Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
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ABSTRACT |
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We describe the
biochemical properties of an eicosanoid-modulated Cl
channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open
probability (Po). After a purification protocol
involving wheat-germ agglutinin affinity and anion-exchange
chromatography, the proteins were sequentially inserted into liposomes,
which were then fused into PLBs. Functional and biochemical
characterization tests confirm that the Cl
channel is a
55-kDa glycosylated monomer with voltage- and Ca2+
concentration-independent activity. 5,6- and 8,9-EET decreased the
conductance of the native channel (control conductance: 70 ± 5 pS
in asymmetrical 50 mM trans/250 mM cis CsCl) in a
concentration-dependent manner, with respective 50% inhibitory
concentration values of 0.31 and 0.42 µM. These regioisomers
similarly decreased the conductance of the purified channel (control
conductance value: 75 ± 5 pS in asymmetrical 50 mM
trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the Po of the native
channel with respective 50% inhibitory concentration values of 0.27 and 0.30 µM but failed to alter the Po of the
purified protein. Thus we suggest that the effects of these EETs on
channel conductance likely result from direct interactions of
EET
anions with the channel pore, whereas the alteration
of Po requires a lipid environment of specific
composition that is lost on solubilization and purification of the protein.
epoxyeicosatrienoic acid; epithelium-derived hyperpolarizing factor; lipid-protein functional rafts; biophysical characterization; hydrophobic interactions
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INTRODUCTION |
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INTEREST IN
TRANSMEMBRANE chloride (Cl) ion movements has
increased notably over the last years. The biophysical characterization of some of the proteins allowing Cl
fluxes is
sufficiently complete that the roles played by the various channel
types can be investigated. Some Cl
channels
(Ca2+-activated or others) have been shown to exert a
modulatory role on the membrane potential of smooth muscle cells and
consequently on muscle tone (9, 33). With an equilibrium
potential more positive than the resting membrane potential of airway
smooth muscle (ASM) cells (1), activation of the
sarcolemmal Cl
currents by a number of agonists or an
elevation in cytoplasmic Ca2+ levels contributes to the
depolarization of the cells, resulting in a graded contraction of the
tissue (8). In contrast, Cl
channel
inhibition leads to membrane re- or hyperpolarization because of the
enhanced relative contribution of the sarcolemmal K+
conductance (38). Alternatively, other Cl
channels are involved in regulatory volume changes (11,
40) or may be activated by membrane stretch (see Ref.
34 for review).
Molecular biology techniques have yielded valuable insight on the
identity and sequence of a number of Cl channels, whereas
electrophysiological and pharmacological studies have unveiled the
properties of several Cl
currents. Already, nine
different genes belonging to the ClC gene family have been identified
in mammals: ClC-1-ClC-7, and ClC-K1 and -K2, with alternative
splicing contributing a greater number of isoforms (5, 7).
In addition, the well-documented Ca2+-activated
Cl
currents (30) have recently been
associated with the expression of CLCA genes (15). In ASM
cells, measurable expression levels have been reported for ClC-2, the
most highly expressed gene encoding an anionic conductance; the
ubiquitous ClC-3 (10); as well as the so-called
pH-sensitive ClC-4 (24). Moreover, the presence of
Ca2+-activated Cl
channels in ASM tissue has
been functionally confirmed in several mammalian species (22,
44).
To date, few Cl channels have been characterized
biophysically and genotypically. This may be attributed to the absence
of specific pharmacological markers for Cl
channels;
inhibitors such as DIDS, SITS, niflumic acid, and tamoxifen discriminate poorly between various Cl
channels and
become effective at concentrations greater than 10 µM (10, 18,
28). The task of characterizing a Cl
channel at
both the molecular and functional levels is a challenging one, best
accomplished through multidisciplinary efforts such as those that
recently reported the functional properties of individual ClC gene
products (11).
Our previous studies focused on the properties of a voltage- and
Ca2+-insensitive channel reconstituted from bovine ASM
sarcolemmal membranes (37). This channel produced unitary
currents distinct from the Ca2+-activated Cl
channels already described (22) and was characterized by
its sensitivity to a family of eicosanoids, the epoxyeicosatrienoic acids (EETs) (38). One of two EET regioisomers tested,
5,6-EET, specifically inhibited the Cl
current with an
IC50 of 0.26 µM. This bioactive lipid thus appears to be
a suitable marker for the channel, in addition to effecting a
physiologically relevant modulation of the membrane potential. Indeed,
recent functional studies have demonstrated the ability of the EETs to
hyperpolarize vascular smooth muscle cells (6). In ASM, a
series of experiments was based on previous observations of an
epithelial modulation of the underlying smooth muscle cells' contractile response to different agonists (46). The
confirmation, at the tissue and molecular levels, of a direct
activation of the large-conductance Ca2+-activated
K+ channels by the EETs (2, 12),
concomitant with an inhibition of this type of Cl
channel
in ASM cells (38) suggests that the EETs exhibit
epithelium-derived hyperpolarizing factor effects in tracheal and
bronchial tissue.
The mechanisms of action of the EET regioisomers on sarcolemmal ionic channels (in vascular or ASM cells) remains ambiguous. It is unclear whether the EETs act via a direct interaction with the protein itself or indirectly via specific membrane receptors and intracellular cascades (17). In plasma membranes, 90% of the EETs are esterified to the sn-2 position of phospholipids (23); in these complete systems, the EETs may act by disrupting the arrangement of lipids surrounding the channels, thus altering membrane fluidity and channel activity. One way to test this hypothesis is to embed the channel into a controlled lipidic environment and examine the effects of the EETs on the channel protein.
In an attempt to address the issues of the biochemical characterization
of the protein carrying this eicosanoid-regulated Cl
channel, as well as to shed some light on the mechanisms underlying regulation of the latter by the EETs, we purified a previously characterized (37) sarcolemmal Cl
channel
from bovine tracheal smooth muscle and examined the effects of three
EET regioisomers on the activity of the native and purified forms of
the channel. A stepwise purification scheme involving functional
recognition of the channel by 5,6-EET was followed by the
reconstitution of the channel into liposomes of fixed phospholipid composition and subsequent fusion into planar lipid bilayers (PLBs). These experiments reveal some of the biochemical characteristics of the
channel and suggest that the EET anions exert part of their dual effect
via an interaction with both the channel pore and the neighboring lipid matrix.
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MATERIALS AND METHODS |
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Preparation of the microsomal fraction.
The preparation of plasma membrane microvesicles derived from bovine
tracheas was performed according to a protocol described previously
(37). In all, 83 fresh bovine tracheas were obtained from
a local slaughterhouse and submitted to this protocol, yielding a total
of 750 g of smooth muscle, from which 1.1 g of crude
microsomal proteins were obtained. This fraction was kept in 0.3 M
sucrose, 5 mM K-PIPES, pH 7.4, at 80°C until used.
Solubilization of the sarcolemmal integral proteins. The thawed crude fraction, typically 70 mg of protein, was incubated at 30°C for 20 min in (mM) 20 Tris-HEPES, 2 MgCl2, 20 µg/ml DNase type 1 (Sigma Chemical, St. Louis, MO), pH 7.2. The enzymatic digestion was stopped by increasing the KCl concentration to 700 mM. The digested fraction was further incubated on a rotary agitator for 30 min at 4°C and then centrifuged at 100,000 g for 60 min at 4°C. The supernatant was discarded. The pellet was resuspended in a solubilization buffer containing 10 mM Tris-MOPS, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or digitonin, pH 8.1, and incubated with agitation at 4°C for 60 min according to a protocol modified from Ref. 13. The material was subjected to fine potter homogenization and then centrifuged at 175,000 g for 60 min at 4°C. The resulting supernatant was set aside, and the pellet was submitted to a second solubilization cycle in the presence of 1% CHAPS or digitonin to optimize the solubilization of membrane proteins. The supernatant resulting from both cycles were pooled, dialyzed overnight against 10 mM Tris-MOPS, 0.1% CHAPS, pH 8.1, in a 14-kDa cut-off dialysis membrane (Spectrum Medical Industries, Los Angeles, CA). The solubilized material was then concentrated approximately 10-fold on polyethylene glycol (PEG8000, Fisher Chemicals, Fair Lawn, NJ), and the protein concentration was estimated using DC assay (Bio-Rad Laboratories, Hercules, CA). Typical solubilization efficiencies were of the order of 35-40% after two cycles, regardless of the detergent used.
Affinity chromatography. The concentrated, solubilized material was then subjected to wheat germ agglutinin (WGA)-affinity chromatography. Briefly, the lectin from Triticum vulgaris (Sigma Chemical) was coupled to a cyanogen bromide-activated Sepharose MT10 from Bio-Rad. After coupling efficiency and stability tests, the WGA-coupled resin was packed into a 3-ml HPLC column, which had been modified to be fitted to a Bio-Rad Biologic HR liquid chromatography system. Aliquots of solubilized material (10 mg) were loaded onto the column by using a Tris · HCl buffer containing (in mM) 10 Tris · HCl, 500 NaCl plus 0.1% CHAPS, pH 8.1. The retained glycoproteins were eluted batchwise using the same buffer, containing 300 mM N-acetyl-D-glucosamine (Sigma Chemical) and collected in 0.5-ml fractions. The flow-through material (the non-N-acetyl-D-glycosylated proteins) would not bind when reapplied to the column. The affinity chromatography run was repeated four to five times, and the corresponding fractions from all runs were pooled and concentrated by using Ultrafree centrifugal filters (Millipore, Bedford, MA) of nominal 30-kDa molecular mass limit. The concentrated proteins were washed twice on the filters with a solution containing (in mM) 50 NaCl, 20 Tris · HCl, 2 MgCl2, 5 Tris-EGTA, 0.1% CHAPS, pH 8.1.
Anion-exchange chromatography. The bulk of the N-acetyl-D-glycosylated proteins was subjected to anion-exchange chromatography. Typically, 2-3 mg of glycoproteins were loaded on a 2-ml Q2 medium-pressure liquid chromatography column (Bio-Rad) by using the washing solution described above. The anionic proteins were eluted with a linear 50-500 mM NaCl gradient, and fractions were collected in the following scheme: 1-ml fractions for the flow-through (i.e., the cationic proteins that did not adhere to the resin) and 0.5-ml fractions while the anionic proteins were being eluted from the column. Corresponding fractions from subsequent runs were pooled and concentrated on Ultrafree filters (NMWL 30 kDa). The proteins were washed with (in mM) 50 NaCl, 20 Tris · HCl, 5 Tris-EGTA, and 2 MgCl2, pH 7.4. They were dissolved in the same buffer at a final concentration of ~0.5 mg/ml.
Formation of proteoliposomes.
Fractions from WGA-affinity and anion-exchange chromatographies were
selected and mixed with phosphatidylcholine to form proteoliposomes before being fused into PLBs. Briefly, phosphatidylcholine dissolved in
chloroform was used to coat the inside of a plastic 1.5-ml tube, while
the chloroform was completely evaporated under a powerful stream of
nitrogen gas. The proteins were added to this lipid coating (1 mg
protein/10 mg lipid) and sonicated immediately, on ice, for 30 s
at strength 2 (Sonic Dismembrator 550, Fisher Scientific, Pittsburgh,
PA), with the temperature of the mixture kept below 22°C. The mixture
was sonicated twice more for 30 s, on ice, at strength 3. The
proteoliposomes were used as such in the reconstitution experiments.
Initially, small-scale experiments revealed what fractions yielded
functional Cl currents. The currents illustrated in
RESULTS were obtained from this small-scale process. The
fractions containing ionic channels were later selected when the scale
of the purification process was increased. The fractions used for
electrophoresis were obtained through larger scale purification.
Bilayer formation and vesicle fusion. PLBs were formed as described previously (37). Briefly, a 200- to 250-µm-diameter hole drilled in Delrin cups was pretreated with a phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in a ratio of 3:2:1, 30 mg lipid/ml decane. Only PLBs with capacitance values ranging from 150 to 300 pF were retained. Aliquots of microsomal fraction or proteoliposomes were added to the compartment denoted cis, in proximity of the PLB. Under standard conditions, both 3-ml chambers initially contained (in mM) 50 CsCl, 10 µM free Ca2+ (as 109 µM CaCl2 and 100 µM EGTA), and 20 Tris-HEPES, pH 7.4. Before injection of the vesicle suspension, a gradient of 50 mM trans/250 mM cis CsCl was created by adding an aliquot of 2 M CsCl buffer in the cis chamber. All solutions were made of analytical-grade reagents, and all experiments were performed at room temperature (22 ± 2°C).
Recording instrumentation and signal analysis. Channel currents were recorded by using a setup described elsewhere (12). Current recordings were played back, filtered at 500 Hz with a four-pole Bessel filter, and sampled at 2 kHz for storage on disks. Further analysis was performed by using specialized software to determine open channel probability values (Po) and obtain time histograms as described in (38).
SDS-PAGE and Western blot analysis.
The protein and membrane samples were solubilized in 2% SDS and
separated on 10% acrylamide gels under reducing conditions (5 mM
mercaptoethanol). All gels were run in parallel with broad range
molecular mass standards (Bio-Rad). Some gels were stained with silver
nitrate, whereas the separated proteins of other gels were
electrotransferred onto a nitrocellulose membrane, which was then
incubated in the presence of a polyclonal antibody raised in rabbit
against a rat-derived ClC-3 fusion protein (Alomone Laboratories,
Jerusalem, Israel). The microsomal fraction, the glycosylated proteins,
and the purified fractions obtained from the anion-exchange
chromatography were probed for the presence of a protein similar enough
to the native ClC-3 to exhibit cross-reactivity with the polyclonal
antibody. Membrane revelation was performed by using anti-rabbit- or
protein A-coupled horseradish peroxidase activity detected by enhanced
chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL). The
widely distributed epitope recognized by this antibody justified our
verification of possible cross-reactivity with the Cl
channel in the fractions tested in this paper.
Statistical analysis. Results are expressed as means ± SE. Statistical significance of the results was verified by either paired or unpaired Student's t-tests or repeated one-way ANOVAs, with a threshold value for significance set at P < 0.05.
Drugs and chemical reagents. Sucrose, K-PIPES, EGTA, protease inhibitors (except Pefabloc), CaCl2, Tris-HEPES, and DIDS were purchased from Sigma Chemical. 8,9- and 14,15-EET were purchased from Cayman Chemical (Ann Arbor, MI). EET injections were performed with a Hamilton syringe and followed by vigorous agitation to facilitate the dispersion of the eicosanoid. Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc) was ordered from Roche Molecular Biochemicals (Laval, QC). CsCl and MgSO4 were obtained from Fischer (Nepean, ON). The chemiluminescence reagents SuperSignal West Pico were purchased from Pierce and used to expose X-ray films purchased from Kodak, Rochester, NY.
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RESULTS |
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Functional reconstitution of the native Cl channel
and effects of 8,9- and 14,15-EET.
As previously reported (38), unitary Cl
channels were functionally reconstituted into PLBs, in the presence of
asymmetrical CsCl solutions. The currents recorded in the presence of
50 mM CsCl in the trans compartment (typically,
intracellular side of the protein) and 250 mM CsCl in the
cis compartment displayed a linear current-voltage
relationship with a unitary conductance of 77 ± 6 pS
(n = 21) and a reversal potential of +33 ± 3 mV, which is close to the Cl
equilibrium potential,
according to the Nernst equation. On abolition of this gradient, the
reversal potential of the unitary current moved to ~0 mV, and the
conductance of the channel measured in these conditions (250 mM CsCl)
increased to 123 ± 7 pS (n = 11). The channel
activity, quantified in terms of Po, was
independent of the applied voltage and remained high (i.e., 0.8) at all
tested potentials (data illustrated in Ref. 37).
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Channel reconstitution after WGA-affinity chromatography.
The first step in the Cl channel purification consisted
of solubilizing the sarcolemmal integral proteins using either 1% digitonin or CHAPS. The solubilized fractions recovered were then tested for the presence of a protein capable of carrying the
Cl
current characterized previously. Injection of the
solubilized fractions in proximity of a lipid membrane did result in
the appearance of the unitary current (not shown). Although CHAPS
yielded a slightly better overall solubilization efficiency (37 vs.
33% for digitonin, in terms of total proteins solubilized), both CHAPS
and digitonin produced functional Cl
currents in our
reconstitution system. The solubilized material was then subjected to
WGA-affinity chromatography to segregate between the non- and
N-acetyl-D-glycosylated proteins. The latter were eluted batchwise by using 0.3 M
N-acetyl-D-glucosamine, and both fractions were
tested in the reconstitution system to identify which fraction
contained the target Cl
channel. Figure
2A illustrates the
chromatogram obtained after passage of the solubilized material onto
the WGA-coupled Sepharose packed into a Bio-Rad MT10 column. The
elution profile reproduced here shows that the glycosylated moieties
represent <7% of the total proteins loaded onto the column. Control
experiments performed in the absence of protein revealed that the
elution buffer (100% B) containing 0.3 M
N-acetyl-D-glucosamine generated a small optical signal within the fractions 20-35. This
nonproteic absorbance could contribute to the shoulder and the tail of
the ultraviolet signal flanking the peak (fractions
23-27) of N-acetyl-D-glycosylated proteins (Fig. 2A).
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Reconstitution of the Cl channels into PLBs after
anion-exchange chromatography.
Each run of the anion-exchange protocol yielded 42 fractions. Initial
reconstitution tests were performed by pooling two 1-ml or four
500-µl fractions together, to yield 2-ml fractions that were
concentrated to 50 µl. All pooled, concentrated fractions were tested
in the reconstitution system. On average, approximately twelve 4-µl
injections of a given 50 µl-fraction in proximity of the PLB without
recording the Cl
current were considered to rule out the
presence of the Cl
channel from that particular fraction.
Whereas no cationic fraction produced measurable Cl
currents on injection of the proteoliposomes next to the bilayer, a
unitary Cl
-selective activity was recorded on fusion of
fraction 24-27 (500 µl of each, pooled and
concentrated to 50 µl) into the PLB. The reconstituted channel
displayed a linear current-voltage relationship and a conductance
similar to that of the native channel (
= 73 pS in 50/250 mM
CsCl trans/cis conditions). It should be noted that ~25% of injections of fraction 24-27 resulted
in current recordings, whereas almost 50% of injections of native
microsomal fraction yielded functional channels of the same type.
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Effects of 8,9-EET on the purified, reconstituted Cl
channel.
Exogenous 8,9-EET was added in increasing concentrations after the
successful reconstitution of the purified channel. Figure 4A (upper trace) illustrates
the current flowing through a single channel at
30 mV, in control
conditions (250 mM CsCl, 10 µM free Ca2+) and after the
addition of 3 and 6 µM 8,9-EET to the cis compartment. Whereas the amplitude of the current was diminished by both
concentrations of 8,9-EET, the Po of the channel
was not affected significantly until a final concentration of 6 µM
8,9-EET had been added (second and third traces). The third trace was
chosen to illustrate the apparent cause of the lower
Po recorded in the presence of 6 µM 8,9-EET;
the latter concentration often induced a rapid flickering of the
channel from open to closed state, over periods longer than 10 s,
before returning to the usual activity characterized by long open
events delimited by much shorter closed events. Even in the presence of
6 µM 8,9-EET, the Po of the reconstituted
channel remained higher than 0.7, and the channel inhibitor DIDS was
added to verify the identity of the channel. The channel
Po of the decreased to 0.31 on channel addition
of 3 µM DIDS to the trans compartment, an effect similar
to that of this same inhibitor on the native channel (Fig.
4A, lower trace) (38).
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Interaction between anti-ClC-3 antibodies and the Cl
channel from bovine ASM.
In the absence of a specific biochemical and/or pharmacological markers
allowing the rapid detection of the protein within the various
fractions isolated in the course of the purification scheme, possible
cross-reactivity of two distinct polyclonal anti-ClC-3 antibodies with
the channel of interest in this study was verified. One of the IgGs
used was an anti-ClC-3 derived from human brain tissue and raised in
rabbit (a generous gift from Dr. Deborah Nelson). The second
antibody was raised in rabbit against 70 residues (positions
592-661) (25) of the rat isoform of ClC-3. In
addition to the full-length ClC-3, the epitope against which this
antibody is raised is known to have significant homology with rat ClC-4 and ClC-5. Given this, and in the light of our previous failed cross-reactivity tests using four different antibodies directed against
a P-glycoprotein from bovine kidney and a Cl
channel from
bovine airway epithelium (generous gifts from Drs. J. Edwards and D. Benos, respectively) dot-type and Western blots were performed with the
anti-ClC-3 antibody, in reducing conditions. Positive signals were
obtained on the dot-blot in fractions 23, 24, 25, and
27, as well as in the well containing the original crude
microsomal fraction (Fig. 5A).
All other fractions failed to display chemiluminescence in our
experimental conditions. Western blots performed after 10% SDS-PAGE
revealed positive signals in those fractions in which Cl
channel activity had been measured in the reconstitution system. Moreover, the addition to the anti-ClC-3 antibody to the cis
(external) compartment of the reconstitution system resulted in a
disappearance of the unitary Cl
current (not shown),
suggesting that the antibody does react with the current-generating
protein studied herein. Interestingly, the
non-N-acetyl-D-glycosylated fraction, as well as
one of the fractions containing cationic proteins (fraction
4-5 of the anion-exchange chromatography), both of which did
not allow the reconstitution of functional Cl
channels,
failed to produce a positive signal. In some lanes containing the
fractions from which functional activity had been recovered, the
antibody detected a major band at an approximate relative molecular
mass (Mr) of 55 kDa and a fainter band at an Mr of 30 kDa. The latter is absent in
fraction 27, from which the Cl
current was
successfully reconstituted. The similarities between the current
generated by this unique band and that generated by the native fraction
suggest that the functional channel is composed of monomers with
molecular mass in the 50- to 60-kDa range. Treatment of fractions
24, 25-26, and 27 with N-glycosidase
did not eliminate the doublet appearance of the 55-kDa band, implying
that the channel is not glycosylated in the "N"
position. Although several Cl
channel of approximate
Mr in the 50-80 kDa range have been
reported (29, 45), none present biophysical
characteristics similar to the channel reconstituted in this study.
Overloaded SDS-polyacrylamide gels such as that illustrated in Fig. 5
display a band at 55 kDa, which could be the protein responsible for
the Cl
channel activity. Notably, the band can be found
in the lanes containing current-allowing fractions and can be seen to
increase in intensity as the purification progresses. Thus the partial purification scheme implemented appears to successfully segregate between sarcolemmal integral proteins and to allow the purification of
a functional Cl
channel from tracheal smooth muscle
cells, whose properties match those of the native channel on functional
reconstitution into PLBs.
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DISCUSSION |
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The insight gained from the purification and reconstitution of a
functional channel into PLBs can be both structural, as was the case
for the purification of the KCa channel (the iberiotoxin receptor; Ref. 13), or mechanistic as in the case of other
channel proteins (35). The short-term goals of this
particular study were mechanistic; it aimed at assessing the role
played by the environment of the channel in conferring the latter
its unique properties: its insensitivity to changes in voltage
and Ca2+ levels, its tonically high
Po, its midrange conductance, as well as its
lack of affinity for the traditional pharmacological markers used
against Cl channels. This was ultimately done by
examining the interactions of the protein with a fixed-composition
membrane, as well as with exogenous eicosanoids known to modulate ASM
electrophysiology and tone (21).
The biophysical properties and pharmacological sensitivities of the
channel could be recovered after the purification protocol described
herein. The procedure was attuned to a variety of methods used to
isolate other ionic channels and transporters (4, 31, 32, 35,
41). It assumed various properties assigned to Cl
channels, such as approximate molecular mass, glycosylation, and
isoelectric point. Prior experiments done in our laboratory had
suggested that the Cl
channel under scrutiny was
glycosylated; WGA, when added in the reconstitution system, inhibited
the channel completely. In addition, attempts to cross-react antibodies
raised against other Cl
channels (see
RESULTS) with isoelectrically separated integral proteins
derived from ASM sarcolemma suggested that the channel would be
negatively charged at slightly basic pH.
Both the native and purified channel displayed a linear unitary
conductance of 70 ± 5 pS in asymmetrical 250/50 mM CsCl
(cis/trans), which puts the channel in the medium
conductance range for Cl channels. Other specific
characteristics include the voltage and Ca2+ independence
of the channel, both of which were comparable in native and purified
forms of the channel; the Po of the channel remained high (>0.75) at all tested voltages and over Ca2+
levels covering the physiological range estimated in ASM cells. These
characteristics, along with unitary conductance, clearly distinguish
the channel under scrutiny here from its Ca2+-dependent
counterpart, expressed in ASM (22) and vascular smooth muscle (30). That these parameters were not altered by
inserting the isolated protein into phosphatidylcholine proteoliposomes suggests that the activity of the native channel is not directly modulated by closely associated G- or cytoskeletal proteins. Thus the
widely accepted model of F-actin stretching does not seem to apply to
this particular case, implying that the channel is not directly
regulated by membrane stretch or cell swelling, modes of action
associated with the clc-2 and clc-3 gene products
(16, 47). Caution must be exerted when working with
reconstitution systems; the activity of the native protein, attached or
associated as it is, may already reflect the loss of these functional
regulatory elements. Yet, the similarities in the properties of other
Cl
channels studied both in intact cells and after
vesicle reconstitution (36) strongly argue against such a loss.
Analysis of the functional and immunological data reported here highlights an apparent paradox; the biophysical properties of the channel suggest that the channel is not a ClC-3 (in addition, the channel is not blocked by tamoxifen, a known blocker of ClC-3), yet the antibodies raised against the rat and human ClC-3 recognize a peptide of ~55 kDa in the purified fraction containing the channel. This could be because of the short and highly conserved epitope against which the ClC-3 antibodies were raised. Far from alleging that the protein isolated here is the clc-3 gene product, it is the cross-reactivity of the antibody with a similar region on the channel studied that was sought.
The absence of adequate pharmacological and immunological markers for
the channel renders its functional electrophysiological imprint the
prime method of identification used in this study. However, the unique
affinity of the protein for the EETs introduces another biochemical
tool for identifying and studying the Cl channel. The
four EET regioisomers are produced by the cytochrome P-450
monooxygenase (27), with 11,12-EET (45%) and 8,9-EET
(44%) being most abundantly produced in rabbit and guinea pig lungs, respectively (26). The regioenantiomers are synthesized
from the arachidonic acid bound at the sn-2 position of the
membrane phospholipids. In an intact cellular membrane, the EETs
produced within the sarcolemma can interact with the underlying smooth muscle cells (14), which have demonstrated an immense
capacity to incorporate EETs within their phospholipids, up to 20 µM
(42). To date, it has been put forth that the in vivo
accumulation of esterified EETs into specific membrane domains could
result in perturbed bilayer packing and account for some functional
effects of the EETs in smooth muscle cells (20).
The interactions of the EETs with the native and purified forms of the
channel were characterized in bilayers of controlled composition. Table
1 quantifies the effects calculated after the addition of the various EETs on both forms of the channel. 5,6-EET
was the regioisomer displaying the greatest effects both on the
conductance of the native, single channel and on its
Po, with IC50 values lower than most
Cl channel inhibitors (IC50 = 0.31 and
0.27 µM, respectively). Controls performed by using acetonitrile (the
solvent for 5,6-EET) proved that the solvent concentrations used in
these experiments had no significant effect on the PLB or the behavior
of the reconstituted channel (38). 8,9-EET and 11,12-EET
followed, with IC50 values for conductance decrease of 0.42 and 1.87 µM, respectively, on the native channel. The
IC50 measured for these two agents with respect to
Po alterations were 0.30 and 1.14 µM on the
native channel. 14,15-EET had no effect on either parameter. It is
noteworthy that this order of potency parallels perfectly the ease with
which each EET regioisomer is inserted into phospholipids; in porcine aortic endothelial and smooth muscle cells, uptake of 5,6-EET is much
more efficient than that of 8,9- (55% of that factor was inserted) and
11,12-EET (35% uptake), factors which in turn are more abundantly
inserted into phospholipids than 14,15-EET (<15%) (43).
Similar orders of EET insertion into phospholipids have also been
measured in murine mast cells (3). This definite order of
lipid insertion represents an elegant way in which the regioisomer
specificity and inhibitory efficiency can be dictated by the molecular
structure of EETs. It also reconciles the otherwise surprising
specificity of a lipid-dependent effect on this Cl
channel.
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We initially assumed that the effect of the EETs occurred partly via a
nonspecific, lipid-dependent mechanism. Indeed, the Cl
channel studied here displayed a significantly lower sensitivity to the
EETs after its insertion into proteoliposomes composed of PC compared
with its sensitivity when surrounded by a mixed lipid-protein
environment. All IC50 values measured in this study were
augmented for the purified channel compared with the data measured on
the native channel (Table 1). It has been shown that all four EET
regioisomers are inserted more efficiently into
phosphatidylethanolamine than phosphatidylcholine (23,
43); thus the lesser effects of the EETs on the purified
Cl
channels could be due to the composition of the
neighboring phosphatidylcholine lipid domains. As well, whereas the
native microsomal preparation contained the enzymes responsible for the
accelerated esterification of the EETs into the phospholipids, this
enzymatic machinery is missing from the purified fraction. The fact
that none of the effects are lost and the concentration-response curves
of all three active EET
hydrophobic anions are
right-shifted suggests that a combination of direct EET
interactions with the pore of channel proteins and lipid-packing modification may be involved. An alternative explanation is based on
the absence of an associated protein, present in the native membrane
preparation and with which the EETs could interact. Well-documented membrane-bound effects suggest that the EETs interact with other structures, such as the adenylate cyclase (39) and
KCa channels (19). Clearly, further binding
studies to determine the extent of the affinity of the protein for the
various EETs and a parallel investigation into the role played by
functional rafts of lipid-protein microdomains in the regulation of
this ionic channel are warranted to verify this hypothesis.
This study documents the partial purification and reconstitution of a
55-kDa monomer exhibiting Cl channel activity and derived
from ASM sarcolemmal membrane. The complete substitution of proteic and
lipidic environment of the Cl
channel resulted in
significant modifications of the sensitivity of the latter to three
exogenous EET isomers. In addition to the insight gained on channel
regulation by a category of eicosanoids, it reports the first steps
toward obtaining a molecular imprint to complement the biophysically
established phenotypic profile of the protein. A greater knowledge of
the structure and regulation of this protein may confirm the proposed
depolarizing role played by the channel in myocyte electrophysiology
and ASM tone.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Gilles Dupuis for reading and discussing the manuscript and to Sonia Proteau for technical assistance.
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FOOTNOTES |
---|
This study was supported by Canadian Institutes of Health Research Grant MT-15173. D. Salvail is the recipient of a Fonds de la Recherche en Santé du Québec (FRSQ)-Fonds pour la Formation de Chercheurs et d'Aide à la Recherche studentship. E. Rousseau is a "national" FRSQ scholar and member of the Health Respiratory Network of the FRSQ.
Present address of D. Salvail: IPS Therapeutique, 3001 12th Ave. N., Sherbrooke, PQ, Canada J1H 5N4.
Address for reprint requests and other correspondence: E. Rousseau, Le Bilarium, Dept. of Physiology and Biophysics, Faculty of Medicine, Univ. of Sherbrooke, 3001, 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4 (E-mail: erouss01{at}courrier.usherb.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00029.2001
Received 19 January 2001; accepted in final form 29 October 2001.
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