Glibenclamide blocks volume-sensitive
Cl
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 |
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 |
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
-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 |
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 M
). Series resistance (<5 M
)
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 |
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.
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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.
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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).
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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
(
f and
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
( 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
( s) at +60, +80, and +100 mV.
Differences between values without and with glibenclamide are
statistically significant (P < 0.05).
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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.
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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.
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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).
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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.
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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).
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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
f and
s), as summarized in Fig.
10A,
top. In contrast, at pH 9.2. glibenclamide (500 µM) dramatically decreased both
f and
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: 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:
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.
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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 |
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
-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
-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.
 |
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