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1 Center for Oral Biology, Aab Institute of Biomedical Sciences, 2 Department of Pharmacology and Physiology, and 3 Eastman Department of Dentistry, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Six ClC-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6. cDNA sequences from these genes suggest that clh-2, clh-3, and clh-4 may code for multiple channel variants, bringing the total to at least nine channel types in this nematode. Promoter-driven green fluorescent protein (GFP) expression in transgenic animals indicates that the protein CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. In an Sf9 cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did not form functional plasma membrane channels. In contrast, both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian ClC2, but which operate over different voltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene family provides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.
nematode; electrophysiology; transgenic; green fluorescent protein
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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EXPRESSION CLONING OF A VOLTAGE-GATED chloride channel gene from the electric organ of Torpedo marmorata provided the first member of the ClC family (16). Subsequently, at least nine genes have been shown to exist in mammals (1, 3, 8, 17, 35, 36, 38, 39). The function of these genes probably includes the control of electrical excitability, transepithelial transport, and the charge compensation necessary for the acidification of intracellular organelles (for review, see Ref. 15). In addition, ClC2 and ClC3 may play a role in cell volume regulation (41, 45). Evidence for the physiological significance of the ClCs includes mutations in the ClC1 muscle chloride channel that leads to myotonia (19, 34), in the ClCKb kidney-specific channel that leads to Bartter's syndrome [associated with severe renal wasting (32)], and in the ClC5 channel that leads to Dent's disease [associated with proteinuria and hypercalciuria (21)]. Furthermore, mice with targeted disruption of the Clcnckl gene display nephrogenic diabetes insipitus (24). ClC-like genes appear to be conserved from mammals to lower organisms including bacteria (23) and yeast (12).
Caenorhabditis elegans is a free-living soil nematode that feeds on bacteria and has a life cycle of ~3 days, although adults can live for several weeks after egg laying (for review, see Ref. 44). The adult hermaphrodite, which comprises the vast majority of the population, has 959 somatic nuclei, for which complete fate maps and lineage determinations exist. Juvenile worms develop into adults through a series of molts, resulting in four distinct larval stages (L1-L4). In addition to a complete wiring diagram being available for the C. elegans neuronal circuitry, the genome is completely sequenced.
The completion of the C. elegans genome sequencing project
led to the prediction of a family of six voltage-gated chloride channel
genes in nematodes. Recently, the cDNA has been cloned for
clh-1 through clh-5, expression patterns have
been determined for clh-2, clh-3, and
clh-4, and recombinant CLH-3 has been characterized electrophysiologically by expression in Xenopus oocytes
(31). In addition, it has been shown that disruption of
the clh-1 gene causes a defect in body width of the adult
and that the clh-1 gene product localizes to seam cells, a
set of multinucleated cells that help to maintain the cuticle that
encases the worm (29). Analysis of these cDNA sequences
confirmed that the CLH proteins share many of the characteristics of
mammalian ClC chloride channels (31): each contains two
conserved cystathione 
-synthase (CBS) domains of unknown function at
the carboxy terminus and multiple membrane-spanning domains containing
a conserved motif GKxGPxxH, which may act as a core structural element
of the pore region (7).
In the present study, the cDNA sequence has been determined for all six clh isoforms, including clh-6. The sequences of the clh-2, clh-3, and clh-4 clones appear to be different from those previously reported (31). The new variant proteins are named CLH-2b, CLH-3b, and CLH-4b. Some of these differences may be attributable to start site selection and/or alternative splicing patterns, and, in the case of the clh-2 variants, suggests the use of entirely nonoverlapping promoters. In contrast to the previously described clh-1 (31), which did not express functional channels, the variant we identified gave rise to voltage-dependent, inward-rectifying chloride currents. We also found that CLH-3b, despite significant differences in the amino acid sequences at both ends of the protein, generated currents that were generally similar to those described previously for CLH-3 (31). Promoter::GFP constructs were used to determine the expression patterns driven by the clh-1, clh-2b, clh-3b, clh-4b, clh-5, and clh-6 genes. For clh-2b, we found that the expression pattern differed remarkably from that described for clh-2 (31), suggesting that the two separate promoter elements may regulate expression of this gene in different cell types. We also detected strong expression from the clh-1 and clh-3b promoters in cells not previously documented (29, 31). Furthermore, clh-5 appears to be expressed ubiquitously, while clh-6 appears to be expressed in most nonneuronal cells in the nematode.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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cDNA cloning.
Basic local alignment search tool homology searches of GenBank with
mammalian ClC sequences yielded multiple expressed sequence tags and 6 genes spread over 10 genomic cosmid clones (clh-1 on T27d12;
clh-2 on B0491 and C33b4; clh-3 on E04f6 and
F32a5; clh-4 on T06f4 and R02e4; clh-5 on C07h4
and T24h10; clh-6 on R07b7). Isoform-specific probes were
generated by RT-PCR and were targeted to sequences that lie near the 5'
end of the predicted coding regions (clh-1, nt 479-848;
clh-2, nt 353-689; clh-3, nt 299-655; clh-4, nt 499-870; clh-5, nt 453-865;
clh-6, nt 428-802). Probes were labeled by random
priming using a Ready Prime DNA labeling kit (Life Technologies,
Rockville, MD) and [32P]dCTP. Nearly full-length coding
regions for six of the ClC homologs were obtained by screening two
C. elegans cDNA libraries: an oligo(dT)-primed cDNA library
and a random-primed cDNA library, 
-ACT-RB1 and -ACT-RB2, respectively (kindly provided by Dr. R. Barstead, University of Wisconsin-Madison). Seven hundred thousand phage of each library RB1
and RB2 were plated onto 24 × 24 cm Nunc plates and a lawn of
LE392 Escherichia coli cells. The plates were plaque-lifted using Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ),
and the membranes were hybridized overnight at 42°C in 5× sodium
chloride-sodium phosphate-EDTA, 50% formamide, 5× Denhardt solution, 0.1% SDS, and 100 µg/ml salmon sperm DNA, containing 5 × 105 cpm/ml of each 32P-labeled
denatured probe. Filters were washed three times for 20 min each in 2×
SSC and 0.1% SDS at the following three temperatures: 42, 64, and
42°C. Initial screening was performed with a mixture of all six
probes for isoforms clh-1 through clh-6. Positive
plaques were cored, dot-blotted on multiple Hybond-N membranes, and
probed with individual isoform-specific probes, using the conditions above. Several clones that hybridized to each isoform-specific probe
were isolated to homogeneity. Cre-lox excision of the pACT plasmid from
each clone was accomplished by transduction into the E. coli strain RB4, which expresses the Cre recombinase. Quiagen quality plasmid DNA was prepared in the RB4 host and used directly for
cycle DNA sequencing with ABI BigDye terminator mix and thermostable DNA polymerase on an MJResearch autosequencer. The reactions were run
by the University of Rochester Core Nucleic Acids Facility. Both
strands of all clones were completely sequenced.
Electrophysiological analysis.
Baculovirus expression constructs were generated for clh-1,
clh-2b, clh-3b, clh-4b, and
clh-5 and for murine ClC2 by amplification of the entire
coding region using pfu DNA polymerase and insertion as
restriction-site tagged products, complete with an Sf9 insect cell
translation initiation consensus sequence, into the pBlueBac4 vector
(Invitrogen, Carlsbad, CA). Subsequently, portions of the PCR-derived
coding sequence were replaced with that from the cDNA clones, and
the inserts were completely sequenced. The PCR-generated clones were
derived as follows: pBB-T27 contains the amplified coding region for
clh-1 inserted at Nhe I and Hind III
sites of pBlueBac4, pBB-C33 contains clh-2b inserted at
Nhe I and Hind III sites, pBB-E04 contains
clh-3b inserted at Nhe I and Bgl II sites, pBB-T06 contains clh-4b inserted at Nhe I
and Bgl II sites, pBB-C07 contains clh-5 inserted
at Nhe I and BamH I sites, and pBB-ClC2 contains
ClC2 inserted at BamH I and EcoR I sites.
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(1) |
10 to 2 mV. In
general, chloride channels are not particularly selective for anions
and even glutamate is sparingly permeant in some channels [e.g.,
Arreola et al. (2)]. With our recording solutions, the
reversal potential expected for a channel permeant to chloride and
glutamate (0.1 the permeability of chloride) is 18 mV. Our more
positive values likely reflect the difficulty of this measurement and
the fact that any leak current will bias the measurements toward less
negative potentials.
We fit a Boltzmann relation to the channel conductance
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(2) |
Construction of clh::GFP promoter fusions and generation of transgenic animals. Nematodes (Bristol N2 strain) were cultured at 14°C or 18°C on NGM agar plates seeded with HB101 or OP50 bacteria from an overnight culture. GFP expression constructs for each clh gene consisted of ~4 kb of promoter sequence from upstream of the ATG translation initiation site, together with 10-12 nt downstream, cloned into expanded multiple cloning site vector pFH6(II) (courtesy of F. Hagen, University of Rochester, Rochester NY), which is a derivative of pPD 95.81 (courtesy of A. Fire, Carnegie Institute of Washington, Baltimore, MD). The inserts were PCR amplified from a genomic template. Both upstream and downstream oligonucleotides were tagged with unique restriction sites for cloning purposes. The downstream oligonucleotide was designed to be an imperfect match such that the genomic start site ATG would be mutated to a TTG in the final construct.
pJP72C07 contains the clh-5 promoter and was amplified using oligonucleotides 5'-GCTCCTGTTGCTCAGCTGAAGAAGACC-3' and 5'-CGACCTGCTCGTTCCAATTTCGGCTGG-3', with Nhe I and BamH I tags, respectively (mutated residue from within the ATG codon is given in bold and underlined). Similarly, pJP72C33 (clh-2) used 5'-ACTACCGAGCATCGCTGCAGGCTTGG-3' and 5'-ACTTTTGCCAATGGATCCAATGTTAAAGGAGTT-3' with a Pst I tag on the upstream oligonucleotide and a BamH I site internal (ORF nt +4) to the downstream primer; pJP72E04 (clh-3) used 5'-CAAATCAAGTGACGCAATCTGACTCGC-3' and 5'-ACCAATACCCAAACTTTTGGAATCCTCG-3' with Nhe I and Pst I tags; pJP72R07 (clh-6) used 5'-GTAGATGGTGATCTGTTTCTGGCTTGTG-3' and 5'-CTGTTACGGGATGTCAACTGAAATGTTG-3' with Nhe I and BamH I tags; pJP72T06 (clh-4) used 5'-CCACATTGGTGGTGCTATGAATTCAGC-3' and 5'-CGCACCGTTCAAACGACAAAATTCAGGCG-3' with Nhe I and BamH I tags; pJP72T27 (clh-1) used 5'-CGGAAATGGCCTTTATTTCCGCGCAC-3' and 5'-GCGTCTTCCAACCTGATGTGCAGAATC-3' with Nhe I and BamH I tags. GFP fusion construct and pRF4, which produces a rol-6 roller phenotype (20), were mixed at 75 µg/ml each in injection buffer, then coinjected into the gonad of young adult Bristol N2 nematodes, as described in Ref. 26. After 4 days, rollers were picked from at least 10 injections to separate plates to look for germ line transmission. The nematodes were imaged on 2% agarose pads using a Nikon Eclipse E800 microscope equipped with a Nikon 60× oil objective under 100 W mercury illumination and a GFP or DAPI filter set, as appropriate. The images were captured using a Spot2 camera and analyzed in Adobe Photoshop (Adobe Systems, San Jose, CA). Due to the intensity of fluorescence from cells that are out of the plane-of-focus, many of the images that we present here are derived from mosaic animals.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The six ClC genes in C. elegans (clh-1 through clh-6) express at least nine distinct channels. Six ClC genes from C. elegans, termed clh-1 through clh-6, have been previously described (29, 31). While the cDNA sequences reported here are similar in many ways to those identified previously, significant differences do occur in three of the five isoforms previously cloned, particularly in terms of start site selection and potential alternative splice patterns. The new mRNA variants described in the present report are denoted as clh-2b, clh-3b, and clh-4b. We also present the first cDNA sequence cloned from clh-6.
Figure 1A illustrates the genomic structure of the six nematode clh isoforms. For the three new variants that we have identified, the differences from the original clones are denoted schematically by a combination of asterisks to indicate a small change, plus or minus signs to indicate new or missing exons, respectively, and boldface type at new ATG and translational stop sites. Amino acid sequence alignments suggest that the most divergent segments of CLH-2, CLH-3, and CLH-4 and CLH-2b, CLH-3b, and CLH-4b, respectively, lie at the amino and carboxy termini of the proteins, rather than in the core transmembrane domain, which is conserved even between isoforms (Fig. 1B).
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Expression of GFP from clh promoters in transgenic animals.
Approximately 4 kb of promoter region from each clh gene,
extending upstream from and including a mutated ATG-to-TTG initiator codon, was cloned as a transcriptional fusion with cDNA encoding a
cytoplasmic form of GFP. These constructs were then injected with a
rol-6 marker plasmid that produces a roller phenotype into the Bristol N2 strain, and transgenic lines were established. Table
1 indicates where each of these
constructs is expressed and incorporates expression data generated from
several other groups as well (29, 31). Figure
2, A and B, is
intended to provide orientation in the form of a general schematic of
the nematode architecture, including the major organs, reproductive system, and neuron cell body locations.
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Functional expression of clh chloride channels in Sf9 cells.
We infected Sf9 cells with viruses encoding the putative C. elegans chloride channels. Of the five putative channels that we
infected, two expressed robust time- and voltage-dependent currents in
Sf9 cells. Examples of currents from cells infected with virus coding
for CLH-1 and CLH-3b channels are shown in Fig. 9. The inset in Fig.
9A contains currents from cells infected with CLH-1 virus in
response to 80-ms voltage pulses to 120, 80, and 40 mV. There was
very little current at 40 mV, but fast-activating currents were
activated at more negative potentials. The steady-state currents at the
indicated potentials are illustrated (closed squares) in the main part
of Fig. 9A. Sf9 cells infected with wild-type virus
expressed currents that were always <0.1 nA and showed no significant
rectification. An example of these currents is included (open circles)
in Fig. 9A.
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120, 80, and 40
mV. CLH-3b channel currents activated more slowly (note time scale differences) and at slightly less negative potentials than CLH-1 channels, as indicated by the measurable current at 40 mV. The steady-state currents at the indicated potentials are illustrated (closed squares) in the main part of Fig. 9B.
To quantitatively assess the kinetics of the chloride channels
expressed in Sf9 cells, we fit a single exponential time function to
the currents in response to a voltage step to 120 mV. An example of
this procedure for the CLH-1 channel currents is shown in the inset of
Fig. 10. The currents (closed circles)
are well described by this simple function (solid line) with a time
constant of 1.25 ms. The average time constant for CLH-1 currents at
this potential was 1.3 ± 0.16 ms (SE, n = 3).
CLH-3b channels activated almost sixfold slower with an average time
constant at 120 mV of 7.4 ± 0.68 ms (n = 3).
The kinetics of ClC2 channels expressed in Sf9 cells were somewhat
variable but were considerably slower than those through CLH-1 or
CLH-3b channels with a mean time constant of 270 ± 69 ms
(n = 3).
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110 mV.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Six voltage-activated chloride channel genes are predicted based on the sequencing of the C. elegans genome. cDNA sequences for five of these have been previously reported, and the respective genes have been named clh-1 through clh-5 (31). We have reported here the cDNA sequence of the sixth and final isoform, clh-6. We have also identified variants of clh-2, clh-3, and clh-4, which we have termed clh-2b, clh-3b, and clh-4b. Comparisons between these variants at the genomic structure and protein levels are summarized in Fig. 1. Alternate translation initiation or stop sites and alternate splicing appear to result in changes, for the most part, to the amino and carboxy termini of the proteins. We have also confirmed the genomic structure of clh-5 and defined the organization of the clh-6 gene.
The nematode genome appears to code for representatives from each of the three branches of the mammalian ClC family. Clustal analysis indicates that protein isoforms CLH-1 through CLH-4 are most closely related to the mammalian ClC2 family, while CLH-5 is more closely related to mammalian ClC3, ClC4, and ClC5, and CLH-6 is related to mammalian ClC6 and ClC7. The clh-5 and clh-6 genes contain fewer introns than clh-1, clh-2b, clh-3b, or clh-4b and both code for proteins of <800 amino acids, which is significantly smaller than the 880-1,084 amino acid length of the other isoforms. They are also expressed nearly ubiquitously, compared with the relatively restricted expression of clh-1, clh-2, clh-3, and clh-4. Despite obvious sequence similarities between clh-1, clh-2, clh-3, and clh-4, the genomic organization (Fig. 1) suggests that there is no conserved intron-exon structure within the clh family.
To study the normal expression pattern of each CLH protein, transgenic nematode strains were created where GFP production is driven from a clh promoter. Because plasmid transmission in nematodes occurs via an extrachromosomal array, a phenomenon known as mosaicism exists (26) whereby the array becomes lost during cell division, resulting in lineage-specific deficits in expression. Mosaicism, combined with a general lack of expression of extrachromosomal arrays in germ line cells and arbitrary determination of what defines the "promoter region," combine to confound the analysis of transgenic nematodes. Expression patterns obtained from transgenes are generally acknowledged to be preliminary until confirmed via antibody staining or in situ analysis. With these caveats in mind, we have presented data generated from at least three stable lines for each clh promoter construct and have indicated in RESULTS which lines are mosaic.
The clh-1, clh-2b, clh-3b, and
clh-4b promoters drive specific patterns of GFP expression,
ranging from one to over a dozen cells labeled. In agreement with
previous results (29) we have shown that the
clh-1 promoter drives expression of GFP in seam cells (Fig.
3). Tc1 transposon-mediated mutagenesis of the clh-1 gene
causes a change in body width of the adult, indicating that this
protein is functionally important for the development of a normal
nematode morphology (29). We have also demonstrated clh-1 promoter-driven expression in the D-cell of the vulva,
neurons in the head of the nematode, posterior cells of the intestine, and cells of the spermathecal structure (Fig. 3; Table 1). In addition
to the morphological phenotype in the Tc1 mutant, there may be
unrecognized functional deficits associated with other cell types
expressing CLH-1. These cells may have been missed in the earlier study
due to the expression construct; the clh-1 promoter drove
expression of a nuclear-targeted -galactosidase enzyme. Given that
seam cells are multinucleate and run the length of the body, the signal
arising from other cells could be masked. Alternatively, the GFP enzyme
that we have used contains multiple synthetic intron sequences that can
stabilize the message and result in increased protein production.
The clh-2b promoter demonstrated a totally different, nonoverlapping cellular expression pattern than the clh-2 promoter (31), as summarized in Table 1. The clh-2 promoter corresponds to sequences contained in the first intron of clh-2b, and, as indicated, the two promoters do not overlap. The fact that there are differences in the amino acid sequences and in the expression patterns for these clh-2 variants suggests that the expressed proteins may have different physiological roles. However, it is difficult to assign physiological roles for these channels, since we did not observe channel activity for CLH-2b in the Sf9 cell expression system and expression of the CLH-2 protein in Xenopus oocytes (31) produced currents that were too small to study.
The cellular distribution patterns for the two clh-3 variants were identical [excretory cell, intestinal cells, rectal muscles, and the hermaphrodite-specific neuron (31); Table 1], except that the clh-3b promoter drove expression in the uterus, as well. The predicted start site for translation of clh-3 differs from that of clh-3b by almost 3 kb. Given the respective promoter fragments used for each study and their corresponding overlap, one of two possibilities exists: either 1 kb of sequence upstream of the clh-3 start site is enough to drive the specific pattern of expression observed or the expression pattern derived from the clh-3 promoter fragment in reality originates from the clh-3b start site. The mutated ATG in the clh-3b promoter is in frame with GFP and may give rise to a translational fusion if translation does begin upstream at the predicted start site for clh-3. Deletion analysis may be required to determine the functional boundaries of the clh-3 promoter.
The expression driven by the promoters of both clh-4 variants is particularly striking, because it occurs only in the excretory cell (Fig. 6 and Ref. 31). Laser ablation studies have demonstrated that the excretory cell is required for maintenance of osmotic balance and internal hydrostatic pressure in the nematode (28). Nematodes lacking an excretory cell bloat and die within 24 h, and it has been shown that the activity of the cell is responsive to changes in external osmolarity (28). The ability of the clh-4 promoter to confine transcription to this single kidneylike cell makes it a useful tool in examining excretory cell defects using reverse genetics and antisense inhibition, especially in cases where a whole organism gene ablation may be lethal.
In contrast to the other isoforms, both clh-5 and clh-6 were expressed in many cells, although clh-6 expression was limited mainly to cells of nonneuronal origin. Thus the physiological role of these ubiquitous chloride channels could reflect a function necessary for all cells.
We have functionally expressed two of the six ClC-like channels from C. elegans, CLH-1 and CLH-3b. Like mammalian ClC2 (38), these channels exhibit strong inward rectification. However, the amino-terminal cytoplasmic domain, which has been implicated in the gating of ClC2, is not conserved among these three proteins (13). While both CLH-1 and CLH-3b are inwardly rectifying, they activate >200- and 30-fold faster than ClC2, respectively. Moreover, CLH-1 activates at more negative voltages than ClC2 and CLH-3b. Previous attempts to functionally express CLH-1 in Xenopus oocytes and HEK-293 cells were unsuccessful (31). This may be due to characteristics of the expression system, because we used Sf9 cells, or to differences arising from a change in the sequence reported by others and ourselves (29) that adds 38 amino acids to the amino terminus of the protein.
CLH-3b appears to arise from a splice variant of an isoform that was recently shown to possess channel activity when expressed in Xenopus oocytes (31). The CLH-3 (31) and CLH-3b variants appear to generate similar current-voltage relations. The physiological role of having two variants with similar properties expressed in the same cells is unknown. Although both proteins share a common amino acid core, significant differences do occur at the amino and carboxy termini of the protein (Fig. 1B), suggesting some individualized function. A better understanding of those functions may first necessitate deciphering how these channels relate to the particular functions endogenous to the cells in which they are expressed.
ClC2 and ClC3 are postulated to act as volume-sensitive chloride channels, which suggests that they may be involved in cell volume regulation (41, 45). Water movement is often coupled to chloride transport, so the expression of CLH-3 channels could mediate the osmoregulatory role of the excretory cell and fluid secretion in the intestinal cells. However, it was noted that CLH-3 did not respond to cell swelling when expressed in Xenopus oocytes (31). We have not examined the responsiveness of CLH-3b to swelling in Sf9 cells, largely due to high background currents. Voltage-gated chloride channels may also be involved directly in regulating the membrane potential or could produce changes in the chloride equilibrium potential, both of which could have secondary effects on the activity of other cell properties (33). The robust expression from the promoters for both CLH-1 and CLH-3b in neurons also suggests an important physiological role for these anion channels. However, to date, these types of channels have not been identified in C. elegans neuronal cells or neuromuscular junctions (11, 30). RNAi or TC1 transposon mutagenesis combined with behavioral studies may help us to reconcile these observations or uncover a role that is difficult to observe through electrophysiology.
Even in mammalian cells, the role of most ClC isoforms in basic cellular function and physiology has yet to be well defined. One of the advantages of C. elegans as a model system is that both genetic and reverse genetic screens are accessible. The ability to rapidly generate cell-specific antisense inhibition of a given chloride channel isoform or to employ RNAi knockdown of message levels along with the evolving techniques involved in in situ patch clamp of the worm (22) may allow us to answer very specific questions about the role of chloride channels in defined cellular events, such as transepithelial transport, neurotransmission, and muscle excitation. In addition, since a complete lineage map and fate determinations are available for every cell in C. elegans, this model system may be used to address the relevant question of the role of chloride channels during development. To this end, the results presented here and in previous work in this area (29, 31) provide the foundation for advancements in our understanding of both nematode biology and channel biology in general.
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ACKNOWLEDGEMENTS |
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We thank Fred Hagen and Karen Gentile for technical support, strains, and comments.
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FOOTNOTES |
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This work was supported in part by National Institute of Dental and Craniofacial Research Grants DE-13539 and DE-O9692 (to J. E. Melvin).
Address for reprint requests and other correspondence: J. E. Melvin, Center for Oral Biology, Univ. of Rochester, Medical Center Box 611, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: james_melvin{at}urmc.rochester.edu).
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.
Received 8 May 2000; accepted in final form 10 July 2000.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) from an Sf9
cell infected with virus coding for CLH-1 measured at the end of the
80-ms pulses at the indicated membrane voltages and currents
(
) from a cell infected with wild-type virus. B,
inset: currents from Sf9 cells infected with virus coding for
CLH-3b in response to 500-ms depolarizations to 
) from an Sf9 cell infected
with virus coding for CLH-1 in response to an 80-ms pulse to 
1, respectively.