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Am J Physiol Cell Physiol 279: C1198-C1210, 2000;
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Vol. 279, Issue 4, C1198-C1210, October 2000

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-2Delta 77-86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Delta 77-86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Delta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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-2Delta 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-2Delta 77-86 and pi 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 MOmega . 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/tau 1) + a2exp(-t/tau 2) + c, where a1, a2, and c are current amplitudes and tau 1 and tau 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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).

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.

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.

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.

Heterologous expression of ClC-2 splice variants. Functional assays of gpClC-2 and gpClC-2Delta 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-2Delta 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-2Delta 77-86 (n = 9), respectively. These currents were observed neither in untransfected cells (Fig. 8B) nor in cells transfected with the pi 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-2Delta 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-2Delta 77-86. E: current-voltage relations for the experiments in C and D (open circle , gpClC-2; triangle , gpClC-2Delta 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-2Delta 77-86 (n = 9).

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-2Delta 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-2Delta 77-86.

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-2Delta 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-2Delta 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-2Delta 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-2Delta 77-86. A: voltage protocol with the respective currents obtained in cells transfected with gpClC-2 or gpClC-2Delta 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-2Delta 77-86, respectively, for the 35 mM chloride. Equivalent numbers for replications at 60 mM chloride were 6 for each variant. Differences between tau 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-2Delta 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-2Delta 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.

                              
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  		Table 1.   Effect of mutations on S78 on the kinetics of activation and deactivation of gpClC-2-mediated currents

Finally, Fig. 11 shows the current responses of gpClC-2 and gpClC-2Delta 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-2Delta 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-2Delta 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-2Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-2Delta 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-2Delta 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-2Delta 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 Delta 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 (pi 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.


    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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