Functional reconstitution of an eicosanoid-modulated Clminus channel from bovine tracheal smooth muscle

Dany Salvail, Martin Cloutier, and Eric Rousseau

Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

The experiments reported aimed at characterizing the interaction of two isomers of the EETs, namely, 8,9- and 14,15-EET, with the Cl- channel. Figure 1A illustrates the current recorded after the reconstitution of a single channel from the crude microsomal fraction. In control, symmetrical 250 mM CsCl and 10 µM free Ca2+, the channel displayed a typically high Po (at -40 mV, Po = 0.91), which was unaffected by the addition of the EGTA (140 µM) to the trans compartment (not shown). The addition of increasing concentrations of 8,9-EET in the cis compartment resulted in a concentration-dependent decrease of both the unitary conductance and Po of the anionic channels such that 3 µM 8,9-EET left relatively rare and shorter open events (Fig. 1A, lower trace). A quantitative analysis of the effects of 8,9-EET on the unitary conductance of the channels appears in Fig. 1B. Significant decreases in current amplitude measured at -40 mV were recorded after the addition of 0.3 µM and 1.0 µM 8,9-EET to the cis compartment, to values respectively 87 ± 7 and 69 ± 8% of control values (submitted to paired Student's t-tests, n = 7-8, P < 0.05). Addition of higher concentrations of 8,9-EET did not cause significantly greater decreases in unitary current amplitude. The IC50 value of 8,9-EET for conductance decrease was estimated at 0.42 µM.


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Fig. 1.   Representative activity of the native Cl- channel and concentration-dependent inhibition by 8,9-epoxyeicosatrienoic acid (EET). A: single-channel recording obtained in symmetrical 250 mM CsCl plus 10 µM free Ca2+ at a holding potential of -40 mV. Open channel events appear as downward deflections from the closed-state level (C). First trace: control conditions; the channel displays a typically high open probability (Po). Second and third traces: addition of cumulative concentrations of exogenous 8,9-EET (1 and 3 µM) to the cis compartment gradually decreases both the amplitude of the unitary current and the channel Po. B: 8,9-EET-induced concentration-dependent decreases in current amplitude recorded at -40 mV in the same experimental conditions as in A (1-way ANOVA, P < 0.05, n = 7-8). Ctrl, control. C: Concentration-dependent decreases in channel activity (Po) were observed after the addition of submicromolar concentrations of 8,9-EET, at a holding potential of -40 mV. *Statistically significant variations from the control conditions (1-way ANOVA, P < 0.05 n = 7-8).

Channel activity was also affected in a concentration-dependent manner (Fig. 1C); the Po decreased to 0.68 ± 0.08, 0.55 ± 0.07, and 0.39 ± 0.07 after the respective additions of 0.3, 1.0, and 3.0 µM 8,9-EET to the cis compartment (n = 7-8, submitted to paired t-tests, P < 0.05). Increasing the total 8,9-EET concentration to 4.0 µM failed to lower the Po any further. The IC50 value of 8,9-EET for channel inhibition in terms of Po was estimated at 0.30 µM. Po values were used to allow the direct comparison of channel activities regardless of the number of channels reconstituted per individual recording. In contrast, 14,15-EET had no effect on either parameters; over the range of concentrations tested (300 nM to 16 µM), 14,15-EET failed to alter the conductance and Po of the native channels. Qualitatively, no modification in channel kinetics could be observed. The effect of the solvent (in both cases ethanol) was verified and measured to be insignificant at the levels used in the experiments reported herein (38).

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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Fig. 2.   Wheat germ agglutinin (WGA)-affinity chromatography of the solubilized, microsomal fraction. A: chromatogram displaying the protocol and separation of the N-acetyl-D-glycosylated moieties from the non-N-acetyl-D-glucosamine-proteins (large peak, fractions 2-10). Protein elution was monitored by ultraviolet (UV) absorption at 280 nm (left axis, solid line), while buffer conductivity was monitored simultaneously (right axis, dashed line). The proteins without N-acetyl-D-glucosamine moieties eluted with a retention time of <25 min (bottom axis). N-acetyl-D-glucosamine (N-AGA; 0.3 M) was then added batchwise in the same buffer (-100% B, dotted line) to detach the glycoproteins bound, which eluted in fractions 20-35. The fractions in which the distinctive populations were collected appear on top axis. Glycoproteins constituted <7% of the total sarcolemmal proteins solubilized. B: reconstitution of the Cl- current after the reincorporation of the glycosylated fraction into planar lipid bilayers (PLBs). At -10 mV, in asymmetrical 50/250 mM CsCl conditions, 2 anion-selective currents were recorded, illustrated here as downward deflections from the zero-current level (C). Upon addition of 40 µM EGTA to lower the free trans compartment Ca2+ concentration to 223 nM, the larger activity disappeared, leaving only the smaller current. C: current-voltage (I/V) relationship of the anionic currents reconstituted from the peak (fractions 23-27) of the N-AGA fraction. In asymmetrical conditions, the small and larger, Ca2+-dependent currents illustrated in B displayed linear I/V relationships.

Attempts to record electrical activity from the flow-through fraction yielded no discernable anionic current (n = 20-25 injections in CsCl buffer). On the other hand, two distinct Cl- currents were recorded after the injection of the peak of N-acetyl-D-glycosylated proteins (fractions 20-27) in proximity of the PLB (Fig. 2B). One consisted of a smaller anion-selective current, whereas another, larger current exhibited a Ca2+ dependence and channel kinetics similar to those of the Ca2+-activated K+ (KCa) channels. Addition of 140 µM EGTA to the trans compartment rapidly abolished the larger current, leaving the small Cl- current unaffected (lower trace). The channels supporting the smaller current had an average unitary conductance of 78 ± 3 pS in 50 mM trans/250 mM cis CsCl (n = 5) and displayed a typically high and Ca2+-insensitive Po. The reversal potential measured for the current recorded in 50 mM trans/250 mM cis was 31 mV and shifted to 6 mV when the CsCl gradient was abolished, suggesting a good selectivity of the channels for Cl- over Cs+ (Fig. 1C). The larger, Ca2+-dependent channel displayed an average zero-current potential above 40 mV and a unitary conductance of 127 ± 8 pS in asymmetrical 50/250 mM CsCl, suggesting that it is an unidentified anion-selective channel also present in ASM cell membranes. Our initial belief that this might be a KCa channel was based on the Ca2+-dependence and kinetics of the channel, as well as on the knowledge that the beta -subunit of KCa channels is glycosylated (13).

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 (gamma  = 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.

Additional chromatographic runs using pooled fractions (e.g., fraction 24-25 from runs 2-6) were carried out. This pooling of the fractions was necessary to obtain the quantity of proteins required for the reconstitution experiments and was made possible by the highly reproducible performance of the anion exchanger (Fig. 3A). Medium conductivity (mS/cm) was used to ensure correspondence of the fractions that were pooled. Fractions 24-25 and 26-27 were then inserted into phosphatidylcholine (PC) liposomes and injected in proximity of the PLB. Fractions 24-25 and 26-27 both yielded discernable Cl- currents, with the current from the former illustrated at -30 mV in Fig. 3B. The second trace was recorded after the addition of EGTA to lower free Ca2+ concentration levels below 100 nM, which did not affect the channel activity. The currents carried by the purified and reconstituted Cl- channel displayed a linear current-voltage relationship, with an average conductance of 75 ± 6 pS (in 50 mM trans/250 mM cis, n = 7-10), statistically indiscernible from that of the native channel (70 ± 5 pS, n = 11) in the same conditions (Fig. 3C, left). In addition, channel Po as a function of free Ca2+ concentration levels (not shown) and voltage remained high at all tested potentials (Fig. 3, right). ANOVAs revealed that there was no significant difference between the Po exhibited by the native channels (n = 8) and that exhibited by the purified channels (n = 5-6) after their reconstitution into PLBs. The anionic conductance, Ca2+- and voltage-independent behavior of the channel contained in fractions 24-25 and 26-27 suggest that it is the channel derived from bovine smooth muscle sarcolemma and previously characterized in its native form (37). Moreover, the insertion of the purified channel into liposomes exclusively composed of PC did not alter the conducting properties of the channel.


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Fig. 3.   Separation of the glycosylated transmembrane proteins by anion-exchange chromatography. A: chromatogram displaying the protocol and separation of the anionic proteins of the glycosylated fraction on an anionic exchanger Q matrix. UV absorption at 280 nm (left axis, solid line) and buffer conductivity (near right axis, dashed line) were monitored as the anionic proteins were eluted with a linear, 50-500 mM NaCl gradient (far right axis, dotted line). All collected fractions were tested for Cl- channel activity in the bilayer reconstitution system. Fraction 24-25 (vertical open bar) represent fractions from which the Cl- channel activity was recovered (retention time, 52.5 min). AU, arbitrary units. B: unitary currents recorded after the insertion of fraction 24-25 into PLBs, in asymmetrical 50/250 mM CsCl, at -30 mV. Open-channel events are displayed as downward deflections from the zero-current level (C). Lowering free Ca2+ levels did not significantly affect the Po of the channel. C: purified channel displayed a linear I/V relationship quantitatively similar to that of the native channel in the same conditions (left). Still, in the presence of 50/250 mM CsCl, the purified channel Po was insensitive to voltage over the range tested (-50 to +30 mV), remaining above 0.75 at all voltages (right). The Po of the purified channel was indistinguishable from that of the native channel.

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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Fig. 4.   8,9-EET and DIDS inhibition of the current carried by the purified channel. A: representative single-channel recording obtained in symmetrical 250 mM CsCl plus 10 µM free Ca2+ at a holding potential of -30 mV. In control conditions, the channel displays a high Po (upper trace). Second and third traces illustrate how the addition of increasing concentrations of exogenous 8,9-EET (3.0 and 6.0 µM) to the cis compartment induced decreases in current amplitude but failed to alter visibly the Po of the channel, despite recurrent changes in channel gating. Third trace illustrates such a temporary change in channel kinetics; despite their frequent occurrence, these episodes were too short in duration to have profound effects on overall channel activity. On the other hand, 3 µM DIDS added to the trans compartment successfully inhibited the current (lower trace), thus verifying that the channel carrying this current was indeed the Cl- channel studied. B: in symmetrical 250 mM CsCl, and at a voltage of -30 mV, concentrations >0.3 µM 8,9-EET caused significant decreases in current amplitude. C: in the same experimental conditions, concentrations <6.0 µM 8,9-EET failed to significantly alter channel Po, measured at -30 mV. *Statistically significant variations from the control conditions (1-way ANOVA, P < 0.05, n = 7-8).

The effects of 8,9-EET on amplitude and Po are expressed quantitatively in Fig. 4, B and C. The average current amplitude measured at -30 mV is illustrated in the presence of increasing concentrations of exogenous 8,9-EET; 1.0, 3.0, 4.0, and 6.0 µM all significantly decreased the amplitude of the current flowing through the purified channel, by 24, 39, 49, and 54%, respectively (n = 4-6). An IC50 value of 1.1 µM was estimated for this lipidic mediator. This decrease in unitary current amplitude resembled the decrease seen after 8,9-EET addition with native channels (Fig. 1B). On the other hand, the lack of effect of 8,9-EET on the Po of the purified channel contrasts with the results obtained with the native channel, whereas 8,9-EET caused a concentration-dependent decrease in native channel activity; it failed to inhibit the purified channel to a significant extent until a total concentration of 6 µM had been added, which induced a 10.5% decrease in channel Po (IC50 = 3.21 µM). It is noteworthy that removing the channel from its native environment significantly affected its sensitivity to 8,9-EET.

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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Fig. 5.   Detection of specific protein bands by ClC-3 antibodies. A: 7.5% polyacrylamide gel on which various fractions of interest were loaded in the presence of ferritin. Combined silver nitrate and Coomassie blue staining reveal a light band at a relative molecular mass (Mr) of 55 kDa that increases in intensity as purification progresses. The final anion-exchanger chromatography yielded bands 7-10, with Cl- currents previously recorded from bands 9 and 10. B: some gels were electrotransferred to a cellulose membrane and incubated by using the same anti-ClC-3 antibody. In reducing conditions, the antibody recognized a predominant band with an approximate Mr of 55 kDa. This band was present in all fractions from which the Cl- activity was reconstituted and notably absent in those from which no activity could be reconstituted (nonglycosylated fraction and cationic fraction 4-5 are illustrated here). Fract, fraction.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Quantified effects of the EETs on the native and purified Cl- channel

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.


    ACKNOWLEDGEMENTS

We are indebted to Dr. Gilles Dupuis for reading and discussing the manuscript and to Sonia Proteau for technical assistance.


    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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DISCUSSION
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