Splice variants of a ClC-2 chloride channel with
differing functional characteristics
L. Pablo
Cid1,2,
María-Isabel
Niemeyer1,2,
Alfredo
Ramírez1, and
Francisco V.
Sepúlveda1,2
1 Instituto de Ciencias Biomédicas, Facultad de
Medicina, Universidad de Chile, Santiago-7; and 2 Centro de
Estudios Científicos, Valdivia, Chile
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ABSTRACT |
We identified two ClC-2 clones in a guinea pig
intestinal epithelial cDNA library, one of which carries a 30-bp
deletion in the NH2 terminus. PCR using primers
encompassing the deletion gave two products that furthermore were
amplified with specific primers confirming their authenticity. The
corresponding genomic DNA sequence gave a structure of three exons and
two introns. An internal donor site occurring within one of the exons
accounts for the deletion, consistent with alternative splicing.
Expression of the variants gpClC-2 and gpClC-2
77-86 in HEK-293
cells generated inwardly rectifying chloride currents with similar
activation characteristics. Deactivation, however, occurred with faster
kinetics in gpClC-2
77-86. Site-directed mutagenesis suggests
that a protein kinase C-mediated phosphorylation consensus site lost in
gpClC-2
77-86 is not responsible for the observed change. The
deletion-carrying variant is found in most tissues examined, and it
appears more abundant in proximal colon, kidney, and testis. The
presence of a splice variant of ClC-2 modified in its
NH2-terminal domain could have functional consequences in
tissues where their relative expression levels are different.
chloride secretion; alternative splicing; guinea pig; channel
deactivation; intestinal epithelium
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INTRODUCTION |
THE EPITHELIUM
COVERING the small and large intestines is a site of absorption
of nutrients, electrolytes, and fluid. Under the action of certain
neurohumoral agents or toxins, it becomes a tissue mediating copious
secretion of fluid and electrolytes. It is now known that secretion and
absorption are the property of different cellular compartments and that
chloride channels play a major role in these functions, often
constituting the rate-limiting step elements in the translocation of ions.
The most numerous family of chloride channel proteins discovered so far
is termed ClC and consists of nine different mammalian members.
Sequence identity between different ClC proteins varies between 30 and
90%, but they share the characteristic of being voltage gated and some
selectivity properties (24). It has been suggested that
the diversity of this channel family might be increased further by
alternative splicing, as demonstrated for the ClC-2 and ClC-6 mRNA,
although the functional consequences of these variations have not been
studied (6, 7, 15). The importance of the ClC chloride
channel family is highlighted by the association of certain human
inheritable diseases with mutation of several of its members. ClC-1
mutations are responsible for myotonia, whereas altered ClC-Kb and
ClC-5 account for two renal diseases, a form of Bartter salt wasting
disease and Dent's proteinuria and hypercalciuria (27, 28,
34).
Recent work has also revealed another family of chloride channels
apparently activated by intracellular calcium through a calmodulin
kinase II-dependent mechanism (17). This family, termed
CLCA or alternatively CaCC, has a high tissue specificity. For example,
human CLCA1 appears specific for intestine, whereas CLCA2 is found
exclusively in lung, trachea, and mammary gland (20, 21).
The function of these channels is unknown, but it is speculated that
they support chloride-linked fluid secretion in epithelia. Evidence has
also been presented for the presence of cystic fibrosis
transmembrane conductance regulator (CFTR), a channel gated by
cAMP-dependent phosphorylation and present in the crypt region of
the intestinal epithelium (38).
One of the ClC family members for which channel function has been
clearly demonstrated is ClC-2. This channel, widely expressed in
mammalian tissues, shows low activity under resting conditions but
opens slowly on hyperpolarization (37). When expressed in amphibian oocytes, ClC-2 can be activated by hypotonic cell swelling, suggesting that it might mediate regulatory volume adjustments. An
important observation regarding the gating mechanism of ClC-2 came from
work suggesting that the NH2 terminus of this channel behaves as an inactivating region (22). Deletion
experiments demonstrated that ClC-2 channels lacking a cytoplasmic
NH2-terminal domain became constitutively active and
independent of cell swelling or hyperpolarization. It is of great
potential interest to understand the possible physiological role of
ClC-2 and particularly the function of the NH2-terminal
domain in determining the contribution of this channel to the membrane
conductance in epithelial and other cells.
In the present work, an intestinal epithelium cDNA library derived from
distal small intestinal crypts has been screened, and two clones have
been identified with an intestinal guinea pig ClC-2 probe. These
transcripts differed in a predicted 10-amino acid deletion, suggesting
alternative splice variants of this chloride channel protein. The
region of intestinal guinea pig ClC-2 containing the putative
alternative splicing site is studied and the existence of the
transcript types confirmed. Examination of the corresponding genomic
sequence is consistent with the existence of three exons and two
introns in the region, with an internal acceptor site in one exon
accounting for the deletion. Potential functional differences between
the splice variants were studied by heterologous expression and
patch-clamp analysis. The only difference detected was on the rate of
deactivation of channels previously activated by voltage. This occurred
at a markedly faster rate in the deletion-carrying variant. The
presence of this splice variant of ClC-2 modified in its
NH2-terminal domain could alter the physiological effect of
these channels in the tissues where their relative expression is different.
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MATERIALS AND METHODS |
Cell and tissue isolation.
Male guinea pigs obtained from the Instituto de Salud Pública
(Santiago, Chile) and weighing 250-400 g were used throughout. Intestinal crypts and villi were isolated by a modification of previously published methods (2, 40). Animals were
deprived of food for 24 h, with free access to water. Guinea pigs
were killed by cervical dislocation. This procedure was carried out by
trained personnel under supervision and was approved by the local
Animal Bioethics Committee. A segment of small intestine, either of
proximal (jejunum) or distal (ileum) origin and ~15 cm in length, was
rinsed with ice-cold phosphate-buffered saline and everted over a
plastic rod. After being filled with a calcium-free solution
(containing in mM: 127 NaCl, 5 KCl, 1 MgCl2, 5 D-glucose, 5 sodium pyruvate, 5 EDTA, 10 HEPES, 1% bovine
serum albumin, pH 7.4), the segment was tied at both ends under
moderate pressure and incubated in the calcium-free solution at 37°C
for two 10-min periods. Fragments of epithelium were released by
mechanical disruption. Villi and crypts were identified and isolated
under a binocular microscope. After the epithelium fractions were
isolated, they were washed in calcium-containing medium (same solution
as described above but without EDTA and containing 1.25 mM
CaCl2). All these procedures were carried out at 4°C.
Colonic epithelium was isolated by scraping with a microscope slide and
was flash frozen in liquid N2. Human jejunum epithelium was
isolated by the same method from surgical specimens removed as
treatment for a gastrointestinal disease at the Universidad de Chile
Clinical Hospital. Tissues from other guinea pig organs were excised,
flash frozen in liquid N2, and stored at
80°C until used.
cDNA library construction.
A cDNA library from crypt epithelium of guinea pig small intestinal
tissue was constructed using the Lambda ZipLox system (Life
Technologies). The library was screened with a guinea pig ClC-2 small
intestinal probe obtained using PCR and degenerated primers from a
conserved amino acid region of members of the ClC family. 3' Rapid
amplification of cDNA ends (3' RACE; Marathon cDNA Amplification kit,
Clontech) was performed to obtain the full-length sequences.
RNA and cDNA preparation.
Total RNA was prepared on average from 80 crypts or 60 villi. The cells
were broken in the presence of guanidinium isothiocyanate and
-mercaptoethanol by homogenization with a Teflon/glass homogenizer. RNA was isolated on a silica gel membrane column (RNeasy kit, Qiagen)
according to the manufacturer's recommendations. A similar approach
was followed to isolate RNA from the human and guinea pig tissues. RNA
concentrations and purity were estimated by ultraviolet spectrophotometry, and RNA integrity was assessed by denaturing agarose
gel electrophoresis. cDNA was prepared from 2-3 µg of total RNA
obtained from crypt or villus preparations or from the other tissues
using the SuperScript system (GIBCO BRL kit), using the oligo(dT)
primer in the presence of RNase inhibitors (RNasin, Promega). RNA from
intestinal epithelial cell preparations was pretreated with DNase
(1).
Genomic DNA preparation and amplification.
Crypts and villi were prepared as a single suspension that was lysed
with Nonidet-40 to isolate nuclei. These were washed, and, after
homogenization with SDS, DNA was isolated and amplified by a
modification of a method used elsewhere for white cell DNA (36).
PCR procedures.
The PCR amplification procedures were carried out using a Perkin Elmer
GeneAmp 2400 thermal cycler. Primers used in RT-PCR experiments are
given in Fig. 2. Standard reaction mixture contained aliquots of cDNA
or genomic DNA (120 ng), 0.2 µM of each primer, 2.5 units
Taq DNA polymerase (GIBCO BRL), 100 µM dNTPs, and 1.5 mM
MgCl2 in a total volume of 50 µl. Conditions were similar
for all primers: initial denaturation at 95°C for 2 min, 30 cycles at
95°C for 30 s, annealing at 58°C for 45 s and extension
at 72°C for 1 min, and final extension at 72°C during 10 min.
For cloning and sequencing, PCR products were resolved by agarose gel
electrophoresis and ethidium bromide staining, and the relevant bands
were excised and extracted for DNA that was cloned into pBluescript
modified to carry out T-A cloning (1). Sequencing was
performed manually by the chain termination method using T7 Sequenase
(Amersham Life Science) or by automatic sequencing.
Site-directed mutagenesis.
Introduction of the point mutations was performed by sequential PCR
steps using Pfu DNA polymerase (Stratagene)
(1). The first PCR step was performed in different tubes
using primers in opposite directions containing the nucleotide changes
necessary to transform the serine-78 into glutamic acid (or glutamine)
and sense and antisense primers designed from the gpClC-2 sequence flanking the region of the mutation. The amplified fragments were then
placed in the same tube and amplified in a second PCR step using the
flanking primers only. The full-length fragment generated was digested
with Sac II and Blp I and subcloned into the
appropriately cut gpClC-2/pCR3.1 (see below). The mutations
were verified by sequencing.
Transient transfections and electrophysiological studies.
The gpClC-2 plasmid used in the electrophysiological studies was
obtained by ligation of a cDNA library clone and the 3' RACE-obtained fragment. This was further subcloned in an expression vector under the
control of the cytomegalovirus promoter (pCR3.1, Invitrogen). For the purpose of expression, a gpClC-2
77-86 was created by introducing a restriction fragment containing the deletion into the
former construct. HEK-293 cells used for transfections were grown in
DMEM/F-12 supplemented with 10% fetal bovine serum at 37°C in a 5%
CO2 humidified incubator. At 60-80% confluence the cells were cotransfected with 1.5 µg of total expression plasmids for
gpClC-2 or gpClC-2
77-86 and
H3-CD8 (kindly provided by Dr. Brian Seed, Massachusetts General Hospital, Boston, MA) in a 3:1 ratio
using Lipofectamine Plus (Life Technologies). Expression of CD-8
antigen was used as a means to identify effectively transfected cells
within the dish (26). After 24-48 h the cells were
incubated briefly with microspheres coated with an antibody against CD8 antigen (Dynabeads). The experiments were performed in bead-decorated cells at room temperature in 35-mm-diameter plastic petri dishes mounted directly on the stage of an inverted microscope. The bath solution contained (in mM) 140 NaCl, 2 CaCl2, 1 MgCl2, 10 sucrose, and 10 HEPES pH 7.4. The pipette
solution (35 mM chloride) contained (in mM) 100 sodium gluconate, 33 CsCl, 1 MgCl2, 1 Na2ATP, 2 EGTA, and 10 HEPES
pH 7.4. Alternatively, a 60 mM chloride solution was made by equimolar
replacement of gluconate.
Standard whole cell patch-clamp recordings were performed as described
elsewhere (12) using an EPC-7 (List Medical, Darmstadt, Germany) amplifier. The bath was grounded via an agar bridge. Patch-clamp pipettes were made from thin borosilicate (hard) glass capillary tubing with an outside diameter of 1.5 or 1.7 mm (Clark Electromedical, Reading, UK) using a BB-CH puller (Mecanex, Geneva, Switzerland). The pipettes had a resistance of 3-5 M
. Voltage and current signals from the amplifier were recorded on a digital tape
recorder (DTR-1204; Biologic, France) and digitized using a computer
equipped with a Digidata 1200 (Axon Instruments) AD/DA interface. The
voltage pulse generator and analysis programs were from Axon
Instruments. Unless otherwise stated, when giving trains of pulses, an
interval of 60 or 80 s between pulses was left at the holding
potential to allow for complete current deactivation.
Time courses for current activation and deactivation were fit to double
exponential plus a constant term equation of the form i(t) = a1exp(
t/
1) + a2exp(
t/
2) + c, where a1,
a2, and c are current amplitudes and
1 and
2 are the corresponding time
constants. The fractional amplitudes A1,
A2, and C were obtained by dividing a1, a2, and c
by the total current.
The conductance as function of voltage was adjusted to a Boltzmann
distribution of the form: G = G0 + Gmax/{1 + exp[(V
V0.5)/k]}, where G,
G0, and Gmax are
conductance as a function of voltage, residual conductance independent
of voltage, and maximal conductance at full activation (extrapolated),
respectively. V0.5 is the voltage at which 50%
activation occurs, and k is the slope factor.
Significance of differences between means was determined by unpaired
t-test.
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RESULTS |
Presence of ClC-2 splice variants in intestinal epithelium.
To explore the presence of ClC-2 transcripts in the guinea pig
intestinal epithelium, we screened a cDNA library derived from distal
small intestinal crypts. A 1,667-bp clone comprising a 5'-untranscribed
region and a partial open reading frame (ORF) reaching membrane segment
D11 in translation (37) was obtained from the library. The
remaining sequence of the ORF as well as the 3'-untranslated sequence
was acquired by RACE-PCR performed on distal small intestinal crypt
mRNA, yielding a final sequence of 3,212 bp (GenBank accession no.
AF113529). This was consistent with the size of the corresponding
transcript observed by Northern analysis (not shown). The deduced
translation of gpClC-2 predicted a 902-amino acid protein having 93.7, 92.6, and 92.5% identity with the respective human, rat, and mouse
ClC-2. A second partial 1,395-bp [nucleotide (nt) 243 to nt 1670]
clone was also identified (AF113530) showing identity with a
corresponding stretch of the gpClC-2 transcript except for a 30-nt
deletion. RT-PCR on distal small intestinal crypt and villus mRNA using
primers within the untranslated 5'- and 3'-regions of the gpClC-2
message confirmed that this deletion-carrying transcript was present as
a full-length ORF.
To confirm further the presence of the two variants of gpClC-2 in
intestinal tissue, a RT-PCR strategy was used to amplify the putative
region of this channel that might carry the deletion in isolated
epithelial cells. The rationale of the approach is described
schematically in Fig. 1. The
primers described as P1 and P2 are designed to
flank the deletion region and are therefore expected to generate two
PCR products of differing lengths. The products of the RT-PCR assay are
shown in Fig. 2A. One fragment detected had an electrophoretical migration consistent with the expected size of 285 nt. A second smaller band was also visible in the
gel corresponding to a smaller amplicon of ~250 nt. The fragments
were present both in crypt and villus preparations from proximal and
distal small intestinal epithelium. The amount of each amplicon was not
quantitated, but, because they result from amplification with the same
primers, it could be assumed that the abundance of the larger one is
greater than that of the short form in all the cellular locations
examined.

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Fig. 1.
Rationale for the design of primers to study splice variants of
ClC-2. The heavy lines represent the ClC-2 transcripts found in guinea
pig tissue with their bp numbers as counted from the initiation codon.
The box represents the 30-bp zone deleted in the short variant of the
transcript. P1, P3, and P4 are the sense primers.
P2 is the antisense primer. The sizes of the expected
fragments are 285 and 255 (P1/P2), 230 (P3/P2) and 215 (P4/P2).
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Fig. 2.
A: agarose gel electrophoresis of PCR products
amplified with P1 (sense) and P2 (antisense)
primers using either villus (V) or crypt (C) cDNA prepared from
proximal (P) or distal (D) small intestinal epithelium. Water
corresponds to amplification without DNA template, and RT( )
corresponds to a reaction without reverse transcriptase. Two lanes with
the 300- (arrow) and 200-bp molecular weight markers are also shown
(M). B and C: agarose gel electrophoresis of PCR
products amplified from cDNA of proximal villus cells or from genomic
DNA. B: cDNA was amplified with variant-specific primers.
Lanes marked P3/P2 and P4/P2 correspond to
reactions carried out with the respective sense/antisense primers. Two
lanes with the 100 bp molecular weight markers are also shown (M, arrow
at 600 bp). C: genomic DNA was used as a template and PCR
performed with primers P1 and P2. M, 100-bp
molecular weight marker ladder. Water indicates a control reaction
without template. Arrow points to the 745-bp migration level.
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The fragments, termed CR1 and CR5 for the long and short amplicons,
respectively, were subcloned and sequenced. The nucleotide sequences
shown in Fig. 3 show identity, except for
a 30-nt deletion in CR5 affecting a region 44 nt downstream from primer
P1. To check the authenticity of the two amplicons detected,
new primers were designed that, should the variants be genuine, ought
to lead to the amplification of single bands in RT-PCR assays. The
rationale in the design of these primers, termed P3 and
P4, is depicted in the scheme of Fig. 1. P3 was
designed to encompass a portion of the deleted fragment and,
consequently, when used as sense primer in conjunction with
P2, should amplify specifically a region of the undeleted
splice variant only. P4 was designed to take sequences
flanking the deletion and, therefore, used in a separate PCR run with
P2 as antisense primer, should generate a single 215-bp
product corresponding to the deletion-carrying variant. Figure
2B shows the RT-PCR results of assays using primers
P3 and P2 or P4 and P2
(sense and antisense primers) in separate reactions. These gave single
bands consistent with the 230- and 215-nt expected products. Similar
RT-PCR experiments (not shown) were conducted, and products were
determined with better resolution in a 10% polyacrylamide
electrophoresis gel. Single bands were obtained confirming the data in
Fig. 2B. Primers for glyceraldehyde-3-phosphate dehydrogenase (based on the GenBank accession no. U51572) included in
the same reaction gave bands (~300 bp) of similar intensity in all
the runs, suggesting efficient amplification in every reaction. Subcloning and sequencing confirmed the expectations of the primer design strategy (Fig. 3).

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Fig. 3.
Nucleotide sequence alignment of amplicons CR1 and CR5. Primers
used to obtain CR1 and CR5 are labeled P1 (sense) and
P2 (antisense) and are shown in bold. P3 and
P4 are sense primers used in conjunction with P2
to obtain amplicons specific of the 2 variants. Amplicon labeled hCR5
was obtained using human RNA isolated from jejunum epithelium and is
aligned with the corresponding cDNA sequence of human ClC-2 (hCR1)
obtained from the published sequence (7).
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To check whether the second, deleted amplicon was present in human
tissue, RNA from human jejunum was used. RT-PCR was performed with
human specific primers hP4 and hP2 derived from
the known human sequence (8) and homologous to guinea pig
P4 and P2. This gave a single product that was
sequenced with the result shown in Fig. 3, bottom. The
amplicon, termed hCR5, when compared with the published sequence
(equivalent fragment termed hCR1), shows a 30-nt deletion that is
analogous to that seen in guinea pig.
To ascertain whether the sequence variations observed could be due to
alternative splicing, an examination of the genomic guinea pig sequence
was done. PCR of genomic DNA using primers P1 and
P2 generated a 744-nt fragment as shown in Fig.
2C. The fragment was subcloned and sequenced with the
results reported in Fig. 4 together with
the sequences of CR1 and CR5 for comparison. Examination of the
sequence suggests the presence of two introns (termed
Ix and Ix+1,
sequence given in lowercase) and three exons (termed
Ex, Ex+1, Ex+2 and given in uppercase; exons
Ex and Ex+2
could be truncated). Both introns Ix and
Ix+1 have the consensus donor and
acceptor sites GTA (and GTG) and CAG at the intron/exon borders. In
addition, an extra acceptor site (TAG) is found at a distance of 27 nt
from the
Ix/Ex+1
border. This suggests that alternative splicing gives rise to the CR1
and CR5 amplicons in the form described by the scheme in Fig.
5.

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Fig. 4.
Nucleotide sequence of genomic DNA corresponding to the fragment
amplified with P1 and P2. Introns are shown in
lowercase and exons in uppercase. Amplicons CR1 and CR5 are shown
aligned with the genomic fragment. Acceptor and donor splice sites are
shown in bold. The deletion occurring in CR5 is underlined, and the
internal donor site (TAG) is shown in bold.
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Fig. 5.
Diagram showing a possible mechanism for alternative splicing
giving rise to variants CR1 and CR5. The structural relationship
between the two RT-PCR fragments and the genomic PCR fragment are
indicated. Donor and acceptor consensus sites are shown. E, exon;
I, intron; G, guanine; U, uracil; A, adenine; R, adenine or guanine; Y,
cytosine or uracil.
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To search for expression of the splice variants in different guinea pig
tissues, the specific sense primers P3 and P4
were used in separate RT-PCR assays in conjunction with the antisense primer P2. Figure 6 is an
array of agarose gels showing the presence of both types of amplicons
(termed P3 and P4) in heart, brain, skeletal muscle, liver, spleen,
lung, kidney, adrenal gland, testis, and epithelium from proximal and
distal colon. In separate experiments (not shown) all these tissues
gave detectable amplification products in RT-PCR experiments with
primers for glyceraldehyde-3-phosphate dehydrogenase. It can be seen
that both sets of primers amplified products of the expected size in
all tissues examined. The smaller (P4) fragments were in general more
faintly stained than the larger (P3) fragments. In liver there was
barely detectable presence of either amplicon. Similar low levels were
observed in skeletal muscle, particularly for P4. Brain tissue
presented a strong amplification, but the P4 amplicon was poorly
represented. The strongest amplification was seen in testis, with the
products probably being at saturation. Kidney tissue gave apparently
similar amounts for both amplicons, whereas, in proximal colon cells
and spleen, amplicon P4 appeared better represented. These observations
will have to await confirmation in quantitative assays.

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Fig. 6.
Occurrence of putative splice variants in different
guinea pig tissues. Variant-specific primers were used (a,
P3/P2; b, P4/P2). Tissues were as follows: 1, heart; 2, brain; 3, skeletal muscle; 4, liver; 5, spleen; 6, lung; 7, kidney; 8; adrenal gland; 9, testis; 10, proximal colon; 11, distal
colon. The molecular weight markers shown correspond to 200 and 300 bp.
Other labels as in legend to Fig. 2.
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Figure 7 gives the predicted amino acid
sequence for CR1 and CR5. The deletion in CR5 determines a
corresponding deletion of 10 amino acids and the change M76I at the
beginning of the deletion without alteration of the reading frame. The
deletion causes the disappearance of three positively charged amino
acids (R80, H82, K83). In addition, with the disappearance of S78, a protein kinase C (PKC) phosphorylation consensus site
(S/T-X-R/K) is deleted in the CR5 form. The equivalent sequence in hCR5
is also shown in Fig. 7. The deleted portion has the same amino acid sequence as in the guinea pig. This also leads to the disappearance of
three positive charges and a PKC phosphorylation site. Unlike in CR5,
there is an S75C change at the beginning of the deletion, but the new
residue is not within a PKC phosphorylation consensus site.

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Fig. 7.
Deduced amino acid sequence for the putative splice variants
corresponding to the CR1 and CR5 amplicons. The
NH2-terminal portion of the sequence is shown down to the
first transmembrane segment. That labeled CR1 corresponds to the guinea
pig full ClC-2 cDNA sequence. Sequence labeled CR5 corresponds to the
same sequence assuming the only change is the 10-amino acid deletion
and the emergence of a novel M. Asterisk indicates an S residue within
a consensus protein kinase C phosphorylation site. Bottom:
deduced amino acid sequences corresponding to hCR1 and hCR5.
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Heterologous expression of ClC-2 splice variants.
Functional assays of gpClC-2 and gpClC-2
77-86 were conducted by
electrophysiological examination of acutely transfected HEK-293 cells.
Figure 8 shows currents elicited by the
voltage protocol (A) for gpClC-2- (C) and
gpClC-2
77-86-transfected cells (D). As described
before for rat ClC-2, currents were small at positive or moderately
negative potentials but activated slowly with strong hyperpolarization.
Figure 8E shows the current-voltage relations for the
experiments in C and D taken at the end of the
main voltage pulse. There was no difference between the variants.
Similarly, there was no change in apparent voltage dependence of
activation, plotted in F as apparent normalized
conductance [proportional to open probability
(Po)] as a function of voltage. These
could be fitted to Boltzmann distributions (see MATERIALS AND
METHODS) with V0.5 values of
101.1 ± 5.3 and
100.8 ± 5.2 mV and k values of 22.98 ± 1.16 and 21.08 ± 1.09 for gpClC-2 (n = 8) and
gpClC-2
77-86 (n = 9), respectively. These
currents were observed neither in untransfected cells (Fig.
8B) nor in cells transfected with the
H3CD-8 plasmid only
(not shown). The absence of endogenous currents activated by
hyperpolarization in HEK-293 cells has been reported before
(30).

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Fig. 8.
Gating properties of gpClC-2 and gpClC-2 77-86. Currents
(i) in response to the voltage protocol in A are
shown in B-D. B: untransfected HEK-293 cell
(notice longer time scale). C: gpClC-2. D:
gpClC-2 77-86. E: current-voltage relations for the
experiments in C and D ( ,
gpClC-2; , gpClC-2 77-86). Same symbols are used in
F for average values (±SE) of conductance as a function of
voltage for gpClC-2 (n = 8) and gpClC-2 77-86
(n = 9).
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To find out whether the channel gating was affected by alternative
splicing, the time dependence of activation was analyzed by fitting
current activation to two exponentials plus a time-independent value.
Figure 9, top, shows examples
of current traces for both gpClC-2 variants together with the fitted
lines, showing that activation can be adequately described by the
double-exponential model. Figure 9, middle, shows the time
constants obtained. They were both voltage dependent, becoming faster
with hyperpolarization. There were no marked differences between the
sets of data for the two splice variants shown side by side in Fig. 9,
middle. Fractional current amplitudes were largely voltage
independent and not markedly different in the two variants, as shown in
Fig. 9, bottom. These experiments were conducted with a 35 mM chloride intracellular solution, but there was no marked difference
when 60 mM chloride was used, at least at the most hyperpolarized
voltage.

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Fig. 9.
Time course of activation of chloride currents expressing gpClC-2
and gpClC-2 77-86. Top: examples of currents elicited
by square pulses to the voltages indicated superposed to lines of best
fit. Time constants and fractional amplitudes shown in the
middle and bottom were obtained by fitting to a
double exponential plus a constant term model, as explained in the
text. Data were obtained with a 35 mM chloride pipette solution except
for closed symbols that correspond to 60 mM intracellular chloride.
Results are means ± SE; n was 11 for gpClC-2 and 7 for
gpClC-2 77-86.
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As shown in the positive afterpulse to the activation protocol in Fig.
8, deactivation was a slow process. To examine the kinetics of
deactivation of gpClC-2 and gpClC-2
77-86, the voltage protocol
in Fig. 10A was used. An
activating pulse to
160 mV was followed by a 6-s pulse at 40 mV. The
current traces have been normalized to the maximal current obtained for
gpClC-2 at
160 mV. As can be seen in Fig. 10A,
bottom, and with better resolution in the inset,
current deactivation occurred at a markedly faster rate for
gpClC-2
77-86 than for gpClC-2-transfected cells. The decay in
current can be described by a two-exponential fit with the time
constants with values shown in Fig. 10B. The slow component of the decay was markedly faster for gpClC-2
77-86 than for
gpClC-2. The fast component had similar time constants for the two
variants.

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Fig. 10.
Time course of deactivation of gpClC-2 and gpClC-2 77-86.
A: voltage protocol with the respective currents obtained in
cells transfected with gpClC-2 or gpClC-2 77-86. The current for
gpClC-2 has been normalized to the last point during the 160- mV
portion of the trace for gpClC-2. Inset: deactivation for
the 40-mV portion of the records at higher resolution. B:
time constants for the fit of the deactivation at 40 mV to a
double-exponential model are shown for intracellular solutions
containing 35 or 60 mM Cl . Numbers are means ± SE
of 14 and 15 separate experiments for gpClC-2 and gpClC-2 77-86,
respectively, for the 35 mM chloride. Equivalent numbers for
replications at 60 mM chloride were 6 for each variant. Differences
between 1 values for the 2 splice variants were
significant at the 1% level.
|
|
To check whether the disappearance of a PKC phosphorylation consensus
site at S78 might be responsible for the altered behavior of expressed
gpClC-2
77-86, this residue was replaced by Q, a nonphosphorylatable residue, or by E to simulate phosphorylation. Expression of either mutant gave rise to sizeable currents with similar
characteristics as observed with gpClC-2. Currents elicited by a
160
mV pulse were
1,825 ± 199 (n = 16),
3,111 ± 612 (n = 7), and
1,251 ± 110 (n = 11) pA for S78E, S78Q, and wild type, respectively. The voltage dependence for the activation of the conductance was also similar in the mutants compared with wild-type channels: V0.5 for the Boltzmann fit of the
conductance vs. V plot gave values of
110 ± 5 mV
(n = 8) and
117 ± 6 mV (n = 5) for S78E and S78Q, respectively. These values were not significantly different from those for gpClC-2 or gpClC-2
77-86. To ascertain whether these mutations affected the opening or closing mechanism, the
kinetics of activation or deactivation of the channel were examined. As
can be seen from the data in Table
1, neither mutation had any
effect on the time constants for activation by a pulse to
160 mV.
Similarly, deactivation at 40 mV after a
160 mV conditioning pulse
occurred with similar kinetic constants for the two mutants as for
wild-type gpClC-2-generated currents.
Finally, Fig. 11 shows the current
responses of gpClC-2 and gpClC-2
77-86 to 12-s square
75-mV
voltage pulses from a holding potential of
25 mV. The interval
between pulses was 7 s. This protocol was intended to simulate
oscillations of membrane potential that might arise in response to
agonist-induced intracellular calcium oscillations in colonic
epithelial cells (9, 11). It can be seen in Fig. 11 that,
at each voltage pulse, inward current was observed for cells expressing
both variants of the channel. The kinetics of current development,
however, differed for second and third pulses for gpClC-2-transfected
cells, with large instantaneous current. This contrasts with the
behavior of the gpClC-2
77-86 variant where successive voltage
pulses led to currents of similar kinetics. When the current evoked
within the first 2 s was integrated, it was seen that, for
gpClC-2, the second and third pulses gave respective increases of 51 and 67% in transported charge with respect to the first one.
Equivalent values for gpClC-2
77-86 were 0.2 and
4.5%.

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|
Fig. 11.
Comparison of the effect of repetitive pulses on
currents elicited through gpClC-2 and gpClC-2 77-86. Cells
transfected with either variant were held at 25 mV, and 12-s square
pulses to 75 mV were given with a 19-s period as shown in the
protocol. Traces show the currents elicited during the main pulse
periods.
|
|
 |
DISCUSSION |
Chloride channels are known to be important in the control of cell
volume as well as in transepithelial ion transport. The function of
ClC-2, a member of the ClC family of chloride channels, is as yet
unknown, although it has been proposed to fulfill a cell volume
regulation role. We identified ClC-2 in a cDNA library from crypt
epithelium of guinea pig small intestinal tissue. Two clones were
isolated, one of which presented a deletion in the NH2-terminal region comprising 30 nt. We used RT-PCR to
demonstrate that these correspond to splice variants, rather than being
the result of a cloning artifact, and are present along the small intestine both in the villus and crypt regions and in proximal and
distal colon. Furthermore, we explored their functional properties by
heterologous expression.
The presence of alternative splice variants within the ClC-family has
been reported for rat ClC-6 and ClC-2 (6, 7, 15). ClC-6
has not yet been proved to function as a channel in heterologous expression experiments, and no physiological consequence of the alternative splicing of ClC-2 has been reported so far. The presence of
an NH2-terminal truncated form of ClC-2 was reported in
rabbit heart, suggesting strongly a variant with open channel
phenotype. This, however, was later demonstrated to be a consequence of
a cloning artifact (18). Our finding of two different
ClC-2 clones in an intestinal epithelium cDNA library could be
interpreted to be the consequence of alternative splicing or could
arise as a consequence of a cloning artifact. To assess these
possibilities, amplicons encompassing the region of the deletion were
obtained with two different strategies. One gave two products differing in size, CR1 and CR5 (see Fig. 2A), and the other led to
specific amplification (Fig. 2B), consistent with the
deletion encountered in the library clones. Analysis of the genomic
sequence corresponding to amplicons CR1 and CR5 gave a structure of
three exons and two introns. An internal donor site occurring within
one of the introns accounts for the deletion in CR5 (Fig. 5) and
strongly supports the idea that the variants of ClC-2 encountered here
originate by alternative splicing.
The alternative mRNA splicing reported here gives rise to functional
changes in the currents they evoke on heterologous expression. ClC-2
has been reported to be activated by hyperpolarization, cell swelling
(when expressed in amphibian oocytes), and low extracellular pH
(22, 25, 33, 37). These gating processes are abolished by
mutation of the NH2-terminal domain and also in a putative cytoplasmic domain. These observations have led to the proposal that
ClC-2 is inactivated by a process akin to the ball-and-chain model,
thought to account for inactivation of potassium and sodium channels
(25). The ball domain in ClC-2, according to these authors, would be within the NH2-terminal region, and the
receptor with which it interacts would be located in the D7-D8 linker
region. Cell swelling, low extracellular pH, and hyperpolarization
would all decrease the affinity of this receptor with the putative ball domain. Although the region deleted in the splice variant described here does not coincide with those previously characterized by mutation,
one could speculate that it might have functional consequences such as
leading to a phenotype where gating is altered to facilitate channel
opening. In fact this does not appear to be the case, because voltage
dependence of conductance (proportional to steady-state Po, Fig. 8F) and the kinetics of
current activation by voltage appear to be unaltered (Fig. 9). Closing
of the channels after activation, as reflected by the time course of
current deactivation, is nevertheless markedly accelerated in
gpClC-2
77-86 (Fig. 10). This effect occurs by a shortening of
the slow time constant of deactivation without altering the fast
component. The gating of ClC-2 has not been elucidated to the degree
that it has been unraveled for ClC-0 and ClC-1 (24). The
slow component of deactivation could be the consequence of the ball
domain returning the channel to the closed state. The deletion would
therefore be affecting this part of the gating of ClC-2 corresponding
to the ball and chain mechanism, perhaps simply owing to a shortening
of the tether in the alternatively spliced product.
ClC-2 has also been cloned from the human colonic T84 cell line
(8) that is capable of organizing in vitro to form a
functional epithelial monolayer. A function for ClC-2 has been proposed
in T84 cells on the basis of characteristics of a
hyperpolarization-activated chloride current inhibited by iodide
(16). These authors propose that ClC-2 could participate
in fluid secretion not associated with CFTR function. ClC-2-like
currents have been reported in pancreatic acinar cells
(4), parotid acinar cells (30), and submandibular duct cells (14), but their function in
transepithelial transport has not been clearly defined. Our
demonstration of ClC-2 transcript in intestinal epithelium also
suggests that a similar transepithelial transport function could be
assigned to this channel for both small intestine and colon. We can
speculate that gpClC-2 is involved in mediating
hyperpolarization-activated secretion when changes in membrane
potential occur in response to secretagogues. A hyperpolarization, as
resulting from the action of, e.g., carbachol, will take membrane
potential from a resting value of around
35 mV to a value of about
80 mV, near the K+ equilibrium potential
through activation of K+ channels
(10). This would result in an activation of the ClC-2 channels from ~5 to 25 of maximal conductance (see Fig.
8F). Modulation of the voltage dependence, as seen for rat
ClC-2 expressed in oocytes after swelling (22) or by
external acidification (25), could further contribute to
enhance such an effect.
It is interesting to mention the analysis of a
hyperpolarization-activated chloride current studied in
Aplysia with similar characteristics to ClC-2-mediated
currents (5). It was noted in that study that this channel
would favor chloride exit and would be enhanced by increased
intracellular chloride. It would therefore "be particularly important
in the case of the apical membrane of chloride-secretory epithelial
cells" (5). We have used two chloride concentrations in
the present work without noticing any effect on ClC-2-mediated
currents. Time constants for voltage-dependent activation and
deactivation were similar at 35 and 60 mM intracellular chloride (see
Figs. 9 and 10), plausible values as measured in intestinal epithelial
cells (see Ref. 19 and references therein). Larger changes in
intracellular chloride have indeed been reported to affect the voltage
dependence of rat ClC-2 expressed in amphibian oocytes
(31), but it remains to be shown whether this holds true
for physiologically significant concentrations.
The deletion in gpClC-2
77-86 eliminates three positively
charged amino acids. The NH2-terminal region of guinea pig
ClC-2 contains 12 negatively charged and 12 positively charged amino acids; therefore, the charge balance is altered in the 92-amino acid
segment up to the first transmembrane domain (see Fig. 7). In addition,
deletion leads to the disappearance of a consensus site for
PKC-dependent phosphorylation and the emergence of a new methionine
without change in the reading frame. There is some evidence that PKC
activation blocks channel activity in dorsal root ganglion neurons
expressing rat ClC-2 (35) as well as the ClC-2-like
currents in T84 cells (16). It has also been suggested that protein kinase A might have an effect on a ClC-2 channel cloned from rabbit stomach (29). We used site-directed
mutagenesis of S78, the consensus PKC-mediated phosphorylation site
within the deletion. Mutant S78E should mimic the negative charge of a
putative phosphorylated state of gpClC-2 at that site (23, 32). Mutant S78Q, on the other hand, should prevent
phosphorylation of the site and was used as a control. Neither of the
mutants reproduced the functional changes observed in
gpClC-2
77-86 because the kinetics of activation and
deactivation remained unchanged, as did the voltage dependence of
conductance, suggesting that the disappearance of this potential
phosphorylation site is not responsible for the functional difference
between the splice variants. A question of charge balance remains to be
explored, but simply, shortening of this putative intracellular region
could account for the functional change.
Various roles for ClC-2 have been suggested. The good evidence for the
activation of ClC-2 expressed in amphibian oocytes by cell swelling
(see Introduction) suggests a function in the regulation of
cell volume. Such a role would be consistent with its wide tissue
distribution. The ion selectivity of ClC-2 and other functional
characteristics, however, do not coincide with those of native
osmosensitive chloride channels (see, e.g., Refs. 13 and 39). In
addition, in T84 cells a possible participation of ClC-2 in regulatory
volume decrease has been considered unlikely (3). In
certain neurons it is postulated that the presence of ClC-2 would
prevent chloride accumulation such that it would alter the effect of
certain neurotransmitters associated with anion conductance
(35). We cannot foresee how the differential expression of
the variants could affect the putative functions described above.
The function of ClC-2 in the transepithelial transport properties of
the small and large intestine has not been studied. Its presence in all
areas along the intestine, as well as in the crypt and villus regions
might be related to specific epithelial transport functions. The fact
that current deactivation of ClC-2 is markedly accelerated in the
77-86 variant would make the activity of this channel more
transient than that of the nondeleted variant after hyperpolarization
activation. Secretagogues such as carbachol are known to hyperpolarize
native intestinal cells and also cultured colonic cells such as T84
cells (10, 40). The changes in membrane potential measured
in the cultured cells have been shown to occur in oscillations
with varying periods, which follow similar intracellular calcium
oscillations. If it is speculated that such a signal is to couple to
pulsatile secretion, channels activated by hyperpolarization and
differing in deactivation rate could mediate very different chloride
fluxes depending on the membrane potential oscillation characteristics
(see Fig. 11). We do not know if this occurs in tissues involved in
secretion evoked by calcium-mediated agonists. Further work with native
cells is needed to test this hypothesis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Brian Seed for generously providing the expression
plasmid for the human CD8 lymphocyte surface antigen (
H3-CD8) and
Jorge González for technical assistance.
 |
FOOTNOTES |
This work was supported by grants from Fondecyt (Chile) 1961208 and
1990939 and the Volkswagen Stiftung (Germany). Institutional support to
the Centro de Estudios Científicos (CECS) from a group of
Chilean private companies (AFP Provida, CODELCO, Empresas CMPC, MASISA SA, and Telefónica del Sur), Fuerza Aérea de Chile
and Municipalidad de Las Condes is also acknowledged. F. V. Sepúlveda was supported by an International Research Scholars
grant from the Howard Hughes Medical Institute and a Cátedra
Presidencial en Ciencias. CECS is a Millennium Science Institute.
Address for reprint requests and other correspondence: L. P. Cid, Centro de Estudios Científicos, Avenida Arturo Prat
514, Casilla 1469, Valdivia, Chile (E-mail:
pcid{at}cecs.cl).
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 28 February 2000; accepted in final form 1 May 2000.
 |
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