1B). In the absence of Mg2+ (5 mM EDTA, no extra Mg2+), run-down was greatly slowed down and inhibition by DIDS (10 μM) was small (~8% after 90 s, Figure 1C). At 100 μM, DIDS inhibited the current by ~40% in an irreversible manner; in addition, channel block by ATP (1 mM) was decreased (Figure 1D). These data show that presence of Mg2+ strongly sensitized the Kir6.2/SUR2B channel to the inhibitory effect of DIDS.

Figure 1
Recordings from inside-out patches showing the inhibition of KATP (Kir6.2/SUR2B) currents by DIDS in the presence (A,B) and absence (C,D) of Mg2+. Holding potential was −50 mV and temperature 22°C. 1 mM ATP, 10 μM (A,C) or 100 ...

The inhibition kinetics of the Kir6.2/SUR2B channel by DIDS were often biphasic, comprising a rapid first component (which was essentially complete after 10 s) and a slower component (Figure 2). The contribution of the rapid phase increased with increasing DIDS concentration and, in the presence of Mg2+, much lower concentrations of DIDS were required to reach an equivalent level of inhibition. Qualitatively similar observations were made with the other recombinant KATP channels containing SUR2A or SUR1. Evaluating the inhibition of the KATP currents in the presence and absence of Mg2+ at 10 s and 90 s after DIDS application, concentration-inhibition curves were constructed. Figure 3 illustrates the inhibition curves for the three channels after 90 s DIDS application; the fitting parameters are listed in Table 1 together with those from the inhibition curves after 10 s. It is seen that the sensitivity of all channel combinations was increased by Mg2+; however, the effect was strongest with SUR2B. Therefore, further studies were concentrated on Kir6.2/SUR2B.

Figure 2
Time-dependent inhibition of the Kir6.2/SUR2B channel by DIDS in the presence and absence of Mg2+. Note the biphasic nature of most inhibition kinetics. Patches with little run-down were selected and responses were normalized with respect to the current ...
Figure 3
Concentration-dependent inhibition of Kir6.2/SUR2B (A), Kir6.2/SUR2A (B) and Kir6.2/SUR1 (C) currents by DIDS in the presence and absence of Mg2+. Currents, measured 90 s after DIDS application, were expressed as percentage of control current prior to ...
Table 1
Inhibition of recombinant KATP channels by DIDS

Proks and colleagues have reported that inhibition of various KATP currents by DIDS (0.7 mM, in the absence of Mg2+) was irreversible (Proks et al., 2001). Figure 1A suggested that this may hold true also for the low concentrations of DIDS required to inhibit the Kir6.2/SUR2B channel in the presence of Mg2+ and, during up to 4 min washout, no significant recovery from DIDS block (10 μM, 90 s, +Mg2+) was observed (n=10, data not shown).

In the [3H]-glibenclamide binding studies presented below, a SUR2B mutant with increased affinity for glibenclamide was used (SUR2B(Y1206S); Hambrock et al., 2001). No differences in channel characteristics between wild type and mutant SUR2B (with the exception of the sensitivity to glibenclamide) have been observed so far (Hambrock et al., 2001; 2002). However, it seemed adequate to repeat the key experiments on the Kir6.2/SUR2B response to DIDS with the mutant. Figure 4A shows that in the presence of Mg2+, DIDS (30 μM, applied for 90 s) produced almost complete current inhibition (97±1%, n=5). In the absence of Mg2+, inhibition was much less pronounced (35±5%; n=5; Figure 4B). These results agree well with the data for the wild type (Figure 3A). In addition, inhibition persisted in both cases during alternating application of MgATP (1 mM ATP, 30 s) and control buffer (30 s) for up to 7 min (Figure 4). These experiments demonstrated that the mutation did not alter the effect of Mg2+ on channel inhibition by DIDS.

Figure 4
Inhibition of KATP (Kir6.2/SUR2B(Y1206S)) currents by DIDS showing Mg2+ sensitization and irreversibility of DIDS-induced block. Experimental conditions were as in Figure 1. ATP (1 mM) and DIDS (30 μM) were applied as indicated.

Investigation into the mechanism of the Mg2+ effect

The effect of Mg2+ on the sensitivity of KATP channels to DIDS could be mediated by Mg2+ binding to Kir or SUR. In order to decide between these possibilities, experiments were performed using a truncated Kir6.2 (Kir6.2Δ26), which shows channel activity in the absence of SUR, whereas Kir6.2 does not (Tucker et al., 1997). The truncated channel expressed poorly and only small currents were obtained (Figure 5A,B). DIDS (30 μM) was only weakly effective and inhibition (up to 50%) was the same regardless of the presence or absence of Mg2+ (n=5, each). Coexpression of Kir6.2Δ26 with SUR2B greatly improved expression and restored the Mg2+ effect (Figure 5C,D): In the presence of Mg2+, DIDS (3 μM) inhibited the Kir6.2Δ26/SUR2B channel by 62±4% (n=4); in the absence of Mg2+, inhibition by DIDS (300 μM) was 88±3% (n=4). Together, these experiments showed that the presence of SUR was required for Mg2+ effect and the truncation of Kir6.2 per se was not responsible for the absence of the Mg2+ effect on the Kir6.2Δ26 channel.

Figure 5
Inhibition of Kir6.2Δ26-containing KATP channels by DIDS in the presence and absence of Mg2+. Mg2+ did not alter inhibition of the Kir6.2Δ26 channel (A,B) but sensitized the Kir6.2Δ26/SUR2B channel for inhibition by DIDS (C,D). ...

Next, the question was addressed whether the effect of Mg2+ required ATP and/or ADP bound to SUR. Although DIDS was applied in the absence of nucleotides, MgATP and/or MgADP may have remained tightly bound to SUR during exposure of the channel to nucleotide-free solutions. In order to eventually displace such nucleotides, the Kir6.2/SUR2B channel was kept in a Mg2+-free solution (5 mM EDTA) and then exposed to the nonhydrolysable ATP-analogue AMP-PCP (1 mM) for 2 min; this manoeuvre induced immediate channel closure (Figure 6A). After washout of AMP-PCP, DIDS (10 μM) was applied in the presence of Mg2+, producing almost complete and irreversible inhibition of the current (Figure 6A; in six experiments, current decrease was 83±3%). As a control, the KATP channel opener P1075 (at the saturating concentration of 0.1 μM and in the presence of Mg2+) was applied in the continued presence of AMP-PCP (Figure 6B). P1075 was unable to open the channel, suggesting that no significant amounts of MgATP/MgADP had remained bound to the channel (n=5). Together, these experiments showed that the potentiating effect of Mg2+ did not require the presence of hydrolysable adenine nucleotides bound to SUR.

Figure 6
Effects of AMP-PCP and of the mutation Kir6.2(G334D) on the inhibition of KATP currents by DIDS. Upper panels: AMP-PCP. (A) After exposure of the channel to AMP-PCP in the absence of Mg2+ for 2 min, the enhancement of DIDS block by Mg2+ was preserved. ...

Interaction of DIDS and MgATP at KATP channels

In the absence of Mg2+, ATP (3 mM) has been shown to afford protection of Kir6.2Δ36 and Kir6.2/SUR1 channels against irreversible inhibition by DIDS (0.7 mM; Proks et al., 2001). It was therefore of interest to examine if this was true also for the Kir6.2/SUR2B channel in the presence of Mg2+. The results of these experiments are summarized in Table 2.

Table 2
Protection by MgATP of the Kir6.2/SUR2B current against irreversible DIDS block

MgATP (30 μM) completely protected against the irreversible block by DIDS (10 μM). Protection by 100 μM MgATP against 30 μM DIDS was only partial but total by 1 mM MgATP. With increasing DIDS concentration (0.1 or 0.3 mM), protection by MgATP (1 mM) was partial or absent. These experiments show that MgATP protected against DIDS also in the presence of Mg2+ and that protection depended on the concentration ratio of ATP : DIDS.

In order to learn more about the interaction between ATP and DIDS at Kir6.2, we studied the effects of the mutation G334D which exhibits about 1000× lower sensitivity to ATP, presumably due to a reduction in ATP binding (Drain et al., 1998). When Kir6.2(G334D) was coexpressed with SUR2B, only small currents were obtained; therefore, experiments were performed with the Kir6.2(G334D)/SUR1 channel which gave better currents. In agreement with Drain et al (1998)., the channel was insensitive to ATP (1 mM, not shown); however, channel activity was greatly reduced by acidification to pH 6 (Figure 6C,D). In the presence of Mg2+, DIDS (10 μM) inhibited the current by 70±3% (n=5). In the absence of Mg2+, no block was observed (Figure 6; n=4) whereas at 100 μM, DIDS inhibited this channel almost completely by 95±1% (n=4, not illustrated). These data are similar to those observed with the native channel (Figure 3C). It is concluded that the mutation G334D did not interfere with the Mg2+-dependent channel inhibition by DIDS.

Radioligand binding experiments: interaction of DIDS with SUR2B(Y1206S)

The electrophysiological experiments in Figure 5 had shown that the Mg2+ effect required the presence of SUR and earlier experiments using the opener, [3H]-P1075, as the radioligand had shown that DIDS interacted with SUR2B (Russ et al., 2000). In order to study the Mg2+-dependence of this interaction, [3H]-glibenclamide was chosen as the radioligand instead of [3H]-P1075, since the latter requires the presence of Mg2+ and ATP for high affinity binding to SUR2 (Hambrock et al., 1998; 1999; Schwanstecher et al., 1998) whereas glibenclamide does not (Hambrock et al., 2001; 2002). To obtain sufficient binding, the mutant SUR2B(Y1206S), which exhibits higher affinity for glibenclamide, was used.

Figure 7A illustrates the inhibition of [3H]-glibenclamide binding by DIDS. In the absence of Mg2+, the inhibition curve was monophasic with an IC50 value of 60 μM (Table 3, 22°C). In the presence of Mg2+, the competition curve was flattened (Hill coefficient=0.74). Two component analysis revealed a smaller component with high affinity (IC50.1=1.4 μM) and a second component with IC50,2=39 μM (Table 3). Analogous experiments were performed also with SUR2B(Y1206S) coexpressed with Kir6.2 (Figure 7B). In the absence of Mg2+, the inhibition curve was monophasic with an IC50 of 39 μM, i.e. similar to that found with mutant SUR2B in the absence of Kir6.2. In the presence of Mg2+, the curve was essentially monophasic (Hill coefficient 0.89±0.05) with an IC50 value of 7.9 μM. This showed that coexpression with Kir6.2 slightly altered the interaction of (mutant) SUR2B with DIDS, once Mg2+ was present. One also notes that in the presence of Mg2+, there is reasonable agreement between the IC50 value determined in the binding assay (7.9 μM) and that of DIDS blocking the Kir6.2/SUR2B channel (2.7 μM).

Figure 7
Inhibition by DIDS of [3H]-glibenclamide binding to (A) SUR2B(Y1206S) alone and (B) coexpressed with Kir6.2: Effects of Mg2+ (0.7 mM) and MgATP (1 mM). Data are means±s.e.mean from 3–4 experiments performed at 22°C. ...
Table 3
Inhibition of [3H]-glibenclamide binding to SUR2B(Y1206S) by DIDS

In order to see whether DIDS inhibited glibenclamide binding by a competitive mechanism, [3H]-glibenclamide binding to SUR2(Y1206S) coexpressed with Kir6.2 was measured in saturation experiments in the presence of Mg2+ and the absence and presence of DIDS (10 μM). The results are illustrated in Figure 8A and the parameters are listed in Table 4. It is seen that DIDS decreased the number of glibenclamide sites leaving the KD unchanged, i.e. inhibition was of a pure non-competitive type. Surprisingly, however, the mechanism of inhibition depended on assay conditions. Analogous experiments were performed with SUR2B(Y1206S) expressed alone, albeit in the absence of Mg2+ and at 37°C, the standard temperature for binding assays in this laboratory. Under these conditions, DIDS (20 μM) left the number of glibenclamide sites unchanged but shifted the KD of [3H]-glibenclamide binding curve from 4.5 to 15 nM, compatible with a competitive mechanism of inhibition (Figure 8B, Table 4). Analysing the rightward shift of the KD value induced by DIDS according to Cheng-Prusoff (eqn (2), which is based on competition of the two ligands for a common binding site) one estimates for DIDS a Ki value of 9.6 (4.8,19) μM, which is in reasonable agreement with the value obtained from the DIDS inhibition curve under these conditions (Ki=17 (15,19) μM, Table 3). One also notes that the (apparent) competition between DIDS and glibenclamide means that under these conditions the interaction of DIDS with SUR2B(Y1206S) is reversible.

Figure 8
[3H]-glibenclamide saturation binding in the absence and presence of DIDS. (A) Kir6.2/SUR2B(Y1206S) in the presence of Mg2+ (0.7 mM) at 22°C. Data show total (BTOT) and nonspecific binding (NSB) in the absence and presence of DIDS ...
Table 4
Effect of DIDS on [3H]-glibenclamide saturation binding in the absence of ATP

Reversibility of inhibition may not hold in the experiments using the Kir6.2/SUR2B(Y1206S) complex in the presence of Mg2+. Under these conditions, inhibition was unsurmountable (Figure 8A) and this could reflect an irreversible interaction of DIDS with the Kir/SUR complex. To test this possibility, membranes were incubated in the presence of DIDS (30 μM) or solvent, then strongly diluted into [3H]-glibenclamide containing solution (1 : 17 fold, to induce dissociation of DIDS from SUR in case of a reversible interaction), and the association kinetics were monitored. Figure 9 shows that glibenclamide binding to complex preincubated with DIDS reached ~90% of the control level. This indicated that the interaction of DIDS with the complex was entirely reversible, the slight 10% loss in binding reflecting inhibition by the DIDS concentration (1.8 μM) remaining after dilution. Figure 9 also shows that the kinetics of glibenclamide binding to complex preincubated with DIDS were significantly slower than that in the absence of DIDS (half-times ~4.0 min (DIDS) vs 2.7 min (control)), indicating that dissociation of DIDS from the complex was rate-limiting.

Figure 9
Kinetics of [3H]-glibenclamide binding to Kir6.2/SUR2B (Y1206S) after preincubation with DIDS. Membranes (3.9 mg protein ml−1) were preincubated with DIDS (30 μM) or solvent in the presence of Mg2+ (0.7 mM) for 45 min at ...

Since MgATP protected against the inhibition of the Kir6.2/SUR2B channel by DIDS it was examined whether the nucleotide (in the presence of Mg2+) affected also the interaction of DIDS with mutant SUR2B. To facilitate comparison with the electrophysiological experiments, [3H]-glibenclamide-DIDS inhibition experiments were performed at 22°C and using SUR2B(Y1206S) coexpressed with Kir6.2. In the presence of MgATP (1 mM), the inhibition curve was biphasic with 33% of glibenclamide binding being inhibited with IC50,1=1.1 μM and the remaining 67% with IC50,2=200 μM (Figure 7B, Table 3). As shown above, the corresponding DIDS inhibition curve in the presence of Mg2+ alone, was (essentially) monophasic with Ki=7.9 μM (Figure 7B). Hence, MgATP induced a heterogeneity in the DIDS sites or in their coupling to the glibenclamide site shifting one part towards ~7 fold higher and the other to 25 fold lower affinity (Table 3). Qualitatively similar observations were made at 37°C for mutant SUR2B expressed alone (Table 3). In the absence of Mg2+, the inhibition curve was monophasic with an IC50 value of 27 μM; MgATP induced a 5 fold leftward shift (IC50=5.1 μM) and a slight heterogeneity (Hill coefficient 0.84±0.03).

Discussion

It has previously been shown that, in the absence of Mg2+, high concentrations of DIDS irreversibly inhibited KATP channels by binding to Kir6.2 (Furukawa et al., 1993; Proks et al., 2001). This study describes two new findings: First, Mg2+ sensitizes the KATP channel for the blocking action of DIDS in a nucleotide independent manner and, second, as sensed by [3H]-glibenclamide, the interaction of DIDS with (mutant) SUR2B is modulated by Mg2+.

Mg2+ modulation of DIDS block

In the absence of Mg2+, the sensitivity of the recombinant KATP channels (Kir6.2/SURx) to inhibition by DIDS decreased with the rank order SUR1>SUR2A~SUR2B (Table 1, values at 90 s). The rank order SUR1>SUR2A agrees with the observation of Proks et al. (2001) and the IC50 value of 180 μM for the Kir6.2/SUR2A channel is in reasonable agreement with that determined by Furukawa et al. (1993) for the KATP channel in guinea-pig cardiocytes (73 μM). These values are in the range of inhibition constants observed for various anion transporter and channels (Furukawa et al., 1993). The presence of physiological concentrations of Mg2+ increased the sensitivity towards DIDS in a manner depending on the SUR subtype with the most dramatic effect occurring with the Kir6.2/SUR2B channel: Here the IC50 value of DIDS was shifted by Mg2+ to 70 fold lower concentrations and the rank order of sensitivity towards DIDS was now SUR2B>SUR1>SUR2A.

This dependence on the SUR subtype raised the possibility that Mg2+ exerted its effect by binding to SUR. More direct evidence for this possibility came from the observation that Mg2+ did not affect the inhibition by DIDS of the Kir6.2Δ26 channel, which forms a channel in the absence of SUR (Tucker et al., 1997); however, sensitivity to Mg2+ was restored by coexpression with SUR2B. These experiments exclude the possibility that, similar to EDTA, Mg2+ forms a complex with DIDS, thereby changing the chemical nature of the blocker. Instead, they show that the presence of SUR is required for Mg2+ to be effective. It seems plausible to assume that Mg2+ binds to SUR to elicit its effect; the ability of Mg2+ to affect DIDS binding to SUR2B(Y1206S) in the absence of Kir6.2 supports this hypothesis. However, more work like mutating the conserved Asp residue in the Walker B motif of the NBFs (Nichols et al., 1996) or other residues possibly involved in coordinating Mg2+ (Gaudet & Wiley, 2001; Urbatsch et al., 2000a,b) must be done to definitively prove this point.

Another point of interest is that Mg2+ did not require the presence of a hydrolysable nucleotide like ATP or ADP in order to affect channel inhibition by DIDS. This is again matched by the results of the binding studies (Table 3). Comparison with sulphonylureas shows similarities and differences: Similar to DIDS, the potency of tolbutamide in blocking the cardiac KATP channel is increased by Mg2+ in the absence of nucleotide (Miyamura et al., 2000); however, unlike in the case of DIDS, Mg2+ alone does not affect glibenclamide binding to SUR2B(Y1206S) (Hambrock et al., 2002) and the presence of the Mg-nucleotide complex is required to inhibit binding. A further point is that in the presence of Mg2+, exposure of the channel even to low concentrations of DIDS ([gt-or-equal, slanted]10 μM) led to irreversible inhibition and that a sufficiently large excess of MgATP afforded protection.

Interaction of DIDS with SUR

It was shown earlier that DIDS inhibits binding of [3H]-P1075 to SUR2B with an IC50 value of 1.6 μM (3 mM MgATP, 37°C; Russ et al., 2000). Here we showed that DIDS inhibited [3H]-glibenclamide binding to (mutant) SUR2B in a manner dependent on Mg2+ and on coexpression with Kir6.2. With mutant SUR alone, Mg2+ induced a leftward shift and a flattening of the DIDS inhibition curve. This means that Mg2+ produced an apparent heterogeneity in the binding sites for DIDS by inducing a new class of sites with higher affinity (>40 fold) and leaving the other part essentially unchanged. Coexpression with Kir6.2 reduced this heterogeneity and reduced the Mg2+ shift to a factor of ~5. In the presence of MgATP, the effect of coexpression was even more prominent: Whereas for mutant SUR2B alone MgATP induced a ~5 fold leftward shift of a homogenous inhibition curve (data at 37°C), it induced strong heterogeneity in the binding curve of the Kir/SUR complex (Table 3).

The biphasic inhibition curve for mutant SUR2B in the presence of Mg2+ and the fact that the relationship between DIDS and glibenclamide has a competitive or a non-competitive appearance depending on the assay conditions (Figure 8) suggested that SUR harbours more than one binding site for DIDS. However, these complexities may also be interpreted assuming that SUR expressed alone forms tetramers (Löffler-Walz et al., 2002; Hambrock et al., 2002) and that site-site interactions between subunits occur. More importantly, the inhibition of [3H]-glibenclamide binding to the Kir6.2/SUR2B(Y1206S) complex by DIDS (30 μM) was entirely reversible with a half-time of 4 min.

Since binding of DIDS and glibenclamide to mutant SUR2B was measured at 22 and 37°C (Table 3), one obtains a rough estimate of the thermodynamics of binding. The Ki values of DIDS (−Mg2+) did not change much with temperature, indicating a negligible change in free enthalpy (ΔH) for DIDS binding to SUR2B(Y1206S). In view of the two negative charges carried by DIDS this is surprising. In contrast, the KD values of glibenclamide binding strongly decreased with decreasing temperature. From the two values in Table 3 one estimates ΔH~−8.5 kcal mol−1 for this interaction in good agreement with the value determined for glibenclamide binding to rat brain cortex and heart (−10.5 and −13.5 kcal mol−1 respectively; Gopalakrishnan et al., 1991).

Does DIDS binding to SUR mediate channel block?

In the absence of Mg2+, the IC50 value of DIDS binding to Kir6.2/SUR2B(Y1206S) was ~5x lower than the IC50 value for block of the Kir6.2/SUR2B channel. This difference can be explained by assuming that there are four equal and independent binding sites for DIDS and that occupation of all of them is required for channel block (Dörschner et al., 1999; Russ et al., 1999). However, preliminary experiments with other SUR subtypes show discrepancies of 10× (SUR1) and 20× (mutant SUR2A; Quast, unpublished results), rendering this explanation unlikely. More importantly, Proks et al. (2001) have demonstrated that in the absence of Mg2+, DIDS blocked recombinant KATP channels by binding to the Kir6.2 subunit at a site interacting with, but different from the ATP site. Our data with the Kir6.2Δ26 channel and the Kir6.2(G334D) mutant confirm and extend these results. One has to conclude that, in the absence of Mg2+, binding of DIDS to SUR is not transduced into channel inhibition.

In the presence of Mg2+, the binding studies involving the Kir/SUR complex and the electrophysiological experiments gave several concordant results. First, Mg2+ moved both inhibition curves leftwards and the IC50 values were similar (7.9 vs 2.7 μM). Second, MgATP (1 mM) shifted most of the binding sites to low affinity (200 μM) which is compatible with the decreasing protection afforded against increasing concentrations of DIDS (30, 100, 300 μM) in the electrophysiological experiments. However, the fact that binding of DIDS (30 μM) to the channel complex as seen by [3H]-glibenclamide was entirely reversible whereas channel inhibition was irreversible clearly shows that the binding site seen by radioligand is not the one which mediates channel inhibition. Therefore one is left with the tentative model that Mg2+, probably by binding to SUR, allosterically affects the binding site for DIDS on Kir6.2, rendering it more accessible and/or more reactive. This allosteric effect depends on the SUR subtype. Obviously, this model could be tested using some of the mutants recently described by Proks et al. (2001).

In conclusion, we have shown here that Mg2+ sensitizes the KATP channel for the blocking action of DIDS in a manner dependent on the SUR subtype. The fact that Mg2+ was effective in the absence of nucleotides highlights its role in signal transduction in KATP channels.

Acknowledgments

L. Gojkovic-Bukarica is a fellow of the Alexander von Humboldt-Stiftung. The study was supported by the Deutsche Forschungsgemeinschaft, grants QU 100/2-4 and QU 100/3-1 (A. Hambrock and U. Quast), the Federal Ministry of Education, Science, Research and Technology (Fö 01KS9602) and the Interdisciplinary Center of Clinical Research (IZKF) Tübingen. The authors thank Drs Y. Kurachi and Y. Horio (Osaka) for the generous gift of the murine clones of SUR2A, 2B and Kir6.2, and Dr C. Derst (Freiburg) for the rat clone of SUR1. The expert technical assistance of Ms C. Müller and many helpful comments of Ulf Lange are gratefully acknowledged.

Abbreviations

AMP-PCP
β-γ-methylene-adenosine 5′-triphosphate
DIDS
4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid
GBC
glibenclamide
HEK cells
human embryonic kidney cells
KATP channels
ATP-sensitive K+ channels
Kir
inward rectifier potassium channel
NBF
nucleotide binding fold
SUR
sulphonylurea receptor

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