Correspondence to: Christopher Miller, Department of Biochemistry, Brandeis University, 415 South Street, HHMI, Waltham, MA 02254-9110. Fax:781-736-2365 E-mail:cmiller{at}brandeis.edu.
Released online: 25 October 1999
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Abstract |
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ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coliderived ClC Cl- channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.
Key Words: yadQ, liposome, chromatography, glutaraldehyde, ultracentrifugation
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INTRODUCTION |
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The phylogenetically ubiquitous Cl- channel proteins of the ClC family are responsible for a multitude of physiological functions in organisms as varied as mammals, elasmobranchs, yeast, and green plants (
Recently emergent genome sequences have revealed in prokaryotes many homologues of ion channels hitherto considered strictly eukaryotic (
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MATERIALS AND METHODS |
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Materials
All chemicals were reagent grade. 36Cl was purchased from DuPont NEN as a 0.51 M HCl solution and was neutralized with NaOH. Working stock solutions were 5060 mM NaCl, 2631 µCi/ml. 3H-Glutamic acid was also obtained from DuPont NEN. Dowex 1x4100, from Sigma Chemical Co. was converted to the glutamate form and stored in water. E. coli lipids (polar lipid extract) were obtained from Avanti Polar Lipids. Glutaraldehyde was Grade I from Sigma Chemical Co.
ClC Searches
Searches for ClC homologues were performed using BLAST 2.0 (
Recombinant DNA and General Biochemical Methods
Oligonucleotides corresponding to flanking regions of the E. coli ClC gene yadQ (
All SDS-PAGE experiments were run under reducing conditions (2% ß-mercaptoethanol) using standard Laemmli solutions (
Expression and Purification
E. coli JM83 cells were transformed with pEriC, grown overnight at 37°C, and diluted 50-fold into room-temperature Terrific Broth (
Membranes were extracted for 2 h (at a concentration equivalent to A280 = 20 in 0.5% SDS) in Buffer BK (95 mM K-Pi, 5 mM KCl, pH 7.0) containing 15 mM dodecylmaltoside (DDM)1. Insoluble material was removed by centrifugation (110,000 g, 45 min). For binding, 1/10 volume of Buffer E (400 mM imidazole-HCl in Buffer BK, 1 mM DDM) was added and the pH was adjusted to 7.8. This extract was incubated overnight with Ni-NTA beads (0.25 ml beads/liter culture; QIAGEN Inc.). The beads were transferred into a column and washed with ~40 vol wash buffer (40 mM imidazole-HCl, 95 mM K-Pi, 5 mM KCl, 1 mM DDM, pH 7.8) until A280 < 0.02. EriC was then eluted with Buffer E, pH 7.0. The 40-mM imidazole present during binding and wash steps suppresses the binding of contaminating E. coli proteins and thereby increases the purity (while decreasing the final yield) of the EriC preparations.
Protein concentration was measured from the absorbance at 280 nm, using a mass extinction coefficient ( = 0.85 cm2/mg) calculated from the sequence (
Protein Reconstitution
Reconstituted vesicles were formed by combining EriC with lipid-detergentmixed micelles, and then removing detergent by gel filtration. Except where noted, all reconstitution procedures and flux assays were performed at room temperature. E. coli lipid was dried under N2 in a glass tube, resuspended in pentane, and redried. The lipid was suspended at 20 mg/ml by sonication in Buffer R [450 mM KCl, 20 mM morpholino ethanesulfonic acid (MES)-NaOH, pH 6.2], 34 mM 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate (CHAPS) was added, and the suspension was sonicated again. After a 2-h incubation, EriC was added to the desired concentration (0.095 µg/mg lipid), along with enough Buffer R to bring the lipid concentration to 10 mg/ml. In control samples, equivalent volumes of Buffer E were added in place of EriC. After 20 min, detergent was removed and vesicles were retrieved using Sephadex G-50 spin columns as follows. Columns (1.5-ml bed volume) equilibrated with Buffer R were prespun in a clinical centrifuge at 1,000 g, 15 s to remove excess solution. A 95-µl sample of the reconstitution mix was applied to each column, and vesicles were collected by centrifuging 700 g, 1 min. Recovery from the spin varied in the range 100150 µl. The samples were frozen in a dry ice/acetone bath and stored overnight in a frost-free freezer at -20°C. Before use, samples were removed from the freezer, cooled in a dry ice-acetone bath, thawed at room temperature, and then sonicated 510 s in a cylindrical bath sonicator.
Concentrative 36Cl- Uptake Assay
Influx of 36Cl- against a concentration gradient was assayed by a three-step procedure, essentially as described (
3H-Glutamate Trapped-Volume Assay
EriC-reconstituted liposomes were prepared as described above with the following variations. Proteoliposomes were formed in Buffer L (20 mM KCl, 20 mM K-glutamate, 20 mM MES-KOH, pH 6.0), and stock solutions of 36Cl- and 3H-glutamate were added to the vesicles (final concentrations of 2 and 7 µCi/ml, respectively). Intra- and extra-vesicular solutions were equalized by freezing and thawing the samples twice, and then sonicating the suspension 510 s. Passive equilibrium-exchange efflux was initiated by diluting the loaded vesicles into Buffer L lacking radioactive tracers. After 4060 min, intravesicular content was determined by spinning through Buffer Lequilibrated G-50 columns as above. Separate experiments under these conditions demonstrated: (a) that insignificant efflux of Cl- occurs in protein-free liposomes, (b) that full equilibration of Cl- is achieved with EriC-containing liposomes, and (c) that negligible efflux of glutamate occurs for liposomes with or without EriC. The crucial measurement in this assay is the fraction of the intraliposomal volume that is inaccessible to external Cl-. This parameter is determined from the fractional trapped Cl- space, f, measured by double-label counting of internal and external tracer concentrations (dpm per gram lipid and per cubic-centimeter solution, respectively, shown in Equation 1a):
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(1a) |
where , the total intravesicular volume (cm3/g lipid), is measured from 3H-glutamate (Equation 1b):
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(1b) |
For vesicles that have been diluted infinitely, the Cl--inaccessible fraction, f0 is (Equation 2a):
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(2a) |
For practical purposes, the samples were diluted not infinitely, but 10-fold, and f0 was correspondingly normalized, by Equation 2b:
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(2b) |
This assay can be used to estimate the fraction, s, of EriC protein that is functionally active as a Cl- channel. The analysis assumes that the protein distributes randomly according to a Poisson distribution into spherical liposomes of uniform size, and that Cl- is retained only in those liposomes that contain no EriC molecules. As the mass, mE, of EriC reconstituted in a fixed mass, mL, of liposomes increases, this Cl--inaccessible fraction will decrease according to
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(3) |
where No is Avogadro's number, is the intravesicular volume (cm3/g lipid), ME is the molecular mass of the EriC channel (51,000 g/mol per subunit), and
sigma}"> is the surface area of lipid in a bilayer (cm2/g).
Chemical Cross-linking
Glutaraldehyde-mediated cross-linking of EriC was performed at room temperature. EriC (6 µg) was diluted to 27.5 µl in Buffer X (150 mM NaCl, 50 mM Na-Pi, 1 mM DDM, pH 7.0), and glutaraldehyde (0.5 µl, 18 mM final concentration) was added. The reaction was quenched with 19 mM Tris for 10 min. Sodium dodecylsulfate (SDS)loading buffer was added, and the sample was analyzed by SDS-PAGE. In some experiments, we varied concentrations of protein (5500 µg/ml), DDM (15 mM), or glutaraldehyde (14160 mM).
Gel Filtration
Samples were chromatographed at 1 ml/min on a Superdex 200 column (10 x 300 mm, 24 ml bed volume; Pharmacia LKB Biotechnology Inc.) equilibrated in buffer G (100 mM KCl, 50 mM Na-Pi, 1 mM DDM, pH 7.0). Elution profiles were monitored at 280 nm.
Analytical Ultracentrifugation
EriC was dialyzed overnight against Buffer U (155 mM KCl, 95 mM K-Pi, 1 mM DDM, pH 7.0) containing 5 mM dithiothreitol (DTT). For control experiments, KcsA protein was dialyzed into Buffer U lacking DTT. To ensure proper blank subtraction, for each dialyzed sample, the solution outside the dialysis cell at equilibrium was collected as the blank. Samples and buffer blanks were loaded into charcoal-Epon two-sector cells. Sedimentation velocity was analyzed using an Optima XL-A analytical ultracentrifuge (Beckman Instruments, Inc.) at 44,000 rpm, 20°C; scans at 280 nm were taken every 5 min. Data were fit to the modified Fujita-Macosham function (
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RESULTS |
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For the past few years, it has been appreciated that ClC channels are represented in microbial genomes (
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Overexpression, Purification, and Reconstitution of EriC
We cloned one of the above ClC genes into an E. coli expression vector and added an NH2-terminal hexahistidine sequence for large-scale production and straightforward purification of EriC, a putative ClC-type channel product of the yadQ gene. After an overnight room-temperature induction of E. coli bearing the pEriC plasmid, a protein band of ~38 kD, absent in uninduced controls, was observed on SDS-PAGE of bacterial membrane fractions (Figure 2). This protein was detergent-extracted from the bacterial membrane fraction and purified on a Ni-chelate column. The apparent molecular mass of ~38 kD is smaller than that calculated from the sequence (51 kD), but the integrity of the full-length protein is indicated by two observations. First, robust interaction with the metal-affinity column indicates that the NH2-terminal hexahistidine tag is present; second, an EriC construct carrying an eight-residue, COOH-terminal 1D4 epitope (
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EriC is unambiguously a ClC-family protein at the level of primary sequence, but does it function as an ion channel? To assess this protein's ability to catalyze passive transmembrane flux of 36Cl-, we reconstituted EriC into liposomes and performed a concentrative influx assay (
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Absolute Estimation of Functional Activity
It is desirable to know if the observed flux activity is due to a major, minor, or minuscule fraction of the purified EriC protein. However, the observed influx kinetics are inadequate to quantify activity in our preparations because the absolute rate of Cl- uptake catalyzed by a single EriC channel is unknown. Instead, we employ an assay that does not require knowledge of absolute flux rates, but merely relies on the assumption that any liposome permeable to Cl- will equalize internal and external concentrations of this ion at thermodynamic equilibrium. Vesicles are loaded to equilibrium with two tracers: 36Cl-, which is permeant exclusively to EriC-containing liposomes, and 3H-glutamate, a trapped volume marker that is impermeant to all liposomes on the experimental time scale. The loaded vesicles are then diluted into tracer-free solution of identical ionic composition, and 36Cl- is allowed to flow out (in exchange for unlabeled Cl-) until equilibrium is achieved. Any vesicle containing one or more functionally active EriC molecules releases 36Cl- and retains impermeant glutamate; vesicles devoid of EriC channels will trap both 36Cl- and 3H-glutamate. Glutamate is thus used as an internal standard to measure the total intravesicular volume to which the Cl--accessible volume can be compared.
As EriC is reconstituted at increasing protein/lipid mass ratio, the fraction of Cl--impermeable volume, f0, falls exponentially from a protein-free value of unity (Figure 4), as demanded by a Poisson distribution governing the random incorporation of NE EriC channels into NL liposomes (, does not change systematically over this range of protein concentration (data not shown), which roughly spans the range of 0.24 channels per liposome. The protein concentration dependence of f0 quantitatively obeys the expectations of Equation 3, where the crucial fit quantity, the fraction of active protein, is s = 1.9. Since we have based our analysis on a utopian model in which protein molecules distribute perfectly into uniform spherical vesicles, it is perhaps not surprising that we reach a physically impossible conclusion of 190% activity. As discussed below, several assumptions in this analysis are expected to lead to uncertainty in the estimation of EriC activity, so we can easily rationalize this absurd value. The important point of this calculation is that a major fraction of the purified EriC proteinperhaps 100%is responsible for the functional activity detected in the assay.
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Ion Selectivity of EriC
The concentrative uptake assay was used in two ways to gauge the ionic selectivity of EriC. In the first set of experiments, internal Cl- was replaced with various test anions. Permeant anions support concentrative uptake, while impermeant anions do not. Of the anions tested (Figure 5 A), only Cl-, Br-, and NO3- are permeant by this criterion. In the second set of experiments, we added test ions to the external solution to see which of these would collapse the liposome membrane potential and thereby impede influx. In this assay, SCN-, and to a lesser extent I- and F- score as permeant in addition to Cl-, Br-, and NO3-. The discrepancy observed with SCN- is not disconcerting; SCN- both blocks and permeates some eukaryotic ClCs (
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EriC Is a Homodimer
The most extensively studied eukaryotic ClC channels, ClC-0 and ClC-1, are both homodimers, a quaternary structure underlying their double-barreled behavior (
Glutaraldehyde, a nonspecific cross-linking agent, has been used convincingly to report the oligomeric state of several membrane proteins (
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To complement the cross-linking experiments, we analyzed EriC by gel filtration chromatography and compared its migration to a reference membrane protein of known size, the K+ channel KcsA, a 74-kD homotetramer (
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Since these gel filtration results cannot distinguish dimers from higher-order oligomers, we also analyzed velocity sedimentation profiles of EriC and again used KcsA as a membrane protein size standard. Qualitatively, EriC sediments more rapidly than KcsA (Figure 8). A fit of the sedimentation data to a Fujita function (
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We attempted to carry out equilibrium sedimentation experiments in neutral-density detergents, to eliminate rigorously the contribution of bound detergent to the measured mass of the protein (
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DISCUSSION |
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At the current early stage of understanding the chemistry of integral membrane proteins, prokaryotes have served as the singular bearers of high-resolution structural information about ion channels. Porins, K+ channels, and mechanosensitive channels from bacteria have been expressed at high levels in E. coli as a prelude to crystallization and structure determination (
We have overexpressed a prokaryotic ClC channel, EriC, the product of one of the two ClC genes in E. coli. The biological role of this channel is unknown, but the present results make it clear that it is in fact a ClC-type Cl- channel. The purified protein promotes the passive, conductive flux of Cl- and other anions across liposome membranes, and the ionic selectivity of this effect is reminiscent of vertebrate ClC channel electrophysiology. We attempted but failed to observe single-channel current fluctuations induced by EriC in planar lipid bilayers; this negative result is disappointing but not surprising in light of the extremely low conductances of many eukaryotic ClC channels (
From a biochemical standpoint, perhaps the most important property to quantify for any new protein preparation, even more important than purity or yield, is functional competence. This measurement is difficult for uncharacterized ion channels reconstituted into liposomes, and our value implying 190% activity (Figure 4) is certainly disconcerting. We have used a limiting Poisson method of "counting" the fraction of liposomes that carry no channels in a sample containing NL liposomes and NE EriC channels, a fraction s of which are functionally active. If the insertion of channels into a uniform population of liposomes is random, then this fraction, f0, must obey a Poisson distribution (Equation 4):
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(4) |
We estimate f0 directly from the fraction of Cl--impermeable liposomes, and NE is known. The experimental samples consist of a known mass of lipid corresponding to a fixed liposome surface area, and the total intravesicular volume is directly measured. If the liposomes were uniform spheres of known lipid surface density, this information would be sufficient to determine NL and hence to derive a functional activity value s.
However, estimation of the number of liposomes is subject to several sources of error. First, the estimated value of activity varies with the cube of the bilayer surface area per mass of lipid, sigma}">, in Equation 3. The reconstituted liposomes used here are formed from a phosphatidylethanolamine-rich, undefined, complex mix of lipids. Molecular surface areas of phosphatidylethanolamines vary substantially (5572 Å2) depending on hydrocarbon chain, lipid composition in mixed bilayers, and other factors (
EriC behaves, as do the muscle-type channels ClC-0 and ClC-1 (
ClC channels carry out many crucial biological functions, but conventional mutagenesis studies have provided only limited glimpses of ClC molecular architecture. Since EriC is functionally active as a Cl- channel and may be obtained in milligram quantities, this protein is an excellent candidate for future structural studies.
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Acknowledgements |
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We thank Lise Heginbotham for E. coli genomic DNA, the KcsA gene in pASK90, and the waiver of consulting fee. We thank Jeff Gelles for essential assistance with analytical ultracentrifugation, and Rob Blaustein, Meredith Lemasurier, and Joe Mindell for comments on the manuscript. Sequence data were obtained from TIGR website at http://www.tigr.org. Sequencing of Methanococcus jannaschii and Archaeoglobus fulgidus was accomplished with support from the Department of Energy.
Sequencing of E. coli was accomplished with support from the National Human Genome Research Institute. Sequencing of Mycobacterium tuberculosis was accomplished with support from the Wellcome Trust. Sequencing of Vibrio cholerae was accomplished with support from the National Institute of Allergy and Infectious Diseases. M. Maduke was supported by a grant from the W.M. Keck Institute for Cellular Visualization.
Submitted: 29 July 1999
Revised: 20 September 1999
Accepted: 27 September 1999
1used in this paper: DDM, dodecylmaltoside; MES, morpholino ethanesulfonic acid; SDS, sodium dodecylsulfate
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