©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a New Outwardly Rectifying Cl Channel That Belongs to a Subfamily of the ClC Cl Channels (*)

(Received for publication, October 17, 1995; and in revised form, February 6, 1996)

Hisato Sakamoto (§) Masanobu Kawasaki Shinichi Uchida Sei Sasaki Fumiaki Marumo

From the Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A new outwardly rectifying Cl channels (ORCC) that belongs to ClC Cl channel family has been identified from rat kidney and designated as ClC-5. ClC-5 cDNA encodes a polypeptide of 746 amino acids, which is indicated by hydrophobicity analysis to have structural features that are common of the ClC family. However, the amino acid sequence was weakly homologous to those of other ClC Cl channels except for ClC-3, which we recently identified as a Ca-sensitive ORCC. Northern blot analysis of rat tissues showed that ClC-5 mRNA was predominantly expressed in the kidney and colon. To characterize the functional properties of ClC-5 by whole cell patch-clamp technique, we established the stably transfected CHO-K1 cell line using intranuclear microinjection technique. The transfected cells induced outwardly rectifying and 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid-sensitive Cl currents on whole cell configuration. Following the identification of two highly homologous ORCCs, ClC-3 and ClC-5, a new subfamily encoding ORCC has emerged in the ClC family. Furthermore, ClC-5 was almost identical to a partial sequence of human cDNA that is related to Dent's disease. The molecular structure and functional properties of ClC-5 will provide an important insight into ORCCs and the pathogenesis of Dent's disease.


INTRODUCTION

Many physiological studies have demonstrated the presence of outwardly rectifying Cl channels (ORCCs) (^1)in a variety of cells(1, 2, 3, 4) . Single channel recordings by patch-clamp technique have shown that they have strong outward rectification in a positive membrane voltage and that their conductance is about 40 picosiemens(1, 2, 3, 4) . Recently, ORCC has attracted special interest in relation to cystic fibrosis transmembrane conductance regulator (CFTR)(5, 6, 7, 8, 9) . Patch-clamp studies in the epithelial cells of patients with cystic fibrosis have shown that cyclic AMP-dependent ORCC is not properly regulated in these patients(5, 6, 7, 8, 9) , indicating the possible importance of ORCC in the pathogenesis of cystic fibrosis. When the CFTR gene was cloned in 1989(10, 11) , it soon became clear that CFTR encodes a small linear Cl channel of 10 picosiemens and not ORCC(12) . This discrepancy has been puzzling investigators, but recent new findings that CFTR acts as an ATP-permeating channel may solve this problem. Schwiebert et al.(13) have shown that CFTR is able to permeate ATP in addition to Cl and that ATP transported from inside to outside of the cell in turn activates the purinergic receptors on the cell surface. Activation of the purinergic receptor then stimulates ORCC. On the basis of these findings, subsequent attention has been focused on the molecular structure of ORCC.

Recently, we have cloned and characterized a Ca-sensitive ORCC, ClC-3, an intriguing member of the ClC family(14, 15) . Only 20-24% of the amino acid sequence encoded by ClC-3 is identical to those of other cloned ClC Cl channels, i.e. ClC-0, -1, -2, -K1, and -K2(16, 17, 18, 19, 20) . These findings led us to the hypothesis that ClC-3 may belong to a new subfamily encoding ORCCs in the ClC family. In the present study, we examined whether a new member of ORCC exists in the ClC family using a homology-based cloning strategy. Here, we report a new cDNA clone encoding an ORCC, ClC-5, isolated from rat kidney. ClC-5 is highly homologous to ClC-3 and ClC-4 and almost identical to a partial human cDNA, which is recently reported as a strong candidate for Dent's disease(21) . We further established the stably transfected mammalian cells and characterized its channel properties by the patch-clamp technique.


EXPERIMENTAL PROCEDURES

Methodology

Reverse Transcription PCR

First, we made degenerate PCR primers that corresponded to the second and third membrane spanning domains of ClC-3: sense strand, CCGGATCCGGNATHCCNGARHTNAARAC and antisense strand, CCGAATTCRTGNACNARNGGNCCYTCYTT (where N = A/C/G/T; H = A/C/T; R = A/G; Y = C/T). Glomeruli microdissected by conventional technique (22) were reverse-transcribed using avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) at 42 °C for 60 min and then heated at 94 °C for 5 min. The synthesized cDNA was used for subsequent PCR in the following profile: 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, 35 cycles. The PCR product was cut with EcoRI and BamHI on both ends, ligated into EcoRI- and BamHI-cut pSPORT1 (Life Technologies, Inc.), and then sequenced. One clone, pH17, had about 74% nucleotide sequence identity with that of the corresponding region of ClC-3.

Isolation of ClC-5 cDNA Clone and DNA Sequencing

An oligo(dT)-primed directional rat kidney cDNA library in gt22A (19) was screened under high stringency (6 times saline/sodium/phosphate/EDTA (SSPE), 50% formamide, 5 times Denhardt's solution, 1% SDS, 100 µg/ml salmon sperm DNA) at 42 °C with a 160-bp PCR clone (pH17) labeled with [alpha-P]dCTP (3000 Ci/mmol, Amersham) (2.5 times 10^9 cpm/µg). The screening yielded four positive clones from 3 times 10^5 plaques. The clone pH1 was cut with NotI and SalI, and a 2.5-kb insert was subcloned into NotI- and SalI-cut pSPORT1 and designated as ClC-5. To sequence the isolated cDNA, nested deletion clones were prepared using the Erase-A-Base system (Promega) and sequenced by T(7) DNA polymerase by the chain termination method or the dideoxy chain termination method using fluorescence labeled primers on an automated sequencer (model 373A, Applied Biosystem, Inc., Foster City, CA). The antisense strand was sequenced using synthetic primers.

Northern Blots

Total RNA was extracted from various rat tissues(23) . Total RNA (20 µg) of each sample was electrophoresed in 1.1% agarose gel containing formaldehyde. Equal loading was additionally checked with ethidium bromide staining. After transfer to nylon membranes, RNA was cross-linked to the membrane by ultraviolet light. Subsequently, the blot was prehybridized in a solution containing 6 times SSPE, 5 times Denhard's solution, 50% formamide, 1% SDS, and 100 µg/ml of denatured salmon sperm DNA at 42 °C and hybridized overnight with 0.8 times 10^9 cpm/µg of a full-length ClC-5 cDNA probe labeled with [alpha-P]dCTP by random priming (Promega). After hybridization, the membrane was washed twice in 2 times SSC, 0.1% SDS at room temperature and once with 0.1 times SSC, 0.1% SDS at 50 °C for 20 min.

Expression of the ClC-5 cDNA in CHO-K1 Cells

Cell Culture

CHO-K1 (JCRB9018) cells were obtained from the Japanese Cancer Research Bank (JCRB)-Cell. Cells were grown in Ham's F-12 medium with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) and incubated in 5% CO(2), 95% air at 100% relative humidity and 37 °C. CHO-K1 cells were cultured 72-96 h prior to microinjection on a 60-mm film-coated dish covered with soft removable culture surface (SR dish, Sumitomo Bakelite Co., Ltd, Tokyo, Japan).

Microinjection of Recombinant Plasmid

The cloned 2.5-kb cDNA of ClC-5 with both the 5`- and 3`-untranslated regions (210 and 117 bp, respectively) was ligated into the cloning site of the mammalian expression vector, pMAM-neo (Clontech), by which expression is under the control of a dexamethasone inducer. CHO-K1 cells were transfected with either pMAM-neo (no insert) or pMAM-ClC5-neo plasmids by intranuclear microinjection using an Eppendorf transjector 5246 and micromanipulator 5171 attached to a Zeiss inverted phase-contrast microscope. CHO-K1 cells were microinjected with approximately 10 fl of solution containing nonlinearized covalently closed circular plasmid into the nucleus. After microinjection, the cells were washed with phosphate-buffered saline, incubated overnight with regular Ham's F-12 medium, and then incubated in Ham's F-12 medium containing G418 (707 µg/ml). When colonies surviving in the G418 treatment grew to 100-200 cells, they were isolated from the dish by cutting the film to which the cells were attached under microscope. Transferred colonies were treated with 0.25% trypsin and EDTA for a few minutes, and the suspended cells were cultured and split after reaching 70-80% confluence.

Northern Blot Analysis of the Transfected Cells

After subselection of the cells resistant to G418 at a concentration of 707 µg/ml, the expression of the transfected ClC-5 cDNA was evaluated by Northern blot analysis. Total RNA was isolated from about 10^7 cells of each G418-resistant clones. To compare the induction of ClC-5 mRNA, each clone was treated with or without 5 mM dexamethasone (DEX) for 24 h. Subsequently, total RNA (20 µg) of each clone was electrophoresed and blotted. The conditions of prehybridization, hybridization, wash, and autoradiography were the same as above.

Electrophysiological Characterization

For whole cell recording, pipettes with a tip resistance of 1-3 M were pulled from hematocrit tubes (Terumo, Tokyo). Currents were measured using a patch-clamp amplifier, EPC-7 (List-Electronic, Darmstadt, Germany), and filtered at 1 kHz. The pCLAMP software (version 6.0, Axon Instruments) was used to generate command potentials and to collect data. The bath solution contained (in mM) 130 mM tetraethylammmonium chloride (TEA-Cl), 1 mM CaCl(2), 1 mM MgCl(2), 1 mM EGTA, and 10 mM PIPES (pH 7.4). In the experiments studying Cl selectivity of the current, 120 mM sodium gluconate replaced 130 mM of TEA-Cl in the bath solution. The electrode solution contained (in mM) 130 mM potassium gluconate, 20 mM KCl, 1 mM EGTA, 10 mM PIPES, and 100 µg/ml pore-forming nystatin (stock solution, 25 mg/ml in dimethyl sulfoxide) (pH 7.20)(24) .

In the study of anion selectivity, the relative anion permeabilities were determined on the basis of the shift of reversal potential after the half-replacement of Cl with other anions using inside-out patch technique (n = 5). The pipette solution contained 120 mM NaCl. The bath solution for fluorine was 60 mM NaCl and 60 mM NaF; for iodine, 60 mM NaCl and 60 mM NaI. In the study where the effect of cAMP on whole cell Cl currents was examined, voltage was applied by step pulse from -30 (100 ms) to 0 (2 s) to +30 mV (100 ms) using a computer software (CLAMPEX, Axon Instruments).

Chemicals were obtained from Sigma unless otherwise noted. All experiments were performed at room temperature (20-25 °C). Data are expressed as means ± S.E. of n observations (where n is the number of individual cells used).


RESULTS

Primary Structure of ClC-5

PCR cloning strategy with rat microdissected glomeruli as a template was adopted to isolate a new ORCC that is homologous to ClC-3 and is predominantly expressed in the kidney. From the PCR products we subcloned and sequenced 32 clones. Sequencing revealed the existence of a PCR clone (pH 17) that was highly homologous to ClC-3 (74% nucleotide identity). Using this PCR clone as a probe, a cDNA clone with a 2568-bp insert designated as ClC-5 was isolated from rat kidney cDNA library. The nucleotide sequence surrounding the translation initiation codon (5`-GAATCATGG-3`) conforms with the Kozak sequences for translation initiation sites (Fig. 1A) (25, 26) and initiates the longest reading frame. The 5`-untranslated sequence does not contain stop codon. The first stop codon occurs at nucleotide 2449, resulting in an open reading frame of 746-amino acid protein (Fig. 1A). The predicted translation product has a calculated molecular mass of 83,089 daltons. A hydrophobicity analysis of the predicted amino acid sequence by the method of Kyte and Doolittle (27) is shown in Fig. 1C. The hydrophobicity profile shows at least 12 hydrophobic regions, which is similar to those of other members of the ClC Cl channel family(28) . This protein has two potential N-glycosylation sites (amino acid positions at Asn-38 and Asn-408)(29) . There are also consensus sequences for phosphorylation by cAMP-dependent protein kinase and protein kinase C(30, 31) . The consensus cAMP-dependent protein kinase phosphorylation sites are located at position Thr-349 and Thr-350, and sites for protein kinase C are located at position Ser-397, Ser-628, Thr-37, Thr-291, Thr-409, Thr-544, Thr-676, and Thr-724.


Figure 1: Amino acid sequence alignment of ClC-5 with ClC-3 and ClC-4. A, nucleotide and deduced amino acid sequence of ClC-5. B, conserved residues are filled in black blocks, putative transmembrane-spanning domains are underlined according to the original topology by Jentsch et al.(16) , potential N-linked glycosylation sites (28) are indicated by asterisks, and PCR primer sites are double-underlined. Sequence data have been deposited in GenBank(TM)/EMBL data library (accession number D50497). C, hydrophobicity profile of ClC-5. The mean hydrophobicity index was computed according to the algorithm of Kyte and Doolittle with a window of 15 residues(27) .



The protein sequence of ClC-5 is highly homologous to that of rat ClC-3 (77%), to those of rat and human ClC-4 (78%) (32) (Fig. 1B), and to that of a partial human cDNA (99%)(21) . In contrast, the overall amino acid identity of ClC-5 with other members of the ClC family is very low (29% amino acid sequence identity with Torpedo channel, ClC-0; 31% with ClC-1, 33% with ClC-2, 27% with ClC-K1). To establish possible evolutionary relationship among these ClC Cl channels, a phylogenic tree was constructed using DNAsis computer software (Mac version 3.2, Hitachi, Yokohama, Japan) (Fig. 2). According to the phylogenic analysis, the eight members of the ClC Cl channel family can be classified into two subfamilies: the ClC-0/ClC-1/ClC-2/ClC-K1/ClC-K2 chloride channels and the ClC-3/ClC-4/ClC-5 chloride channels.


Figure 2: Evolutionary relationship between the ClC Cl channels. The phylogenic tree shows the relationship between different eight members of the ClC family derived from rat except for ClC-0. The phylogenic tree was constructed using the computer software DNAsis.



Tissue Distribution

In Northern blot analysis of a variety of rat tissues, the full-length ClC-5 cDNA probe hybridized with a band at approximately 9.5 kb, as shown in Fig. 3, A and B. In a Northern blot where total RNAs were electrophoresed, the expression of ClC-5 was detected only in the kidney and colon (Fig. 3A). In contrast, transcripts of the same size were also noted in lower amounts in the heart, brain, lung, and testis, in addition to the kidney on a blot prepared with poly(A) mRNA (Fig. 3B).


Figure 3: Northern blot analysis of ClC-5 expression in different rat tissues. Total RNA (20 µg/lane) (A) and poly(A) RNA (approximately 2 µg/lane) (B) from various rat tissues were loaded in each lane and subsequently hybridized with the full-length ClC-5 cDNA probe. Equal loading of RNA was confirmed by staining of 28 S ribosomal RNA by ethidium bromide (A) or hybridization with beta-actin probe (B). Markers of transcript size (in kilobases) are indicated.



Functional Expression

To characterize the function of ClC-5, we transfected the coding sequence of cloned ClC-5 cDNA into CHO-K1 cells using dexamethasone-inducible mammalian expression vector (pMAM-neo)(33) . Transcription of the insert is under the control of the Rous sarcoma virus promoter and the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. One stably transfected cell clone (J2702) selected by resistance to G418 for 3 months showed the induction of ClC-5 mRNA at 3.6 kb in response to DEX (5 µM for 24 h) (Fig. 4). In contrast, no bands were detected in any of the mock-transfected cells with or without DEX treatment. These findings indicated the isolation of the stably transfected cell line (J2702) expressing ClC-5.


Figure 4: Northern blot analysis of transfected cells. Control indicates that total RNA was isolated from the mock-transfected cells; DEX (+), pretreatment with 5 µM dexamethasone for 24 h; DEX(-), without the pretreatment. Each lane contained 20 µg of total RNA, and equal loading was confirmed by hybridization with beta-actin (bottom).



To examine whether ClC-5 actually acts as a Cl channel, the whole cell patch-clamp technique was applied to J2702 cells. Fig. 5shows a typical trace of the whole cell currents following the changes in holding membrane potential between -75 and +25 mV (A) and their I-V relationship (E). The transfected cells generated large, time-dependent and outwardly rectifying currents (1, 562 ± 128 pA at + 25 mV membrane potential, n = 12), in contrast to the mock-transfected CHO-K1 cells (70 ± 8 pA at +25 mV, n = 10; Fig. 5, A and B). The I-V curve based on the currents at the end point of voltage pulses revealed an outwardly rectifying current-voltage relationship (Fig. 5E). The extracellular partial Cl replacement with gluconate reduced the overall current and resulted in a shift of the reversal potential toward the positive direction, thus indicating that the current was Cl selective (Fig. 5E). Also the current was inhibited by extracellular addition of Cl channel blockers; 1 mM 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid (DIDS) (65 ± 3%, at +25 mV, 5 min after the addition of DIDS, n = 4) or 0.5 mM diphenylamine carboxylic acid (DPC) (86 ± 4%, at +25 mV, 5 min after the addition of DPC, n = 5) (Fig. 5, C and D). To determine anion selectivity of ClC-5, we preliminary applied the inside-out patch technique to the wild-type and the ClC-5 transfected CHO-K1 cells. In the wild-type cells, patched membranes occasionally contained Cl channels (34%, 12 of 35), and these channels did not show any rectification as shown previously(15) . In the ClC-5 transfected cells the patches always contained Cl channels as a cluster (100%, 40 of 40), and they showed outward rectification. When the transfected cells were treated with DEX, the number of Cl channels contained in the patch increased (10 per patch), indicating that these Cl channels were mediated by ClC-5. Using these cluster Cl channels, we determined anion selectivity of ClC-5. When we replaced a half of Cl with other anions in the bath solution, the reversal potential for I move to negative direction (-5 ± 2 mV, n = 5), and for F it moved to positive direction (10 ± 4 mV, n = 5). The sequence of relative anion permeability was I > Cl > F.


Figure 5: Electrophysiological characterization of ClC-5 expressed in CHO-K1 cells. A and B, the representative typical traces of whole cell currents from the cells transfected with pMAM-ClC-5-neo (A) or pMAMneo alone (B). C, a representative typical trace of whole cell currents 5 min after extracellular addition of 1 mM DIDS. The holding potential was changed from -75 to +25 mV. Inset, voltage-clamp program. D, the percent inhibition of whole cell currents with 1 mM DIDS and 0.5 mM DPC in the transfected cell. Currents were measured 5 min after extracellular addition of the each blocker (n = 5, mean ± S.E., at +25 mV). E, current-voltage relationships of conductance expressed from ClC-5 cDNA, in TEA-Cl solution (130 mM Cl) (closed circle), in low chloride (10 mM Cl, 120 mM gluconate) (open triangle).



Effect of cAMP on ClC-5 channel was examined in J2702 cells using the whole cell patch-clamp technique. Fig. 6shows the effect of cAMP (CTP-cAMP, 2 mM) on the whole cell Cl currents. The currents were measured in response to voltage pulse of ±30 mV. When the patched cells were exposed to cAMP, the magnitude of outward currents did not change as shown in Fig. 6(n = 4).


Figure 6: Representative trace of whole cell currents in ClC-5 expressing cells in response to cAMP treatment. Labeled bar indicates application of a membrane permeable cAMP (CTP-cAMP, 2 mM). The holding potential was clamped at ±30 mV.




DISCUSSION

The molecular structures of ORCCs have not yet been identified because of the lack of their molecular cloning. In this study, we successfully isolated ClC-5, a new ORCC which is highly homologous to ClC-3, by a sequence homology-based strategy. We further established the stably transfected mammalian cultured cell line expressing ClC-5 using the intranuclear microinjection technique.

The predicted amino acid sequence of ClC-5 is highly homologous with not only ClC-3 (77%), but also with ClCN4 (78%), and its rat counterpart ClC-4 (78%), which has recently been isolated from Xp22.3 region using positional cloning strategy (28, 32) (Fig. 1B). Phylogenic analysis indicated the presence of two subfamily in the ClC family (Fig. 2). A partial deletion of ClCN4 is known to cause delays in psychomotor functions and mental retardation. Furthermore, the amino acid sequence of ClC-5 is almost identical (99%) to a human sequence predicted from a partial cDNA (780 bp) recently isolated as a candidate molecule of Dent's disease (an X-linked hereditary renal tubular disorder) from Xp11.22 region using positional cloning strategy (21) . (^2)It is tempting to speculate that this subfamily of ClC Cl channel is related to human disease. Further clarification of physiological function of these channels is needed to elucidate the pathogenesis of these disease.

In the motif analysis, ClC-5 has two potential N-glycosylation sites. One of the N-glycosylation sites (Asn-408) is located in the segment between D8 and D9, and this glycosylation motif is well conserved among all ClC channels known so far. Kieferel et al.(34) have shown recently that ClC-0, ClC-1, ClC-2, and ClC-K1 channels are glycosylated at this segment in in vitro translation experiments. Therefore, this glycosylation site between D8 and D9 should be located outside of the cells. In addition, there are two consensus sequences for phosphorylation by cAMP-dependent protein kinase in ClC-5 cDNA (see ``Results''). This finding suggest that ClC-5 protein itself might be modulated by cAMP-dependent protein kinase-mediated phosphorylation. However, any change in the whole cell current was not induced in response to cAMP in the present study (Fig. 6). This result revealed that cAMP-mediated signaling does not directly modulate the channel properties. However, it cannot be neglected that cAMP-mediated signaling indirectly modulates the function of ClC-5. A lack of channel regulator(s) in the transfected cells may disrupt the pathway of cAMP-mediated signaling.

Now, eight members of the ClC family have been cloned. Some of these Cl channels have been functionally characterized by transient expression system using Xenopus oocytes. However, the functional expression in Xenopus oocyte may not be ideal for characterization of ClC-3, -4, and -5 channels. For unknown reasons, the expression of these channels has been difficult in the oocytes except for ClC-3 (for review, see (28) and (35) ). Also a Ca-dependent Cl channel is present in the oocytes (36, 37) and sometimes disturbs the detection of the exogenously expressed Cl currents. We thought that mammalian cells were more suitable for proper functional expression of ClC-5, which was obtained from the rat. Accordingly, we stably transfected ClC-5 cDNA into a mammalian cultured cell line, CHO cells, using the intranuclear microinjection technique. This transfection system has the following advantages over others: 1) the stable transformed cell line can be obtained more frequently and more easily because the transfection efficiency of intranuclear microinjection is higher than those of other indirect methods (0.2 versus 0.001)(38, 39) , 2) the dexamethasone-inducible expression vector (pMAM-neo) is useful to induce the overexpression of the transformed full-length gene, 3) a patch-clamp study has demonstrated that only a small linear Cl channel is endogenous in CHO cells(40) .

In the stably transfected mammalian cells with ClC-5, the whole cell currents showed a typical outward rectification that was time-dependently activated following depolarization of membrane voltage (Fig. 5E). The profile and amplitude of the currents (1 nA at +25 mV, Fig. 5A) were different from those of the endogenous small linear Cl channel in the wild-type CHO cells(40) . Several lines of evidence suggest that these currents were induced by a Cl-selective channel. First, Cl was the predominant current-carrying ion under the conditions of patch-clamp experiments. Second, the replacement of Cl with gluconate in the bath solution reduced the currents and caused a positive shift in the reversal potential (Fig. 5E). Third, the currents were inhibited by Cl channel blockers, DPC and DIDS. They act as open channel blockers probably by decreasing the open probability of ORCC from outside of the cells(2) , so that their inhibition of ClC-5 is also consistent with that of ORCC. In addition, based on the shift of zero potential after the half-replacement of Cl with other anions, we determined the anion permeability sequence as I > Cl > F (see ``Results''). This sequence is compatible with Eisenman's series 1(41) , indicating that ClC-5 channel may have the same anion selectivity as that of previously demonstrated ORCCs(42) . These electrophysiological characteristics of ClC-5 are different from those of other ClC Cl channels, ClC-0, ClC-1, and ClC-2. They do not show outward rectification and have relatively less I permeability(28) . In contrast, ClC-3 has outward rectification and a rather high I permeability, which are consistent with ClC-5(15) , indicating that these two channels belong to same category in terms of channel characteristics. However, Steinmeyer et al. most recently isolated the identical Cl channel cDNA from rat brain and showed that it was not sensitive to DIDS and DPC and also has a anion conductivity sequence of Cl > Br > I using Xenopus oocyte expression system. (^3)Although reason for functional discrepancies between theirs and the present study is not clear, the expression system (Xenopus oocyte versus mammalian cultured cells, CHO-K1) and experimental conditions for anion selectivity study (whole cell versus inside-out patch configuration) are different. It is possible that the expressed Cl channel is modulated by endogenous factor(s) in whole cell system. Accordingly, further studies are needed to characterize the electrophysiological properties of ClC-5.

The tissue distribution of ClC-5 mRNA is different from that of this human partial clone. In Northern blot analysis of rat tissues, ClC-5 is abundantly expressed in the kidney and colon and in lesser amounts in the heart, brain, lung, and testis. Although the expression in the colon has not been examined, the human partial clone was expressed only in the kidney. This may represent some discrepancy in the tissue distribution among species, and a similar discrepancy was observed for ClC-4(28, 32) .

ClC-5 is almost identical to a human partial cDNA, which was recently isolated as a strong candidate for Dent's disease(21) . This disease is a renal tubular disorder which is associated with low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure(43, 44, 45) . Although the primary mechanism responsible for this disease has not been clarified, the feature of low molecular weight proteinuria indicates the dysfunction of proximal tubule. The previous physiological studies have shown that the acidification of endosomal compartment is a limiting factor for the uptake of filtered proteins via active endocytotic pathway in the proximal tubule, in which the Cl conductance in endosomal membrane is responsible for the acidification of endosomal compartment(46, 47, 48, 49) . The Cl channel expressed in endosomes has been isolated from rat kidney cortex and has been shown to be voltage-dependent and DPC- and DIDS-sensitive(50) . The further studies on ClC-5 may help to evaluate the hypothesis that the defect of the Cl channel is responsible for Dent's disease.

In summary, a new molecule of ORCC was isolated from rat kidney and functionally characterized. Following the elucidation of two structurally and functionally homologous ORCCs (ClC-3 and ClC-5), the existence of a subfamily encoding ORCCs in ClC Cl channel family has emerged. Although ClC-4 has not yet been functionally characterized as a Cl channel, it could belong to the same subfamily as ClC-3 and ClC-5. The analysis of the structure and function of these ORCCs proteins will provide us with the framework to evaluate their detailed regulatory mechanisms at a molecular level.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D50497[GenBank].

§
To whom correspondence should be addressed: Second Dept. of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-358035216; Fax: 81-358030132; hsakamoto.med2{at}med.tmd.ac.jp.

(^1)
The abbreviations used are: ORCC, outwardly rectifying Cl channel; CFTR, cystic fibrosis transmembrane conductance regulator; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); DEX, dexamethasone; TEA, tetraethylammmonium; PIPES, 1,4-piperazinediethanesulfonic acid; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; DPC, diphenylamine carboxylic acid; CHO, Chinese hamster ovary.

(^2)
During the submission of this draft the full-length human cDNA, which is implicated in Dent's disease, has been published as CLCN5 by Fisher et al. (Fisher, S. E., Bakel, I. D., Lloyd, S. E., Pearce, S. H. S., Thakker, R. V., and Craig, I. W.(1995) Genomics29, 598-606).

(^3)
During the revision of this draft, an identical chloride channel cDNA isolated from rat brain has been published as CLC-5 by Steinmeyer et al. (Steinmeyer, K., Schwappach, B., Bens, M., Vandewall, A., and Jentsch, T. J.(1995) J. Biol. Chem.270, 31172-31177).


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