SPECIAL TOPIC
Model Organisms: New Insights
Into Ion
Channel and Transporter Function. Caenorhabditis
elegans ClC-type chloride channels: novel variants and
functional expression
Keith
Nehrke1,
Ted
Begenisich2,
Jodi
Pilato1, and
James E.
Melvin1,3
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 |
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 |
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 |
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.
Additional sequence information at the 5' end of clones for
clh-1, clh-3b, clh-5, and
clh-6 was derived from 5' rapid amplification of cDNA
ends using the SL1 trans-spliced leader sequence as an anchored
primer and sequence corresponding to the 3'-most 22 nucleotides of the
probes described above as an isoform-specific primer.
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.
The final replacement expression vectors (in bold) were derived from
those above as follows: an Xho I-Mlu I fragment
of clh-1 (nt 355-1957) in pBB-T27 was replaced with
that from a lambda cDNA clone to generate pB3-T27. pBB-C33,
the clh-2b expression clone, was sequenced completely and
found to contain a single nucleotide deletion, which was repaired using
a mutagene protocol with a wild-type oligonucleotide. The final
construct, pB3-C33, was also sequenced completely. pB3-E04
contains a lambda-derived Xba I fragment of
clh-3b (nt 669-2780), while pB4-E04 further replaces a BstB I-Sal I (nt 2875- MCS) fragment
of pB3-E04 with a BstB I-Xho I fragment from the
lambda clone. pB3-T06 contains a lambda-derived Age
I-Xho I fragment of clh-4b (nt
940-2990). pB3-ClC2 contains an Nco I
fragment of mClC2 (nt 339-2080) from the vector m6B-6 (kindly
provided by G. Borsani, Telethon Institute of Genetics and Medicine,
Milan, Italy). Finally, pBB-C07, the first clone derived,
was not substituted, but was sequenced completely to determine the
extent of PCR-induced errors (of which there were several, all in the
wobble position of the codon).
Using standard methods for the maintenance and transfection of Sf9
cells and the identification, amplification, and plaque assay of
recombinant baculovirus (Invitrogen), the clones were expressed at a
multiplicity of infection of 10 in Sf9 cells plated onto 5-mm
glass coverslips. At 24 or 48 h postinfection, whole cell
patch-clamp recordings were done at room temperature (20-22°C) using an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA).
The measured junction potentials for the solutions used were <1 mV, so
no correction for these was applied. The holding potential was 0 mV.
Data acquisition was performed using a 12-bit analog/digital converter
controlled by a personal computer.
The standard external solution contained (in mM) 140 N-methyl-D-glucamine (NMDG) chloride, 2 CaCl2, 2 MgCl2, and 20 mM HEPES, pH 7.1 (with
NMDG). The standard internal solution contained (in mM) 60 chloride, 60 glutamic acid, 120 (NMDG), 3 MgATP, 10 EGTA, and 10 HEPES, pH of 7.2 (with NMDG). Shortly after achieving whole cell mode with these
solutions, an outward rectifying current developed in uninfected as
well as infected Sf9 cells. This is likely the volume-sensitive organic
osmolyte/anion channel (VSOAC; e.g., see Ref. 5). With
these solutions, the activation of this current was transient: it
disappeared within 10-15 min. The elimination of this current was
accelerated if 30 mM mannitol was added to the standard external
solution. Currents through the expressed channels CLH-1, CLH-3b, and
ClC2 were the same with or without mannitol in the external solution.
Since NMDG is impermeant to most channels, currents measured with these
solutions are most likely carried by chloride ions.
As described in RESULTS, several different equations were
fit to the data. These were done using the Simplex algorithm
(4), either in our own implementation or that incorporated
in the Origin software package (version 5.0; Microcal Software,
Northampton, MA). When given, error limits for the fitted parameters
are the estimated errors from the fitting routines.
To provide a quantitative measure of the voltage dependence of
activation [G(Vm)], we converted
the measured ionic current (I) to conductance
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(1)
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where Vm is the membrane potential and
Vrev the current reversal potential. The
rectifying nature of the expressed channels and the rapid deactivation
kinetics of, for example, CLH-1, complicate the accurate measurement of
the reversal potential. Both C. elegans channels and ClC2
had negative reversal potentials in the range
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)
|
This equation allows a nonzero, likely leak conductance,
A2, at positive potentials where these channels
are not activated. The voltage at which half the channels are activated
is given by the V1/2 parameter and the voltage
sensitivity is governed by the k parameter.
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 |
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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Fig. 1.
A: gene organization of the C. elegans
clh family. Arrows represent the coding regions of each gene and
are drawn to scale with the intervening introns. The predicted
translation start sites are indicated by an ATG and stop sites by a
"stop." Transmembrane helices D1 and D12 are indicated to
demonstrate the boundaries of the highly conserved transmembrane
domain. Likewise, the start of CBS domains 1 and 2 are indicated. Those
isoforms where an SL1 trans-spliced leader sequence was identified are
denoted as such. The differences between clh1, clh-2b,
clh-3b, and clh-4b and the originally identified
isoforms (31) are displayed by using an asterisk (*) to
denote a small insertion, deletion, or nucleotide change, a minus sign
( ) to denote a missing exon(s), and a plus sign (+) to indicate a new
exon(s). New start or stop sites are indicated by slightly larger, bold
text. B: protein comparison of CLH-2, CLH-3, and CLH-4
variants. For each set of variants, a Clustal W algorithm [Thompson et
al. (38a)] was used to align the two amino acid
sequences. Regions where the sequences differ are presented in bold in
the alignment, with the regions of similarity presented in either
normal typeface or represented as arrows that connect the areas of
divergence. In general, these differences represent alternative exons,
translational start sites, or stop sites, as indicated
above.
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In addition, there were several small differences between the cDNA
sequence that we found for clh-1 and a previously published sequence (31). A single nucleotide insertion that both we
and Petalcorin et al. (29) predict occurs early in the
transcript and results in the addition of 38 amino acids to the protein
and may have functional consequences on the expression of CLH-1,
because a previous attempt to express this protein was unsuccessful
(31). This single nucleotide difference is not likely due
to alternative splicing, and thus we do not refer to clh-1
in terms of two variants.
Clh-2b contains an exon at the 5' end that is not present in
clh-2; an in-frame ATG adds 31 amino acids to the amino
terminus of the predicted protein (indicated by a plus sign in Fig.
1A). Genomic sequence comparison indicates that the first
intron is nearly 7 kb long (Fig. 1A). This was confirmed by
amplification of a full-length clone from cDNA by RT-PCR. Moreover, we
identified 15 additional nucleotides at the beginning of exon 11 in
clh-2b (indicated by an asterisk), resulting in the addition
of five amino acids within the putative transmembrane domain of this
protein. Maturation of many nematode transcripts frequently includes
the acquisition of an SL1 or SL2 trans-spliced leader (14,
40). While an SL1 splice site was found in clh-2
(31), we were unable to identify an SL1 or SL2 leader for
clh-2b. Amino acid alignments of clh-2 and
clh-2b are found in Fig. 1B.
Clh-3b lacks the first three exons of clh-3
(indicated by a minus sign in Fig. 1) and contains a novel SL1
trans-spliced leader, resulting in use of an alternative translational
start site and consequently the loss of 71 amino acids from the amino
terminus. Multiple differences between clh-3 and
clh-3b were found within the carboxy terminus of the protein
as well. Exons 11 and 12 of clh-3b are not present in
clh-3, and clh-3 ends after exon 14 of
clh-3b, in what is predicted to be intron sequence
(indicated by three plus signs in Fig. 1). This results in the use of
an alternative stop site, as well as differences in the last 6 amino acids of the CBS2 domain, which occurs only 3 amino acids short of the
stop codon in clh-3 compared with 163 amino acids before the
stop codon in clh-3b. A comparison of the results of these changes to the amino acid sequence of clh-3 and
clh-3b is shown in Fig. 1B.
We also found several differences between clh-4 and
clh-4b. The last exon in clh-4 was found spliced
to a new exon in clh-4b (exon 20, indicated by a plus sign
in Fig. 1). This resulted in the substitution of the final 10 amino
acids in clh-4 with 42 amino acids from the new exon and the
use of a new stop codon. There was also a 3-nt deletion at position
2155 of clh-4b between exons 13 and 14 and a 9-nt insertion
at position 2453 between exons 15 and 16 (asterisks in Fig. 1),
resulting in a net gain of two amino acids. Although the mRNA sequence
at the 5' end for clh-4 and clh-4b is identical,
the predicted amino acid sequence is not. This is due to the use of
different predicted start codons. We suggest here that the
clh-4b protein initiates at the first ATG in exon 1, near
the predicted 5' end of the mRNA. A previous report suggested that
clh-4 initiates at an internal ATG in exon 2, at 217 nucleotides from the 5' end of the mRNA (31).
While the cDNA sequence that we identified for clh-5 is
identical to that described earlier (31), the cDNA
sequence that we identified for clh-6 has not previously
been published. The intron-exon boundaries for clh-6 are
shown in Fig. 1. The coding regions for both clh-5 and
clh-6 are shorter than the other isoforms and contain less
introns. This is reflected in a lesser sequence homology compared with
the other isoforms.
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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Fig. 2.
A: schematic of the major anatomical structure of the
hermaphrodite nematode. B: the position of the neuronal cell
bodies. [Adapted, with permission, from Sulston and Horvitz
(37) and courtesy of L. Salkoff.]
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Clh-1 promoter-driven expression has previously been
localized to seam cells, which extend along the lateral sides of the body to form two seam syncytia in adults that remain separate from the
main body syncytium (29, 42). Seam cells secrete collagens
to form the extracellular cuticle of the nematode. Seam cells are also
responsible for the shrinkage that occurs during the dauer molt
(28), a process of facultative diapause that follows
pheromonal or environmental cues (9, 10). Mutations in
cuticle collagen genes and the process of dauer formation can cause
changes in the nematode's body morphology, and disruption of
clh-1 has been shown to result in a nematode with increased body width (29). This suggests that CLH-1 may be important
for certain seam cell functions. In addition to seam cells, we found that clh-1::GFP drove expression in both neurons
and hypodermal cells in the head of the nematode, in the D-cells of the
vulva, which connect the uterus to the body syncytium, and in posterior cells of the intestine (Fig. 3,
A-D). In addition, in late larval and adult stages, as the
worm reached sexual maturity, a fluorescence signal was detected in the
spermatheca (Fig. 3A). As hermaphrodites, nematodes are able
to fertilize their own eggs. The spermatheca, which lies between the
oviduct and the uterus, is the site of fertilization. Finally, in
recently hatched L1 larvae, expression appeared to be limited to seam
cells, seen as rows of 10 blast cells that run along each lateral line,
and the head neurons (Fig. 3, E and F). Some
autofluorescence is detectable in the gut of these and other transgenic
animals, which is mainly due to ingested bacteria.

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Fig. 3.
Green fluorescent protein (GFP) expression directed by the
clh-1 promoter in transgenic nematodes. Fluorescent
expression patterns for nematodes expressing GFP as a transcriptional
fusion from a 4-kb clh-1 promoter (A-C) and
the corresponding differential interference contrast (DIC)
photomicrographs (D-F). The nematode in A
and D is facing with its head upward and to the right. In
this late L4/adult animal, expression is observed in the hypodermal
cells of the head (HYP), unidentified cells of the spermathecal
structure (SPT), seam cells (SC), the D-cell of the vulva (D-cell), and
posterior intestinal cells (IC), as depicted both in the main figure
and the magnified inset of the vulva area (A). Expression is
also observed in neurons of the adult head and their associated
processes (B) and in neurons and seam cells in a recently
hatched L1 larvae, shown with its head facing down and on the right
(C).
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A clh-2b promoter element directed expression to body wall
muscle, which occurs in four quadrants on the dorsal and ventral sides
of the animal (Fig. 4, A and
B). These muscles are involved in locomotion; the backward
propagation of a contractile wave along a dorsal quadrant is balanced
by an antiphase wave along the opposing ventral quadrant, resulting in
the sinusoidal movement characteristic of C. elegans.
Expression from the clh-2b promoter was also observed in
cell bodies of the head and tail ganglia (Fig. 4, A-C).
C. elegans contains 302 neurons out of a total of 959 total
somatic nuclei. Of these, ~50 are involved in maintaining awareness
of the environment through exposure to soluble compounds and volatile
odorants (chemotaxis), heat and cold (thermotaxis), or touch avoidance
(mechanosensation). The cell bodies of these neurons, as do most, map
generally to either a site surrounding the pharyngeal bulb, with axons
extending to the amphid sensilla near the mouth or to a region near the
anus, extending processes to the phasmid sensilla in the tail (Ref.
43; see Fig. 2, A and B).

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Fig. 4.
GFP expression directed by the clh-2b promoter in
transgenic nematodes. Fluorescent expression patterns for nematodes
expressing GFP as a transcriptional fusion from a 4-kb
clh-2b promoter (A-D) and the corresponding
DIC photomicrographs (E-H). Expression is observed in
the adult body wall muscle (BWM), posterior IC, tail neurons (TN), and
head neurons (HN) of the nematode lying with its head to the left in
A. Higher magnification images of fluorescent adult head and
tail neurons are shown in B and C, respectively.
D: a recently hatched L1 larvae (head facing down and coiled
inside) expressing GFP in body wall muscles as well as head and tail
neurons. Whether these neurons are the same as those in which the
clh-2 promoter directs expression in the adult is unknown.
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Clh-2b::GFP is also expressed in the posterior
cells of the intestine, apparently coincident with expression from the
clh-1 promoter (Fig. 4D, compare to Fig. 3). The
earliest larval expression of clh-2 appears at the L1 stage,
predominantly in the muscle and neuronal cells (Fig. 4, G
and H). Although GFP expression from the clh-2
promoter has previously been examined (31), that promoter
is contained entirely within intron 1 of clh-2b and does not
overlap the 4 kb of sequence used here, nor do the expression patterns overlap (see Table 1).
The clh-3b promoter caused GFP expression in the excretory
cell. The excretory system consists of three cells: a large, H-shaped excretory cell, a duct cell, and a pore cell (27). The
excretory cell body is situated ventrally, near the terminal bulb of
the pharynx, and extends two side arms along the lateral lines. It is
the largest mononucleate cell in the body and is coupled to a gland
cell via desmosomes. Laser ablation studies suggest that the excretory
cell is important for osmoregulation and the maintenance of internal
hydrostatic pressure (28).
We also observed expression in the anterior four epithelial cells of
the intestine, in enteric muscles and in select neurons, including the
hermaphrodite-specific neurons, which are required for normal egg
laying (Fig. 5, A, B, E, and
F). This pattern corresponds well with that reported
previously for clh-3 (31), despite the use of
separate, but overlapping, promoter elements. In addition, we noted
expression in a structure surrounding the developing embryos, which is
most likely the uterus (Fig. 5, C and D). We observed this fluorescence most strongly in worms that contain only a
few embryos, and the signal intensity diminished as the number of
embryos increased (data not shown). We also found that the intestinal
cells and tail neurons expressed GFP at the earliest larval stage (Fig.
5, E and F).

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Fig. 5.
Expression pattern of a clh-3::GFP promoter
fusion in transgenic nematodes. Fluorescent (A-C) and
DIC photomicrographs (D-E) illustrate expression of GFP
from a 4-kb fragment of the clh-3b promoter in the following
cells: anterior intestinal epithelial cells (IC), vulval cells (VL),
the excretory cell (EC), the hermaphrodite-specific neuron (HSN), and
rectal muscles (RMG) of the late L4/adult facing right in A;
VL, uterine cells (UT), and EC of the adult in B; and RMG
and anterior IC of the L1 larvae facing with its head up in
C.
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As reported previously for clh-4 (31), the
clh-4b promoter drove expression of GFP in a single cell,
the H-shaped excretory cell (Fig. 6),
where it overlapped with expression from the clh-3 and
clh-5 promoters (Figs. 5 and
7; see below). The convergence of three
separate CLH proteins within the excretory cell suggests that these
genes may be of critical importance for normal function of this cell,
i.e., osmoregulation.

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Fig. 6.
Expression of GFP from the clh-4b promoter is restricted
to a single cell. A 4-kb fragment of the clh-4b promoter
drives strong GFP expression in the excretory cell, an H-shaped
canal-containing cell that runs the length of the nematode and
underlies the seam cells, as shown by fluorescent (A) and
DIC (B) photomicrographs. The cell body of the excretory
cell is positioned anterior, ventral to the terminal bulb of the
pharynx.
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Fig. 7.
Ubiquitous expression of GFP from the clh-5
promoter transgene illustrated by fluorescent (A-E) and DIC
photomicrographs (F-I). Animals harboring a 4-kb
clh-5::GFP promoter fragment construct expressed
GFP in nearly all of the cells of the body, as shown in A.
Although specific expression occurs in the PHX, hypodermal and muscle
cells of the head (HYP), seam cells (SC), and body wall muscle (BWM),
the overall high levels of fluorescence necessitate the presentation of
individual organs and cells via the analysis of mosaic animals.
B and C more closely examine two planes of focus
in a single mosaic animal to demonstrate that the excretory cell (EC)
and seam cells (SC), the vulva (VL), the ventral nerve cord (VNC), and
the BWM all express some level of GFP. The bright spots in B
arise from autofluorescent bacteria in the gut. Mosaic animals
expressing GFP in cells of neuronal lineage in the tail (TN) and head
(HN) are depicted in D and E, respectively. In
addition, we observed GFP expression in germ line cells of the ovaries
during the F1 and F2 generations of the transgenic animal (data not
shown).
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A clh-5::GFP fusion directed expression to nearly
every cell in the nematode (Fig. 7). At low magnifications, many of the transgenic nematodes from the clh-5::GFP lines
appeared to fluoresce everywhere, making an exhaustive discrimination
of specific structures unrealistic, as shown (Fig. 7A). For
orientation purposes, we have indicated in Fig. 7A several
of the most prominent features observed in this transgenic strain. By
examining mosaic animals, however, which have lost the injected array
from cells of specific lineages (see DISCUSSION), we were
able to more carefully delineate specific areas of expression. We show
here that the excretory cell and overlying seam cells express GFP (Fig.
7C), as do the body wall muscles and ventral and dorsal
nerve cords (Fig. 7D). Fluorescence was seen in neurons both
in the head and tail (Fig. 7, F and H), as well
as in their associated processes. We also observed expression in gut
and distal tip cells, as well as in germ line cells in the ovaries of
F1 and F2 generation transgenic animals (data not shown). Although we
have not documented here every cell in which we observe GFP expression,
these results indicate that clh-5 expression may be ubiquitous.
The clh-6 promoter directed expression of GFP to a
wide variety of cells as well (Fig. 8).
We observed intestinal fluorescence, predominantly in the most anterior
and posterior segments (Fig. 8A), as shown for
clh-1 (Fig. 3), clh-3b (Fig. 5), and
clh-5 (Fig. 7). The coincidence of these four isoforms in
various cells of the intestine suggests an important role for chloride
flux in this organ. The main body of the intestine in C. elegans consists of a tube of 20 cells, each bearing a dense layer
of microvilli on their apical surface. The primary function of the
intestine is probably to secrete digestive enzymes into the lumen and
to absorb the processed nutrients. The intestine can serve as one of the primary storage points for protein, carbohydrate, and lipid granules in the body, as well as yolk proteins, and plays a major role
in the nurture of germ cells (18).

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Fig. 8.
The clh-6 promoter drives GFP expression in many of the
muscle cells of the nematode. As with the clh-5 promoter,
expression of GFP by the 4-kb clh-6 promoter resulted in
highly fluorescent animals, as well as a high level of mosaicism.
Therefore, the fluorescent (A-E) and corresponding DIC
(F-J) photomicrographs were drawn from representative
animals where GFP is expressed in only a subset of the cells normally
visualized. Several of those cells are shown, as follows. A:
the gut (GT) and anterior and posterior intestinal cells (IC) are
fluorescent; B: the pharnyx (PNX) expresses GFP;
C: body wall muscles (BWM) are labeled; D: vulval
muscles (VM) fluoresce, as do the anal depressor muscle (ADM) and IC in
E. We do not observe either seam cells or excretory cell
expression. However, several neurons can be visualized under GFP
optics, but expression levels are small compared with the nonneuronal
cells.
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Fluorescence also occurred in nearly all of the muscle cells, including
body wall (Fig. 8C), vulval (Fig. 8D), pharyngeal (Fig. 8B), and excretory muscles, such as the anal depressor
muscle depicted in Fig. 8E. In the C. elegans
hermaphrodite, the expulsion step of defecation depends on the
coordinated contraction of three enteric muscle groups: the anal
depressor muscle, the intestinal muscles, and the sphincter muscle.
These muscles are activated by excitatory GABA neurotransmission
(25) and receive synaptic input only from the DVB ring
interneuron (43). We did not, however, observe strong
fluorescence from the clh-6 promoter in any neurons, which
differs from most of the other family members except the excretory
cell-specific clh-4.
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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Fig. 9.
Expression of C. elegans chloride channels.
A, inset: currents from Sf9 cells infected with
virus coding for CLH-1 in response to 80-ms depolarizations to 120,
80, and 40 mV (largest to smallest) from a holding potential of 0 mV. A, main figure: currents ( ) 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 120, 80, and 40
mV (largest to smallest) from a holding potential of 0 mV.
B, main figure: currents measured at the end of the 500-ms
pulses at the indicated membrane voltages.
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Examples of currents from cells infected with virus coding for CLH-3b
channels are shown in Fig. 9B. The inset contains
currents in response to 500-ms voltage pulses to
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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Fig. 10.
Voltage dependence of activation of CLH-1.
Inset: current ( ) from an Sf9 cell infected
with virus coding for CLH-1 in response to an 80-ms pulse to 120 mV.
Line: fit of a single exponential time function to the data with a time
constant of 1.25 ms. Main figure: conductance (obtained from currents
measured at the end of 80-ms pulses) at the indicated membrane
potentials. Mean values (n = 3) and SE limits. Line:
fit of the Boltzmann relation (Eq. 2) with
V1/2 and k values of 100 ± 1.9 mV and 13 ± 0.82 mV 1, respectively.
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We estimated the voltage sensitivity of expressed channel activation by
an analysis of the channel conductance (see EXPERIMENTAL PROCEDURES). The voltage dependence of CLH-1 channel conductance is illustrated in the main part of Fig. 10 (closed squares). These data
were fit with the Boltzmann relation (Eq. 2 and solid line in Fig. 10). From similar fits to the conductances of CLH-1, CLH-3b, and ClC2 channels, the midpoint of channel activation
(V1/2) was determined, and these are listed in
Table 2. Of the three channels, ClC2 had
the most positive V1/2 value. The value for
CLH-3b channels was somewhat more negative, but this difference was not
significant. CLH-1 channels activated at significantly more negative
potentials than CLH-3b and ClC2 channels with a
V1/2 value of
110 mV.
The parameter k in the Boltzmann relation (Eq. 2)
reflects the steepness of the conductance-voltage relationship. The
properties of the expressed chloride channels also differed in this
parameter (Table 2). CLH-3b and ClC2 channels had a similar voltage
sensitivity, but CLH-1 channels had a substantially greater sensitivity.
 |
DISCUSSION |
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.
 |
ACKNOWLEDGEMENTS |
We thank Fred Hagen and Karen Gentile for technical support,
strains, and comments.
 |
FOOTNOTES |
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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