Glibenclamide blocks volume-sensitive Clminus channels by dual mechanisms

Yan Liu, Shigetoshi Oiki, Takehiko Tsumura, Takahiro Shimizu, and Yasunobu Okada

Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585; and Department of Internal Medicine, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To study the mechanisms of glibenclamide actions on volume-sensitive Cl- channels, whole cell patch-clamp studies were performed at various pH levels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl- currents in the entire range of voltage examined (-100 to +100 mV) and accelerated the depolarization-induced inactivation at pH 7.5. When glibenclamide was applied from the intracellular side, in contrast, no effect was observed. At acidic pH, at which the weak acid glibenclamide exists largely in the uncharged form, the instantaneous current was, in a voltage-independent manner, suppressed by the extracellular drug at micromolar concentrations without significantly affecting the depolarization-induced inactivation. At alkaline pH, at which almost all of the drug is in the charged form, glibenclamide speeded the inactivation time course and induced a leftward shift of the steady-state inactivation curve at much higher concentrations. Thus it is concluded that glibenclamide exerts inhibiting actions on swelling-activated Cl- channels from the extracellular side and that the uncharged form is mainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.

sulfonylurea; pH; swelling-activated chloride current; inactivation; intestinal epithelial cell

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GLIBENCLAMIDE IS A MEMBER of the sulfonylurea family, which is used for the treatment of non-insulin-dependent diabetes mellitus. The mechanism of the sulfonylurea drug-induced stimulation of insulin secretion is that it interacts with the sulfonylurea receptor (SUR) and inhibits ATP-sensitive K+ channels (KATP) in pancreatic beta -cells (3).

Recently, several Cl- channels have also been reported to be blocked by sulfonylureas. Wangemann et al. (36) first reported that glibenclamide inhibits Cl- currents in the basolateral membrane of the thick ascending limb of the loop of Henle. Sheppard and Welsh (25) demonstrated the inhibition of epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in NIH/3T3 cells by glibenclamide and several other sulfonylurea drugs. Similar glibenclamide-induced inhibition was then found for endogenously expressed Cl- channels activated by cAMP in human epithelial T84 cells (11), dogfish rectal gland cells (6), and guinea pig ventricular myocytes (28, 37). Single-channel studies in CFTR-transfected cells showed that glibenclamide exerts a reversible flickery type open-channel blocking action on the CFTR Cl- channel (23, 24). Glibenclamide was also observed to inhibit outwardly rectifying Cl- channels (ORCC) in M-1 mouse cortical collecting duct cells (35) and in human colonic carcinoma cell line HT-29 cells (22). The effect on single ORCC also appeared to be due to reversible flicker block. For both CFTR and ORCC, glibenclamide was known to be effective by application from either the outside or inside of the cells (6, 22).

More recently, volume-sensitive outwardly rectifying (VSOR) Cl- currents were also reported to be sensitive to glibenclamide in M-1 mouse cortical collecting duct cells (19) and in guinea pig atrial myocytes (37). In the present work, whole cell recordings were performed in a human small intestinal epithelial cell line (Intestine 407) to answer the following questions. Is glibenclamide able to inhibit the VSOR Cl- channel in human epithelial Intestine 407 cells? From which side, if effective, can glibenclamide inhibit the VSOR Cl- channel, from the outer or inner surface of the membrane? Which form of the weak organic acid, which exists in both charged and uncharged forms at physiological pH, is effective?

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. A human intestinal epithelial cell line, Intestine 407, was cultured in monolayer, isolated to single spherical cells by detachment from the plastic substrate, and cultured in suspension with agitation for 10-150 min, as reported previously (16). For patch-clamp studies, the cells were placed in a chamber (0.5 ml) and perfused with a CsCl solution at a flow rate of ~5 ml/min. For cell volume measurements, the cells were suspended in normal Ringer.

Patch clamp. Whole cell recordings were performed, as reported previously (16), using wide-tipped electrodes (~2 MOmega ). Series resistance (<5 MOmega ) was compensated (to 60-75%) to minimize voltage errors. The time course of current activation and recovery was monitored by repetitively applying (every 15 s) alternating step pulses (2-s duration) from a holding potential of 0 mV to ±40 mV. To monitor the voltage dependence of the whole cell current, stepping pulses (2-s duration) were applied from a prepotential at -100 mV (0.6-s duration) to test potentials of -80 to +100 mV in 20-mV increments after attainment of steady activation of swelling-induced current. The amplitude of instantaneous current was measured at 2.5 ms after the step-pulse onset. Steady-state current levels were evaluated by fitting the time course of inactivation to a double exponential function, when time-dependent inactivation took place.

Currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Current signals were filtered at 1 kHz using a four-pole Bessel filter and digitized at 4 kHz. pCLAMP software (version 6.0.2; Axon Instruments) was used for the command pulse control, data acquisition, and analysis.

All experiments were carried out at room temperature (23-26°C). Data are given as means ± SE. Statistical differences of the data were evaluated by Student's t-test and were considered significant at P < 0.05.

Cell volume measurements. The mean cell volume was measured with an electrical sizing technique using a Coulter counter, as reported previously (10), at room temperature.

Solutions and chemicals. The solutions employed in patch-clamp experiments are as follows. Isotonic (325 mosmol/kgH2O) CsCl bathing solution (pH 7.5) contained (in mM) 110 CsCl, 12 HEPES, 8 Tris, 5 MgSO4, and 100 mannitol (pH 7.5, osmolality 325 mosmol/kgH2O). Cell swelling was induced by hypotonic (260 mosmol/kgH2O) CsCl solution in which mannitol was reduced to 40 mM. Alkaline solutions were prepared by replacing HEPES with 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) on an equimolar basis. Acidic solutions were prepared by replacing HEPES with MOPS and decreasing the Tris concentration. These buffers (at 12 mM) by themselves did not affect VSOR Cl- currents. The intracellular CsCl solution (290 mosmol/kgH2O) contained (in mM) 110 CsCl, 2 MgSO4, 1 Na2ATP, 15 Na-HEPES, 10 HEPES, 1 EGTA, and 50 mannitol (pH 7.45).

For cell volume measurements, normal Ringer solution was employed as the isotonic solution. The composition was (in mM) 137.5 NaCl, 4.2 KCl, 0.9 CaCl2, 0.5 MgCl2, 20 mannitol, 6 Na-HEPES, and 8 HEPES (pH 7.3). The hypotonic solution (60%) was prepared by diluting the isotonic solution with distilled water.

The following agents were added to solutions: 0.94-1,000 µM glibenclamide (Hoechst), 0.5 or 2 mM chlorapropamide (Sigma), and 0.5 or 2 mM tolbutamide (Nacalai Tesque). Stock solutions of glibenclamide (0.2 M), chlorapropamide (0.8 M), and tolbutamide (0.8 M) in DMSO were diluted to the desired final concentrations just before use. DMSO alone (<= 0.5%) had no effect on the VSOR Cl- currents in Intestine 407 cells. A variety of total concentrations of glibenclamide were selected within the limitation of reduced solubility at lower pH. When necessary, the concentrations of uncharged ([G]) and charged ([G-]) forms were calculated by the equation pH = pKa + log([G-]/[G]), with the assumption that pKa = 5.3 (27).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of sulfonylurea drugs on VSOR Cl- currents. During exposure to hypotonic solution (82% osmolality), Intestine 407 cells exhibited large activation of outwardly rectifying Cl- currents with osmotic swelling under whole cell voltage clamp, as reported previously (16). Extracellular application of glibenclamide (200 µM) potently blocked the current (Fig. 1). The effect of extracellular glibenclamide was rapid in onset and reversible.


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Fig. 1.   Effects of extracellular glibenclamide on whole cell volume-sensitive outwardly rectifying (VSOR) Cl- currents. A: representative record before and after osmotic cell swelling in presence (horizontal line) or absence of 200 µM glibenclamide in bath solution during application of alternating pulses from 0 to ±40 mV or of step pulses from -100 to +100 mV in 20-mV increments (at asterisks). Gain of chart recorder was changed by one-half at 1st and 3rd asterisks from left. B: expanded traces of current responses (a and b in A) to step pulses (protocol shown in inset) in the absence (a) and presence (b) of 200 µM glibenclamide. Arrowheads, zero-current level.

Figure 2 depicts the concentration-inhibition relations for the instantaneous currents. The total glibenclamide concentration for half-maximal inhibition (IC50) and the Hill coefficient were 261.0 ± 8.3 µM and 3.0 ± 0.4 at -40 mV and 232.4 ± 7.0 µM and 2.9 ± 0.3 (n = 4-10) at +40 mV, respectively, exhibiting slight voltage dependence.


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Fig. 2.   Concentration-inhibition relations of extracellular glibenclamide. Whole cell instantaneous currents (Iinst) in presence of glibenclamide recorded at -40 mV (squares) and +40 mV (triangles) were divided by those measured before addition of glibenclamide, and these relative current values were plotted against total glibenclamide concentration (means ± SE of 4-10 observations). Dotted lines are best fits to Hill equation.

Only a slight voltage dependence of the glibenclamide effect was also seen in the current-voltage (I-V) relations for instantaneous currents, as shown in Fig. 3A, top. The glibenclamide effect was evident over the entire range of membrane potentials examined.


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Fig. 3.   Current-voltage (I-V) relationships in absence (open symbols) and presence (filled symbols) of glibenclamide. A: effects of extracellular glibenclamide (200 µM) on Iinst (top) and steady-state currents (Is-s; bottom) (means ± SE). B: effects of intracellular glibenclamide (500 µM) on Iinst (top) and Is-s (bottom) (means ± SE; n = 7).

The VSOR Cl- current exhibited time-dependent inactivation at large positive potentials (see Fig. 1B). The I-V curve for steady-state currents (Fig. 3A, bottom) shows its voltage dependence. Glibenclamide augmented the depolarization-induced inactivation from the extracellular side.

In contrast to extracellular glibenclamide, no effects of intracellular glibenclamide (500 µM incorporated in the pipette solution) were found on either the instantaneous or steady-state currents of VSOR Cl- channel (Fig. 3B).

The time course of inactivation could be fitted to a double exponential function (Fig. 4A), as reported previously (30). Extracellular glibenclamide accelerated depolarization-induced inactivation. As shown in Fig. 4, B and C, 200 µM glibenclamide greatly decreased both the fast and slow time constants (tau f and tau s, respectively) at 200 µM. Similar effects were also observed with application of 100 µM glibenclamide (n = 4, data not shown).


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Fig. 4.   Effect of extracellular glibenclamide (Gliben) on depolarization-induced inactivation of VSOR Cl- currents. A: inactivation time courses during application of steps to +100 mV in absence (control) and presence of 200 µM glibenclamide. Smooth curves are biexponential fits. B: fast time constants (tau f) of inactivation of volume-sensitive Cl- currents in absence (control) and presence of glibenclamide (200 µM) at +60, +80, and +100 mV (means ± SE; n = 7). Differences between values without and with glibenclamide are statistically significant (P < 0.05). C: slow time constant (tau s) at +60, +80, and +100 mV. Differences between values without and with glibenclamide are statistically significant (P < 0.05).

Steady-state inactivation curves were shifted to the left by extracellular glibenclamide in a concentration-dependent manner (Fig. 5). In the presence of glibenclamide, the inactivation kinetics became prominent at lower positive potentials, indicating that glibenclamide affects voltage-dependent inactivation. The midpoint of inactivation (half-inactivation potential) was shifted from +76.6 ± 3.9 mV to +55.4 ± 7.1 mV, +30.4 ± 11.3 mV, and -1.6 ± 5.9 mV (n = 4-10) by glibenclamide at 100, 200, and 300 µM, respectively.


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Fig. 5.   Steady-state inactivation curves of VSOR Cl- currents. Means ± SE of ratio of Is-s to peak current (Is-s/Iinst) from 4-10 different cells in absence (open circles) or presence (filled symbols) of 100 (triangles), 200 (inverted triangles), or 300 (diamonds) µM glibenclamide. Curves are fits of data (excluding noninactivating components) to Boltzmann function.

In contrast to glibenclamide, extracellular application of chlorapropamide or tolbutamide had no effects on the whole cell VSOR Cl- current at 200 µM (n = 3 or 4; data not shown). The instantaneous current recorded at +40 mV was not significantly reduced by 500 or 2,000 µM chlorapropamide (to 93.7 ± 6.4 and 88.5 ± 4.4%, respectively; n = 4, P > 0.34). At 500 and 2,000 µM tolbutamide reduced, although not statistically significantly (P > 0.13), the instantaneous current to 80.2 ± 8.1 (n = 7) and 64.7 ± 7.6% (n = 4), respectively, at +40 mV.

Effects of sulfonylurea drugs on regulatory volume decrease. Intestine 407 cells exhibited volume regulation after transient osmotic swelling during a hypotonic challenge (Fig. 6), as reported previously (10). Extracellular application of glibenclamide (200 and 500 µM) impaired regulatory volume decrease (RVD) dramatically (Fig. 6). However, tolbutamide impaired RVD very little at 500 µM (Fig. 6). Therefore, it is likely that glibenclamide impairs RVD, at least in part, via inhibition of VSOR Cl- current.


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Fig. 6.   Effects of glibenclamide and tolbutamide (Tolbut) on volume regulation of Intestine 407 cells after hypotonic challenge (means ± SE; n = 5). A hypotonic challenge (60% osmolality) was applied at time 0 (arrow). Cell volume was normalized to that before hypotonic challenge (2,188 ± 94 µm3, n = 20). Open squares and filled symbols represent relative cell volume in absence (control) and presence of sulfonylurea drugs, respectively. Triangles, inverted triangles, and circles represent relative cell volume in presence of 500 µM glibenclamide, 200 µM glibenclamide, and 500 µM tolbutamide, respectively.

Voltage-independent effects of uncharged glibenclamide on instantaneous VSOR Cl- currents. Glibenclamide is a weak organic acid, which thus exists in solution in either undissociated or anionic form, depending on the pH. To examine which form, uncharged or negatively charged, of the sulfonylurea is more effective in suppression of the instantaneous peak current, pH was varied from 7.5 to 5 in bath solution, since glibenclamide exerts its action at a site accessible from the extracellular solution.

In the absence of glibenclamide, the acidic pH itself did not significantly alter the instantaneous currents (from 5.4 ± 0.5 nA at pH 7.5 to 5.0 ± 0.5 nA at pH 5; n = 6; +40 mV; P > 0.46), although the instantaneous current was increased transiently on reduction of pH (Fig. 7A). These findings are consistent with reports on other cell types that there were essentially no effects of pH on the instantaneous VSOR Cl- currents (1, 15, 32, 34).


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Fig. 7.   Effects of extracellular glibenclamide at pH 5 on VSOR Cl- currents. A: representative record before and after osmotic cell swelling at pH 7.5 and 5.0 in presence (horizontal line) or absence of 1.88 µM extracellular glibenclamide during application of alternating pulses from 0 to ±40 mV or of step pulses from -100 to +100 mV in 20-mV increments (at asterisks). Gain of chart recorder was changed by one-half at asterisks. B: expanded traces of current responses (a, b, and c in A) to step pulses in absence (a and b) and presence (c) of glibenclamide at pH 7.5 (a) and pH 5.0 (b and c). Arrowheads, zero-current level. C: concentration-inhibition relation. Whole cell Iinst values recorded at +40 mV in presence of glibenclamide were divided by those measured in absence of glibenclamide, and these relative current values were plotted against total glibenclamide concentration (means ± SE of 4-8 observations). Curve is best fit to Hill equation. D: I-V relationships for Iinst (triangles) and Is-s (circles) at pH 5 in absence (open symbols) and presence (filled symbols) of 2.82 µM extracellular glibenclamide (means ± SE; n = 5 or 6).

At pH 5, at which glibenclamide existed largely in the uncharged form (twice that of the charged form, if pKa = 5.3), instantaneous VSOR Cl- currents were markedly suppressed by glibenclamide even at 1.88 µM, as shown in Fig. 7, A and B. In Fig. 7C, the concentration-inhibition relation for instantaneous currents at pH 5 was plotted. The IC50 and Hill coefficient values were 3.2 ± 1.4 µM and 1.4 ± 0.3 (n = 4-6). This result indicates that uncharged glibenclamide is effective at micromolar concentrations.

The I-V curves (Fig. 7D) showed that the suppressive effects of 2.82 µM glibenclamide at pH 5 on the instantaneous and steady-state currents were essentially independent of voltages between -80 and +100 mV.

As shown in Fig. 8, the inhibiting effects of glibenclamide at various pH levels between 7.5 and 5 fall into the same curve (with IC50 of 1.53 µM and Hill coefficient of 1.99) as a function of uncharged concentration calculated under the assumption that pKa = 5.3. This is similar to the effect of quinine or quinidine on swelling-activated Cl- currents in bovine endothelial cell lines (33).


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Fig. 8.   Concentration-inhibition relations of extracellular uncharged glibenclamide at different pH levels. Uncharged glibenclamide concentrations were calculated using a pKa value of 5.3. Whole cell values Iinst in presence of glibenclamide recorded at +40 mV were divided by those measured before addition of glibenclamide, and these relative current values were plotted against concentration of uncharged glibenclamide (means ± SE of relative Iinst values from 4-10 observations). Curve is best fit to Hill equation. IC50 and Hill coefficient values were 1.53 ± 0.06 µM and 1.99 ± 0.21, respectively.

It is concluded that glibenclamide, preferentially in the uncharged form, inhibits instantaneous Cl- currents in an essentially voltage-independent manner.

Voltage-dependent effects of charged glibenclamide on depolarization-induced inactivation of VSOR Cl- currents. An alkaline shift of bath pH in the absence of glibenclamide transiently decreased the instantaneous current, as shown in Fig. 9A. However, the alkaline shift did not significantly change the current in the steady state after the recovery from the transient decrease [from 4.3 ± 0.5 nA (n = 8) at pH 7.5 to 4.4 ± 0.5 nA (n = 7) at pH 9.2; +40 mV; P > 0.83].


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Fig. 9.   Effects of extracellular glibenclamide at pH 9.2 on VSOR Cl- currents. A: representative record before and after osmotic cell swelling at pH 7.5 and 9.2 in presence (horizontal line) or absence of 500 µM extracellular glibenclamide during application of alternating pulses from 0 to ±40 mV or of step pulses from -100 to +100 mV in 20-mV increments (at asterisks). Gain of chart recorder was changed by one-half at asterisks. B: expanded traces of current responses (a, b, and c in A) to step pulses in absence (a and b) and presence (c) of glibenclamide at pH 7.5 (a) and pH 9.2 (b and c). Arrowheads, zero-current level. C: concentration-inhibition relation. Whole cell Iinst values recorded at +40 mV in presence of glibenclamide were divided by those measured in absence of glibenclamide, and these relative current values were plotted against total glibenclamide concentration (means ± SE of Iinst values from 4-8 observations). Curve is best fit to Hill equation. D: I-V relationships for Iinst (triangles and dotted lines) and Is-s (circles and solid lines) at pH 9.2 in absence (open symbols) and presence (filled symbols) of 500 µM extracellular glibenclamide (means ± SE; n = 5 or 6).

At pH 9.2, at which almost all of the glibenclamide (>99.98%, if pKa = 5.3) existed in the uncharged form, the sulfonylurea suppressed the instantaneous VSOR Cl- currents only at a much higher concentration (Fig. 9, A and B). At pH 9.2, the concentration-inhibition relation could be fitted with IC50 and Hill coefficient values of 532.3 ± 7.5 µM and 1.3 ± 0.1 (n = 4-8), respectively (Fig. 9C). The I-V curve (Fig. 9D) shows that glibenclamide (500 µM) more markedly affected the steady-sate current than the instantaneous current at pH 9.2.

As reported previously in other cell types (1, 15, 32, 34), extracellular acidification was found to accelerate the depolarization-induced inactivation (see Fig. 7Bb), whereas extracellular alkalinization markedly slowed the inactivation time course (see Fig. 9Bb). At pH 5, glibenclamide (2.82 µM) did not significantly change the inactivation time course (both tau f and tau s), as summarized in Fig. 10A, top. In contrast, at pH 9.2. glibenclamide (500 µM) dramatically decreased both tau f and tau s, as shown in Fig. 10B, top.


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Fig. 10.   Effects of extracellular glibenclamide mainly in uncharged form (A) or mainly in charged form (B) on depolarization-induced inactivation of VSOR Cl- currents. A: effects of 2.82 µM glibenclamide at pH 5. B: effects of 500 µM glibenclamide at pH 9.2. Top left: tau f of Cl- current inactivation in absence and presence of glibenclamide at +80 mV (means ± SE). Differences between values without (control) and with glibenclamide are statistically significant for B (P < 0.001) but not for A (P > 0.45). Top right: tau s at +80 mV. Differences between values without (control) and with glibenclamide are statistically significant for B (P < 0.022) but not for A (P > 0.87). Bottom: steady-state inactivation curves in absence (open circles) and presence (filled circles) of glibenclamide (means ± SE of Is-s/Iinst). Curves are fits of data (excluding noninactivating components) to Boltzmann equation. Midpoints with and without glibenclamide were +36.5 ± 2.3 and +46.4 ± 1.1 mV (n = 4) for A and +9.9 ± 1.1 and +97.1 ± 1.0 mV (n = 5) for B, respectively.

In the absence of glibenclamide, the steady-state voltage dependence of depolarization-induced inactivation was affected by pH changes in bath solution, as found previously in glioma cells (15), Xenopus oocytes (32), and BC3H1 myoblasts (34). The steady-state inactivation curve was shifted little further by 2.82 µM glibenclamide, which existed mainly in the uncharged form, applied to the bath at pH 5 (Fig. 10A, bottom). In contrast, at pH 9.2, the steady-state inactivation curve was prominently shifted to the left by 500 µM glibenclamide, which existed mostly in the charged form (Fig. 10B, bottom).

In all, it appears that negatively charged glibenclamide facilitates inactivation kinetics in a voltage-dependent manner and causes a leftward shift in the steady-state voltage-dependent inactivation.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The VSOR Cl- channel is known to be ubiquitously expressed in animal cells and plays an essential role in the cell volume regulation of swollen cells (21). The present study indicated that glibenclamide is a potent inhibitor of swelling-activated VSOR Cl- channels in the physiological voltage range and of cell volume regulation after osmotic swelling (RVD) in human epithelial cells. However, two other members of the sulfonylurea family, tolbutamide and chlorapropamide, were much less effective. The binding site of glibenclamide is located at a domain accessible from the extracellular, but not the intracellular, side of the cells. This is in contrast to the cases of CFTR (6), ORCC (22), and KATP (9).

The existence of intracellular ATP is a prerequisite to VSOR Cl- channel activity in many cell types (21), including Intestine 407 cells (20). Because a high concentration of glibenclamide (500 µM) was reported to induce marked depletion of ATP within skate hepatocytes (4) and human hepatoma cells (5), it is possible that the sulfonylurea drug inhibited VSOR Cl- channels via intracellular ATP depletion. However, this possibility can be safely ruled out because 1) glibenclamide was found largely to inhibit both the channel activity and RVD at much lower concentrations (<= 200 µM), 2) glibenclamide could not block the channel activity when applied directly into the cytosol via patch pipettes, and 3) during whole cell patch-clamp experiments the intracellular ATP level was maintained at 1 mM by the pipette solution.

The sulfonylurea drug is a blocker of KATP channels in pancreatic beta -cells, myocardium, and smooth muscle cells (3). A component of the KATP channel was identified as the SUR, which is a member of ATP-binding cassette (ABC) transporter superfamily (12-14). Tolbutamide has been shown to interact with the nucleotide-binding domain on SUR1, thereby inhibiting KATP channels (8). Also, sulfonylurea drugs are known to block another ABC transporter member, CFTR Cl- channel (23, 25, 31). CFTR has recently been shown to confer glibenclamide sensitivity on ROMK2 channel (18). KATP, CFTR, and VSOR channels share some properties, such as sensitivity to sulfonylurea and dependence on ATP. Thus one may imagine that the sulfonylurea drug interferes with ATP binding at an intracellular site of the VSOR Cl- channel. However, the fact that intracellular application of glibenclamide failed to block VSOR Cl- channels is at variance with this inference.

The present study was performed to determine, through application of glibenclamide at a variety of pH levels, which form of the weak acid glibenclamide is effective in suppressing the VSOR Cl- channel. Glibenclamide was found to inhibit VSOR Cl- channels by dual mechanisms; the neutral form exerts the action at micromolar concentrations by suppressing the instantaneous current in a voltage-independent manner, whereas the anionic form does so at submillimolar concentrations by promoting depolarization-induced inactivation due to a shift in the voltage dependence of steady-state inactivation. The effects of glibenclamide could not be ascribed chiefly to effects of pH changes on the channel activity per se because changes in bath pH affected the instantaneous Cl- current peak only slightly and because glibenclamide effects were observed in addition to effects of pH change on steady-state inactivation of the Cl- channel. In light of the fact that the effects of un-ionized glibenclamide at different pH levels fell into the same curve using a pKa value for glibenclamide, it is likely that pH-dependent glibenclamide effects on instantaneous currents are largely mediated by ionization of glibenclamide rather than by protonation of the glibenclamide-binding site on the channel.

The exact mechanism of facilitatory effect of charged glibenclamide on depolarization-induced inactivation awaits further study, especially at the single-channel level. It is possible that extracellular anionic glibenclamide acts as an open-channel blocker by itself, because the VSOR Cl- channel is known to have a large extracellular vestibule accessible to large anions, which may thereby plug the pore and prevent Cl- permeation (21). Alternatively, there remains the possibility that glibenclamide binding may allosterically alter the inactivation gating.

The sulfonylurea drug glibenclamide blocks KATP channels at nanomolar concentrations, thereby promoting insulin secretion from pancreatic beta -cells (2). It is thought that the lipophilic undissociated form of sulfonylureas is effective in inhibiting KATP channels, on the basis of the observation of equipotency of the drug applications from extracellular and intracellular sides (9, 26, 29, 38) and also on the observation of reduced potency at increased pH values (7, 38). Glibenclamide has been demonstrated to be a potent blocker of the CFTR Cl- currents (6, 11, 23-25, 28, 37), with an IC50 of 10-40 µM, and it exerts a flickery open-channel block (23, 24). A recent single-channel study (24) concluded that the anionic form of glibenclamide inhibits CFTR Cl- channels by an open-channel block mechanism producing flickery events. The present study showed that both uncharged and charged forms of glibenclamide are effective for inhibition of VSOR Cl- channel, although the effective concentration ranges and the mechanisms appear to differ for the two forms.

VSOR Cl- channels exhibit broad specificity to blockers for many types of Cl- channels (21). If a glibenclamide derivative that specifically blocks the VSOR Cl- channel could be discovered, it would be a useful probe for purification of the channel protein.

    ACKNOWLEDGEMENTS

We are grateful to A. F. James (King's College London) for reading the manuscript, to R. Z. Sabirov for discussion, and to A. Miwa and M. Ohara for technical assistance.

    FOOTNOTES

This work was supported by Grants-in-Aid for Scientific Research 06404017 and 07276104 (Priority Areas of "Channel-Transporter Correlation") from the Ministry of Education, Science, and Culture of Japan, by CREST of JST, and by a grant from the Uehara Memorial Foundation.

Preliminary accounts of a part of these results were given in abstract form (17).

Address for reprint requests: Y. Okada, Dept. of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan.

Received 24 November 1997; accepted in final form 15 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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