Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
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ABSTRACT |
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We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS (n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 µM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.
endothelium; open-channel properties
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INTRODUCTION |
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VOLUME-REGULATED ANION channels (VRAC) control numerous cell functions, as described in several reviews (4-6, 9, 15). In contrast, with the increasing interest in this channel and its obvious role in diverse biological functions, the elucidation of its gating mechanism, a direct biophysical approach of its open-pore properties, and its molecular identification have so far been largely unsuccessful (5).
In contrast to other ion channels (e.g., the voltage-dependent Na+ channel) in which specific high-affinity ligands have played a pivotal role in the characterization, purification, and cloning of the channel, there are at present no specific high-affinity ligands available for VRAC. Compounds such as tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and dideoxyforskolin inhibit VRAC only in the micromolar concentration range; their mode of action is unclear, and they may also affect other systems (for review, see Ref. 5). A search for VRAC blockers with greater potency and specificity is therefore a necessary step in the development of molecular tools that could be used to probe selective channel features such as open-pore properties.
Recently, a potent block of outwardly rectifying
Cl channels (ORCC) by
compounds belonging to the family of calixarenes was described. The
calix[4]arene used in this study consists of four para-substituted phenols conjugated by
methylenes to form macrocyclic basket (calix)-like molecules (14).
Calix[4]arenes are open-channel blockers of ORCC
incorporated into planar black lipid membranes (13, 14) and blockers of
native ORCC in airway epithelial cells (10). The apparent inhibition
constant of this block in the subnanomolar range prompted
us to investigate the effects of these calix[4]arenes on
the swelling-activated Cl
current
(ICl,swell) in
endothelial cells.
Our results show that calix[4]arenes induce a voltage-dependent inhibition of ICl,swell, probably by an occlusion of the pore region at moderately positive potentials, which is relieved at more positive potentials at which the negatively charged calixerenes permeate the channel. The pH dependence of the calix[4]arene effect suggests a role for positively charged residues, presumably histidine, in the channel pore.
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MATERIALS AND METHODS |
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The methods used in the present experiments have been described in detail elsewhere (7). Endothelial cells from an established cell line from calf pulmonary artery (cell line CPAE, American Type Culture Collection CCL-209) were grown in DMEM with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 20% fetal bovine serum at 37°C in a fully humidified atmosphere of 10% CO2 in air. Cells were detached by exposure to 0.05% trypsin in a Ca2+- and Mg2+-free solution and plated on gelatin-coated coverslips for electrophysiological experiments.
Whole cell membrane currents were measured using an EPC-9
(Heka-Electronics, Lambrecht/Pfalz, Germany) patch-clamp amplifier and
were routinely sampled at 4-ms intervals (2,048 points/record, filtered
at 100 Hz). The following standard voltage protocols were used:
1) a "ramp" protocol that was
applied every 15 s from a holding potential of 0 mV and consisted of a
step to 80 mV for 0.6 s, followed by a step to
150 mV for
0.2 s and a 2.6-s linear voltage ramp to +100 mV;
2) a "step" protocol applied
from a holding potential of
50 mV consisting of 2-s steps to
voltages ranging from
100 to +100 mV in increments of +10 mV;
the time interval between steps was 6 s. Because the swelling-activated currents are much larger than the background currents, the current traces were not corrected for baseline currents.
For the analysis of current fluctuations, the current at +30 mV was
sampled at 2 kHz and filtered at 1 kHz. Mean current
(I) and current variance
(2) were calculated from
consecutive 512-point sweeps. These quantities are related by the
expression (11, 12)
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(1) |
The standard extracellular solution was a Krebs solution containing (in mM) 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES, triturated with NaOH to pH 7.4. The inwardly rectifying K+ current, which is present in CPAE cells, was inhibited by substituting K+ by Cs+ in the Krebs solution. The osmolarity, as measured with a vapor pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 320 ± 5 mosM.
After seal formation, the normal Krebs solution was replaced with another isotonic solution that contained (in mM) 105 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, and 90 mM mannitol, triturated with NaOH to pH 7.4. VRAC was activated by superfusing the cell with the same solution without mannitol, resulting in a 25% hypotonicity.
The pipette solution contained (in mM) 40 CsCl, 100 cesium aspartate, 1 MgCl2, 5 EGTA, 1.93 CaCl2, 4 Na2ATP, and 10 HEPES, triturated with CsOH to pH 7.2. The calculated free Ca2+ concentration of the pipette solutions was 100 nM.
TS-calix[4]arene (p-tetrasulfonato-calix[4]arene) and TS-TM-calix[4]arene (5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy calix[4]arene) were kindly provided by Dr. R. J. Bridges (Pittsburgh, PA). TS- and TS-TM-calix[4]arene are negatively charged because of the presence of four sulfonate groups.
Diethyl pyrocarbonate (DEPC, Sigma) was used at concentrations between 0.001 and 1 mM.
All experiments were done at room temperature (20-22°C). Data were analyzed in Origin (MicroCal Software). Pooled data are given as means ± SE. Level of significance is 0.05.
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RESULTS |
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Voltage-dependent inhibition of VRAC by
calix4]arenes.
Figure 1 shows the effect of
100 µM TS-TM-calix[4]arene on the amplitude of the
ICl,swell in a
CPAE cell challenged with a 25% hypotonic solution. Figure
1A shows the time course of the current at different potentials. The current amplitudes at +100, +50,
and 100 mV were obtained from repetitively applied voltage ramps. TS-TM-calix[4]arene exerts a fast and reversible
inhibition at positive potentials (+50 and +100 mV) but is virtually
ineffective at negative potentials (
100 mV). The fraction of the
current blocked at +100 mV is obviously smaller than that at +50 mV
(Fig. 1A). This voltage dependence
of the inhibition is more apparent from a comparison of the
instantaneous current-voltage
(I-V)
curves derived from the voltage ramps during and after application of TS-TM-calix[4]arenes (see Fig. 1,
A, filled symbols, and
B): current amplitudes at positive
potentials are substantially suppressed, whereas those at negative
potentials are hardly affected at all. It is also obvious that the
current trace in the presence of TS-TM-calix[4]arene is
much noisier in the potential range at which the compound exerts its
inhibitory action. To quantify the effect of
TS-TM-calix[4]arene, we have calculated from these
I-V
curves the fraction of the current that was inhibited at each potential
V
[INH(V)], i.e.
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(2) |
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Kinetic analysis of the inhibition by calix[4]arene. The voltage dependence of the block and the increased current noise at positive potentials at which the calix[4]arenes exert their blocking effect (see Fig. 1) suggest that these compounds may act by blocking the open pore. The classical Woodhull analysis of the time and voltage dependence of the block cannot be applied however to our data because the blocking efficacy decreases at strong positive potentials. We have therefore analyzed our data by assuming that the calix[4]arenes not only block the open VRAC channel but also permeate it to some extent at positive potentials, as shown by the kinetic scheme
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(3) |
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(4) |
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pH dependence of the calix[4]arene block. An alternative explanation for the relief of inhibition at strong positive potentials could be that the binding of calix[4]arenes occurs at a protonated site inside the channel, which is accessible to extracellular protons only. Positive potentials will impede this access and reduce the fraction of protonated binding sites and consequently the inhibitory action of the calix[4]arenes. These effects would result in a more potent block at low pH and shift the peak inhibition to more positive potentials.
Figure 5 A shows instantaneous I-V curves obtained from voltage ramps in the presence and absence of 10 µM TS-calix[4]arene at various extracellular pH values. The corresponding fractional current inhibition at each pH is given in Fig. 5B. It is obvious that changes in extracellular pH strongly affect the blocking efficacy of TS-calix[4]arene. Block at positive potentials is reduced by an increase in the extracellular pH and potentiated at acidic pH. Similar effects of extracellular pH were observed in the presence of TS-TM-calix[4]arenes. Changes in extracellular pH also shifted the potential at which maximal inhibition occurs, from 34.4 ± 1.2 mV at pH 6.0 (n = 11) to 49.5 ± 1.5 mV at pH 7.4 (n = 8) and 77.5 ± 1.9 mV at pH 9.0 (n = 4), which is in a direction opposite to that predicted by the above model. Maximum current inhibition, measured at the peak of the inhibition-voltage curve, decreased from 46.1 ± 3.3% (n = 11) at pH 6.0 to 33.0 ± 1.5% (n = 8) at pH 7.4 and 2.5 ± 0.6% (n = 6) at pH 9.0.
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Calix[4]arene-induced current fluctuations.
It was obvious from Fig. 1 that TS-TM-calix[4]arene
substantially increases the current noise at the positive potentials at which it effectively blocks VRAC. We have therefore analyzed the current fluctuations induced by calix[4]arene block.
Currents were activated by challenging the cells with the hypotonic
solution, and 30 µM TS-calix[4]arene was added after
maximal activation was reached. At this time, all available channels
are supposed to be in the open state with an open probability close to
1, resulting in a low current noise level of the macroscopic current
(2, 16). The cells were clamped at +30 mV at an external pH of 6 to
maximize the calix[4]arene-induced current fluctuations
(Fig. 6A). It
is obvious that application of TS-calix[4]arene rapidly inhibited the current (bottom trace)
and increased the variance of the current (top
trace). Figure
6B shows the current
variance as a function of I = Icalix
Icontrol, i.e.,
the reduction of the macroscopic current relative to its value in the
absence of TS-calix[4]arene. In Fig.
6B, the solid line shows the fit of the data points to Eq. 1. The average
values from nine cells with a mean membrane capacitance of 38.8 ± 2.9 pF are as follows: i = 0.51 ± 0.07 pA, and N = 1,322 ± 115. The
decrease in open probability calculated from the above parameters and
the reduction of macroscopic current is 0.46 ± 0.07. The
single-channel conductance (
) calculated from the single-channel
current and the driving force (V
Cl
equilibrium
potential) is 9.3 ± 2.1 pS. To further validate this noise analysis
procedure, we have compared the single-channel current amplitude
obtained from this analysis with single-channel measurements. From
three patches, excised after full activation of VRAC, we obtained
single-channel current amplitudes of 0.37 ± 0.02 and 0.76 ± 0.04 pA at potentials of +20 and +40 mV, respectively.
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DISCUSSION |
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VRAC, which regulate several cell functions, are not yet identified at the molecular level. A search for pharmacological tools that bind to VRAC with high affinity may therefore be helpful for the purification and molecular identification of these channels as well as their functional characterization. Calix[4]arenes have been reported to represent a class of high-affinity blockers of the ORCC channel. These compounds, which form basket- or calixlike structures and occupy a space of ~7.5 × 12.5 Å (14), are water soluble and induce a fast block of ORCC within milliseconds.
We could confirm in the present experiments that these substances also induce a fast and reversible voltage-dependent block of VRAC channels in endothelial cells. In contrast with the findings on ORCC channels, we did not observe a significant difference in potency between the TS- and TS-TM-calix[4]arenes. Also, the affinity of these compounds for ICl,swell in endothelial cells seems to be much lower than that reported previously for ORCC channels (10, 13, 14).
From the time and voltage dependence of the calix[4]arene action, we were able to deduce a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 from the outer membrane surface and permeate the cell membrane at rather strong depolarizations. The pH dependence of the block is difficult to explain by pH effects on the calix[4]arenes, since the pKa of the sulfonate groups is around two. We can also exclude a pH effect on the OH group of the phenol, since there were no pH-dependent differences between TS-TM- and TS-calix[4]arenes. The alternative possibility is that calix[4]arenes bind to a protonated, positively charged site inside the VRAC pore. The sharp drop in block efficacy between pH 6.0 and 9.0 suggests the involvement of one or more histidine residues, since this amino acid is 80% protonated at pH 6 but only 15 and 1% at pH 7.4 and 9, respectively. Consistent with the protonation at histidine residues is the finding that DEPC, a rather selective histidine reagent, irreversibly inhibits VRAC currents.
It has been reported that nonstationary noise analysis underestimates the single-channel current because activation of ICl,swell consists of the recruitment of channels rather than of an increase of open probability (1-3). Because we first fully activated the current in our experiments and then applied calix[4]arenes to reduce the channel open probability without affecting the number of channels, we could apply the well-established Sigworth analysis to estimate single-channel current amplitude and number of channels from the parabolic relation between current amplitude and current variance (Eq. 1). The similarities of the single-channel current amplitude from this analysis and that from single-channel measurements support the hypothesis of Jackson and Strange (1-3) that cell swelling recruits additional VRAC channels but also lends further support to our contention that calix[4]arenes act by blocking the open-channel pore.
In conclusion, calix[4]arene-related compounds might be a useful tool to probe open-channel properties of VRAC. However, their low-binding affinity for VRAC makes them less suitable for use in biochemical isolation and identification of this hitherto unknown channel.
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ACKNOWLEDGEMENTS |
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We thank Drs. G. Buyse, M. Kamouchi, C. Maertens, A. Mamin, D. Trouet, and L. Wei for helpful discussions.
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FOOTNOTES |
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This research was supported by European Union Grant BMH4-CT96-0602 and Nationaal Fonds voor Wetenschappelijk Onderzoek Grant G0237.95.N from the Flemish Government.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. Droogmans, Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Received 2 March 1998; accepted in final form 28 May 1998.
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