A novel p64-related Clminus channel: subcellular distribution and nephron segment-specific expression

John C. Edwards

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Several closely related proteins that have been implicated as chloride channels of intracellular membranes have recently been described. We report here the molecular cloning and characterization of a new member of this family from human cells. On the basis of sequence similarity, we conclude that this new protein represents the human version of a previously described protein from rat brain named p64H1. The human version of p64H1 (huH1) is a 28.7-kDa protein that shows an apparent molecular mass of 31 kDa by SDS-PAGE. A single 4.5-kb message is detected on Northern blots and is present in all tissues probed. The protein is expressed in an intracellular vesicular pattern in Panc-1 cells that is distinct from the endoplasmic reticulum, fluid-phase endocytic, and transferrin-recycling compartments, but which does colocalize with caveolin. In human kidney, huH1 is highly expressed in a diffuse pattern in the apical domain of proximal tubule cells. huH1 is expressed less abundantly in a vesicular pattern in glomeruli and distal nephron.

caveolin; caveolae; p64H1; chloride intracellular channel 2; NCC27; proximal tubule


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHLORIDE CHANNELS are present in the limiting membranes of a variety of intracellular organelles where they contribute to the regulation of intraorganellar electrolyte composition (1). The most clearly described role for intracellular chloride channels is to short-circuit electrogenic cation transport mechanisms: anion permeability is essential for acidification of intravesicular spaces by the electrogenic vacuolar proton ATPase and is necessary for the KCl influx that precedes exocytosis in certain secretory vesicles.

The role of chloride channels in acidification is of particular interest. Each intracellular compartment has a characteristic pH that is tightly controlled (16). Maintenance of the appropriate pH in various compartments has been shown to be essential to a variety of cellular functions (1). Acidification of intracellular compartments is accomplished by the parallel actions of the electrogenic vacuolar proton ATPase and a chloride channel. The proton ATPase provides the proton-motive force to generate a pH gradient while the chloride channel short-circuits the membrane potential generated by the pump and allows proton transfer. In some intracellular membranes, the chloride conductance not only permits acidification but also may regulate it. In clathrin-coated vesicles (17), kidney endosomes (2), and secretory granules of perifollicular cells of the thyroid (3, 24), acidification appears to be actively regulated by regulation of the chloride conductance.

If acidification of intracellular compartments is at least partially controlled through regulation of the chloride conductance, and because the pH of different compartments is independently regulated, it follows that compartment-specific chloride channels exhibiting different patterns of regulation must be expressed in the appropriate intracellular membranes. Thus it seems likely for there to be a number of subcellular compartment-specific chloride channels expressed simultaneously in a cell.

Over the past several years, a number of genes responsible for plasma membrane chloride channels have been identified, but little is known about genes responsible for intracellular chloride channels. p64 is a chloride channel originally identified by biochemical purification of chloride channel activity from bovine kidney microsomes (14). cDNA clones encoding p64 have been cloned and characterized (15). p64 is a 48-kDa protein that migrates with an apparent molecular mass of 64 kDa by SDS-PAGE. Hydrophobicity analysis reveals the presence of two hydrophobic segments that could be transmembrane domains, both of which are in the COOH-terminal half of the molecule. The protein is selectively expressed in the limiting membranes of what appear to be regulated secretory vesicles in bovine kidney proximal tubule cells and in the human colon cancer cell line T84 (20). Expression of p64 in HeLa cells results in the appearance of a new outwardly rectifying chloride conductance (6).

p64 is a member of a closely related gene family. A 62-kDa protein antigenically related to p64 has been identified as the chloride channel of osteoclast ruffled border (22). NCC27, a 27-kDa protein that shows high homology with the COOH-terminal half of p64, has been reported to be a chloride channel of nuclear membranes in CHO cells (26). We have independently isolated and characterized cDNA encoding this 27-kDa protein and found that, like p64, it shows aberrantly high mobility by SDS-PAGE, running at an apparent molecular mass of 34 kDa (25). We found NCC27 to be expressed in an intracellular vesicular compartment in cultured cells and to be expressed at high levels in the apical domain of renal proximal tubule cells. A third p64 homolog named chloride intracellular channel (CLIC) 2 was found by a genomic sequencing approach (11). Finally, a fourth p64 family member named H1 that was cloned from rat brain was recently reported to be present in endoplasmic reticulum (ER) (5).

In this study, we describe the sequence and immunolocalization of the human version of the previously reported rat protein H1. We find that the human H1 cDNA encodes a 27-kDa protein that, like the other members of the gene family, shows aberrantly low mobility on SDS-PAGE. The protein is quite similar to the COOH-terminal half of p64 and is missing any sequence homologous to the NH2-terminal half of p64. Immunolocalization studies show that H1 is present in an intracellular vesicular compartment that partially colocalizes with caveolin and is distinct from ER, the fluid-phase endocytic compartment, and the recycling compartment. In human kidney, H1 is highly expressed in the apical pole of proximal tubule cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell culture. Panc-1 cells were obtained from the American Type Culture Collection (Manassas, VA). HeLa cells and recombinant vaccinia stock were obtained from Dr. Andrey Shaw (Washington University, St. Louis, MO). Both cell types were grown in DMEM with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. OK cells were obtained from Dr. Keith Hruska (Washington University) and were grown in DMEM with 10% heat-treated serum plus antibiotics as above.

cDNA cloning. A randomly primed cDNA library was prepared from poly(A)+-selected RNA from Panc-1 cells with the Zap-cDNA synthesis kit and inserted into the lambda Zap vector (Stratagene, La Jolla, CA). The library was screened by hybridization at low stringency with the large Pst I fragment from clone H2B (15), which contains the entire coding region of bovine p64. A number of overlapping individual clones were isolated, and sequences were determined with the sequenase system (US Biochemical, Cleveland, OH). Sequences were analyzed with the Wisconsin GCG software (Wisconsin Package version 9.1; Genetics Computer Group, Madison, WI) and with MacVector. Clone pPH3 was found to contain the entire coding region of a novel homolog of p64.

Northern blots. Human multiple-tissue Northern blot was purchased from Clontech (Palo Alto, CA). Poly(A)+ RNA was prepared from Panc-1 cells by standard methods. Three micrograms of poly(A)+ RNA were separated by denaturing electrophoresis, blotted, and probed as described by Landry et al. (15). Both blots were probed at high stringency with a 1,000-bp Pvu II-Sal I fragment from clone pPH3, which contains the entire p31 coding region (positions 1-1017 of the pPH3 sequence in Fig. 1).

Derivation and purification of antisera. Some of the antisera used in this study have been previously described. AP95 (15) was raised against a beta -galactosidase-p64 fusion protein and affinity purified over the same fusion protein immobilized on an AminoLink column (Pierce, Rockford, IL) following the procedure provided by the manufacturer. AP823 (25) was raised against a COOH-terminal peptide derived from NCC27 and was affinity purified over His-tagged full-length NCC27 immobilized on an AminoLink column.

To generate an antiserum specific for H1, the coding region from pPH3 cDNA was inserted into the multiple cloning site of the vector pGEX-KG (9), resulting in a construct encoding a fusion protein containing the entire H1 protein fused with glutathione-S-transferase and separated by a thrombin cleavage site. The fusion protein was expressed in bacteria, purified by affinity to glutathione-agarose, and used to immunize a rabbit. A high-titer antiserum named 1058 was obtained. To affinity purify the 1058 antisera, a His-tagged version of human H1 was generated by inserting the coding region from pPH3 in the pQE-30 vector (Qiagen, Chatsworth, CA). The tagged protein was expressed in bacteria, purified by affinity to Ni-agarose, and immobilized on cyanogen bromide-activated Sepharose. The antiserum was purified by affinity to the immobilized His-tagged protein by standard methods (10). The affinity-purified antiserum is designated AP1058.

Western blots. Cultured cells were solubilized in 150 mM NaCl, 50 mM Tris · HCl, pH 8, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. Bovine, mouse, and human kidney microsomes were prepared as previously described (19). To prepare membrane fractions, Panc-1 cells were scraped from the culture plate into ice-cold 250 mM sucrose, 10 mM imidazole, pH 7.0, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride and homogenized with a Dounce tissue homogenizer followed by 10 passages through a 25-gauge needle. Nuclei and cellular debris were removed by centrifugation at 4,000 rpm for 10 min at 4°C in a Eppendorf microcentrifuge. The supernatant was then centrifuged at 40,000 rpm (100,000 g) for 1 h at 4°C in a Beckman Ti70.1 rotor, yielding a membrane pellet and a soluble fraction. Alkali-washed membranes were prepared by resuspending the membranes in 100 mM Na2CO3, pH 11.5, and incubating on ice for 15 min, followed by centrifugation at 40,000 rpm for 1 h as before.

SDS-PAGE was carried out by standard methods (10). Low-range molecular weight standards from Bio-Rad (Richmond, CA) were used. Proteins were electroblotted to nitrocellulose membranes and probed with antibodies with the Supersignal chemiluminescent system (Pierce). Protein concentrations were determined with the bicinchoninic acid assay (Pierce).

In vitro translation. Plasmid PH3 was linearized with BamH I and transcribed with T7 RNA polymerase as previously described (15). Resulting capped RNA was translated using a reticulocyte lysate (Promega, Madison, WI) in the presence of [35S]methionine. Products were separated by SDS-PAGE, stained with Coomassie blue to detect molecular weight standards, dried, and detected by autoradiography.

Overexpression of proteins in HeLa cells. A vaccinia-T7 RNA polymerase driven system was used to overexpress p64 family proteins in HeLa cells as described (6). HeLa cells were plated in 3.5-cm dishes at 5 × 105 cells/dish in DMEM with 10% FCS. The next day, cells were rinsed with serum-free medium and infected with T7 RNA polymerase-encoding vaccinia at a multiplicity of infection of 10 in 0.75 ml of medium and incubated at 37°C for 30 min. Five micrograms of plasmid DNA encoding p64, NCC27, or huH1 downstream of a T7 promoter were mixed with 15 µl of lipofectase (GIBCO-BRL, Life Technologies, Bethesda, MD) in 0.75 ml of medium and added to the infected cells. The infected and transfected cells were incubated at 37°C in 5% CO2 overnight, and proteins were prepared for Western blotting as described above.

Immunofluorescence. Panc-1 cells were grown on glass coverslips, rinsed with PBS, and fixed in 4% paraformaldehyde at room temperature for 15-30 min. Cells were permeabilized and blocked with 0.05% saponin in PBS-5% goat serum-0.02% fish gelatin (PBSFG) for 30 min. Cells were exposed to the primary antiserum by inverting the coverslip on a 20-µl drop of antiserum diluted in PBSFG plus 0.05% saponin and incubated at room temperature for 2 h. AP1058 was used at a 1:20 dilution. Monoclonal antibodies to BiP (Stressgen, Victoria, BC, Canada) and caveolin (Transduction Lab, Lexington, KY) were diluted 1:200 and 1:50, respectively. Coverslips were washed four times with 10-min exchanges of PBSFG and then exposed to Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG (Jackson Laboratories, West Grove, PA) diluted 1:100 in PBSFG for 1 h at room temperature. Samples were again washed four times with PBSFG, mounted in Vectashield (Vector Laboratories, Burlingame, CA), and photographed with a Zeiss Axiophot epifluorescence microscope or imaged with a Bio-Rad confocal microscope.

Labeling of endocytic compartments. For albumin endocytosis, cells grown on glass coverslips were rinsed with prewarmed HEPES-buffered saline (HBS; in mM: 135 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 2 Na2PO4, 5 glucose, and 10 HEPES, pH 7.4) with 20% FCS and then incubated in the same solution with 10 µg/ml TRITC-albumin (Molecular Probes, Eugene, OR) at 37°C for 15 min. Cells were then washed with four 5-min exchanges of ice-cold HBS with 20% fetal bovine serum, fixed with 4% paraformaldehyde, and stained with the AP1058 antibody as in Immunofluorescence except that FITC-conjugated secondary antibody was used. To label the transferrin-recycling compartment, cells grown on glass coverslips were rinsed with serum-free medium and incubated in serum-free medium at 37°C for 10 min to clear the surface receptors of transferrin. The coverslips were then rinsed with prewarmed HBS with 1% ovalbumin and incubated in HBS with ovalbumin plus 100 µg/ml FITC-transferrin (Molecular Probes) for 15 min at 37°C. The coverslips were rinsed with four 5-min exchanges of ice-cold HBS with 1% ovalbumin, fixed with 4% paraformaldehyde, and stained with the AP1058 antibody and Cy3-conjugated secondary antibody as described in Immunofluorescence.

Human kidney sections were stained with AP1058 antibody and nephron segment-specific lectins as previously described (25) except that a Cy3-conjugated secondary antibody was used.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA cloning. A randomly primed cDNA library derived from Panc-1 cell poly(A)+ RNA was constructed and screened at low stringency with a fragment containing the entire coding region of bovine p64. A number of overlapping individual recombinants were isolated, and the sequences were determined. Clone pPH3 was found to contain the entire coding region of a novel protein similar to but distinct from both bovine p64 and human NCC27. The sequence of the cDNA (GenBank accession no. AF097330) is shown in Fig. 1 along with the derived amino acid sequence from the longest open reading frame. The cDNA consists of 3,729 bp. The sequence does not include a poly(A)+ tail and is not expected to extend to the true end of the message because the library was randomly primed. There is a trinucleotide AGC motif in the 5'-flanking region that is directly repeated nine times. The first ATG in the sequence occurs at nucleotide 139 and the surrounding nucleotides form a good translation start site with guanosines at positions -3 and +4 (12). The reading frame following the initial ATG is open until the first in-frame nonsense codon at position 900. pPH3 thus encodes a 253-amino acid protein with a predicted molecular mass of 28,674 Da.


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Fig. 1.   Sequence of the pH3 cDNA along with derived amino acid sequence from longest open reading frame. Underline, repeated AGC motif; overline, initiation and termination codons. GenBank accession no. is AF097330. Numbering of nucleotide and amino acid sequences are on the right.

The coding region is very similar to the rat H1 coding region, showing 88% nucleotide identity. Lower levels of similarity are evident between the pPH3 coding region and those of other p64 family members. Amino acid sequences derived from the coding regions of pPH3, p64, NCC27, and CLIC2 are aligned in Fig. 2A. The derived amino acid sequence is 97% identical with rat H1, 77% identical with the COOH-terminal 228 amino acids of p64, 67% identical with NCC27, and 66% identical with CLIC2. The sequences are particularly tightly conserved through the hydrophobic domains. Kyte-Doolittle plots demonstrating the strongly conserved hydropathy profile of these proteins are shown in Fig. 2B. A family tree derived from homology data showing the relatedness among these family members is shown in Fig. 2C. Because of the near identity of rat H1 and pPH3, we concluded that pPH3 is the human version of H1.


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Fig. 2.   Comparison of p64 family protein sequences. A: alignment of sequences. Sequences are numbered according to p64. Uppercase, amino acids conserved in at least 2 family members; underline, positions where human H1 (huH1) is not identical to rat H1 (raH1). Terms in parentheses, proposed nomenclature. B: Kyte-Doolittle hydropathy plots. Hydrophobic regions are represented as positive deflections. Values were determined using a window of 15 amino acids. C: family tree. Sequences were compared and relationships were plotted with pileup program of Wisconsin GCG package. CLIC1-CLIC4, proposed nomenclature (see DISCUSSION).

The encoded sequence was searched for known motifs. There is a single consensus protein kinase A phosphorylation site at the COOH terminus that is conserved between H1 and p64. There are four consensus protein kinase C phosphorylation sites at positions 38, 108, 174, and 175, the first and third of which are also present in p64 and NCC27. All of the consensus phosphorylation sites are present in both human and rat H1.

Northern blots. A DNA fragment from the coding region of pPH3 was used to probe RNA from Panc-1 cells and from a series of human tissues as shown in Fig. 3. A single transcript of ~4.5 kb is expressed in all tissues probed. The expression is particularly prominent in heart, placenta, and skeletal muscle and is very prominent in the Panc-1 cells.


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Fig. 3.   Northern blot analysis of pPH3 transcripts. A human multiple tissue Northern blot containing 2 µg of poly(A)+ RNA from various tissues and a blot containing 3 µg of Panc-1 poly(A)+ RNA were probed with pPH3 at high stringency. Lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; and 9, Panc-1 cells. Molecular weight standards in kb.

In vitro translation of pPH3. Capped mRNA was generated from linearized pPH3 plasmid and used to direct synthesis of protein in vitro using a reticulocyte lysate in the presence of [35S]methionine. The products were separated by SDS-PAGE and detected by autoradiography as shown in Fig. 4, lanes 1 and 2. The primary translation product migrates with the 31-kDa molecular mass marker despite the predicted molecular mass of 28.7 kDa. Thus, like p64 and NCC27, the human H1 protein (huH1) shows aberrantly slow mobility on SDS-PAGE gels that is not due to posttranslational modification.


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Fig. 4.   Lanes 1 and 2: in vitro translation of pPH3. Products of cell free translation performed with no exogenous RNA (lane 1) or with T7 transcript for plasmid pPH3 (lane 2) were separated by SDS-PAGE and detected by autoradiography. Lanes 3-6: detection of H1 in Panc-1 cells. Various fractions (30 µg) of Panc-1 cell lysates were probed with AP1058 antibody raised against recombinant huH1. Lane 3, whole cell lysate; lane 4, postmembrane supernatant; lane 5, crude membranes; lane 6, alkali-washed membranes. Molecular mass standards in kDa.

Characterization of antibodies. A high-titer antiserum was raised against recombinant huH1 and affinity purified. This affinity-purified antiserum, named AP1058, was used to probe Panc-1 cell proteins by Western blotting (Fig. 4, lanes 3-6). Whole cell lysate (lane 3), postmembrane supernatant (lane 4), crude membrane pellet (lane 5), and alkali-washed membranes (lane 6) were separated and probed. The AP1058 antibody recognizes a 31-kDa protein that has mobility on SDS-PAGE identical to that of the in vitro translation product. Like NCC27, this protein is present in both membrane and soluble fractions in standard cell fractionations. The fraction of the total that is sedimented with membranes is resistant to extraction with alkali (100 mM Na2CO3), consistent with the behavior of an integral membrane protein.

We have used three antibodies to characterize expression of the p64 family proteins and to test specificity of the antisera. AP1058 is the preparation raised against huH1 described above. Antibody AP95 is the affinity-purified antibody raised against a beta -galactosidase-p64 fusion protein that was previously described (15). AP823 is an affinity-purified antiserum that was raised against a peptide derived from the COOH-terminal domain of NCC27 (25). The specificities of these antibodies are characterized over a panel of antigens in Fig. 5. Each Western blot in Fig. 5 contains (lanes 1-9, respectively) bovine, human, and mouse kidney microsomes, Panc-1, OK, and HeLa cells, and HeLa cells overexpressing p64, NCC27, or huH1 using the vaccinia-T7-driven expression system.


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Fig. 5.   Characterization of antisera. Crude microsomal membrane preparations (30 µg) from kidneys or whole cell lysates from cultured cells were separated on 3 identical gels and probed with AP1058 (A), AP95 (B), or AP823 (C). Lanes: 1, bovine kidney; 2, human kidney; 3, mouse kidney; 4, Panc-1 cells; 5, OK cells; 6, HeLa cells; 7, HeLa cells expressing p64; 8, HeLa cells expressing exogenous NCC27; 9, HeLa cells expressing exogenous huH1. Molecular mass standards in kDa.

Figure 5A shows samples probed with the anti-huH1 antibody AP1058. It recognizes a 31-kDa protein in bovine, human, and mouse kidney microsomes and in Panc-1 and OK cells. No band is visible in the HeLa cells (lane 6), although long exposures revealed that the 31-kDa protein is present in these cells as well (data not shown). A 31-kDa signal indistinguishable from the endogenous signal in Panc-1 cells became easily apparent when huH1 was overexpressed in HeLa cells (lane 9). The antibody did not react with native NCC27 in Panc-1 or HeLa cells or NCC27 overexpressed in HeLa cells (lane 8). AP1058 did not detect p64 in bovine kidney microsomes but did weakly react with p64 when it was overexpressed (lane 7). Very long exposures of this blot failed to reveal any endogenous p64 in Panc-1 or HeLa cells (data not shown).

Figure 5B shows samples probed with the anti-p64 antibody AP95. This antibody recognized native p64 in bovine kidney microsomes (lane 1) and exogenous p64 when overexpressed in HeLa cells (lane 7). It did not recognize a homolog of p64 in human or mouse kidney, and it did not cross-react with native or overexpressed huH1 or NCC27.

Figure 5C shows samples probed with the antibody raised against NCC27, AP823. This antibody recognized native NCC27 in human and mouse kidney, as well as in Panc-1 and HeLa cells. The 34-kDa signal became dramatically more prominent when NCC27 was overexpressed in HeLa cells. This antibody did not react with native or exogenous huH1 or p64 and did not recognize a NCC27 homolog in bovine kidney.

Our conclusions from these data are as follows. 1) Despite the similarity among this family of proteins, these antisera show good specificity. In particular, AP1058 does not cross-react with NCC27, and AP823 does not cross-react with huH1. 2) Our current p64 antisera only react with bovine p64. Whether a homolog of p64 exists in human kidney is not apparent. 3) The endogenous 31-kDa protein detected with AP1058 is huH1.

Immunolocalization. Panc-1 cells were fixed with 4% paraformaldehyde and stained with AP1058 or with control antibody as shown in Fig. 6. The antibody stains very small punctate structures in the center of the cell plus larger peripheral vesicular structures. Because the antibody only recognizes H1 on Western blots of these cells, we concluded that the pattern of staining represents the distribution of H1. However, we had noted that AP1058 can react weakly to p64 (see Fig. 5A). To be certain that the staining pattern with AP1058 is not due to cross-reaction with p64, we also stained Panc-1 cells with AP95, (specific for p64) and found no staining above background (data not shown). To characterize the distribution of H1 more fully, cells were colabeled with AP1058 and with markers of various subcellular compartments. Images were obtained with confocal fluorescence microscopy as shown in Fig. 7. The ER was identified by staining with monoclonal antibodies against BiP (4, 23), a fluid-phase endocytic compartment was labeled by uptake of TRITC-conjugated albumin, and a recycling endosomal compartment was labeled by uptake of FITC-conjugated transferrin (27). Figure 7 shows a sample stained for an intracellular compartment (A, D, and G), a sample stained for H1 (B, E, and H), and a merged image (C, F, and I). Figure 7, A-C, was stained for BiP and H1. The distribution of the ER marker and H1 are quite distinct and there is little overlap. Thus, in contrast to the previous report of immunolocalization of rat H1, huH1 does not appear to be primarily in the ER in Panc-1 cells. Figure 7, D-F, shows TRITC-albumin endocytosis and H1 staining, and G-I shows FITC-transferrin uptake and H1 staining. There is no colocalization of H1 with either of these markers.


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Fig. 6.   Distribution of H1 in Panc-1 cells. Cells were fixed and stained with AP1058 (A) or irrelevant control antibody (B) and photographed with ×100 objective on a Zeiss axioscope epifluorescence microscope. Bar, 25 µm.


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Fig. 7.   Costaining of Panc-1 cells with AP1058 and markers of intracellular compartments. Left column (A, D, G) shows staining with markers for individual intracellular compartments; center column (B, E, H) shows immunolocalization of H1 with affinity-purified (AP) 1058; right column (C, F, I) shows both images superimposed. A-C: lack of colocalization of ER marker BiP (A) with H1 (B). D-F: lack of colocalization of endocytosed albumin (D) with H1 (E). G-I: lack of colocalization of endocytosed transferrin (G) with H1 (H). Images were taken from approximate midpole of cultured cells with confocal microscopy. Bars, 25 µm.

Figure 8 shows Panc-1 cells costained for H1 and caveolin. Caveolin (or VIP21) is a 21-kDa protein that is present in plasma membrane invaginations called caveolae (8, 21) and is also present in some trans-Golgi network vesicles (13). Two confocal images of a single cell at different planes of focus are shown in Fig. 8: the cell stained for caveolin (A and D), the cell stained for H1 (B and E), and a merged image (C and F). Figure 8, A-C, shows images from the very basal pole of the cell, and Fig. 8, D-F, shows images from the midportion of the cell. The caveolin and H1 clearly colocalized in large distinct structures that are present in the very base of the cell. In the midportion of the cell, caveolin and H1 are expressed in a very similar, overlapping, small vesicular pattern, although only a fraction of the vesicles clearly stained with both antibodies.


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Fig. 8.   Costaining of Panc-1 cells for H1 and caveolin. Confocal images of a single cell taken at 2 different planes of focus are shown. A-C are from very basal pole of cell, and D-F are from midpole of cell. A and D: staining for caveolin. B and E: staining for H1. C and F: combined images. Bar, 25 µm.

Thus the intracellular compartment labeled with H1 antibodies is not ER or fluid phase or recycling endosomes, but does partially colocalize with caveolin, particularly in large structures that are present at the base of these cells.

Distribution of H1 in kidney. Human kidney sections probed with the AP1058 antisera are shown in Fig. 9. Pairs of immunofluorescence and corresponding phase-contrast images are shown. Virtually all cells in the kidney show low-level staining with the antibody. Figure 9, A and B, shows a low-power view of renal cortex with scattered staining in tubules and glomeruli. A subset of cortical tubules shows more prominent staining that is concentrated at the apical pole of the cells (e.g., Fig. 9A, bottom right). Nonimmune staining of a comparable field is shown in C and D. E and F show staining of medulla. Scattered tubules clearly stain above background (Fig. 9, G and H). Panels I and J show a higher power view of a glomerulus, demonstrating punctate staining in cells throughout. A high-power view of medullary cells is shown in panels I and J, revealing primarily staining of intracellular punctate structures.


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Fig. 9.   Human kidney sections stained for H1. Pairs of panels showing immunofluorescence and corresponding phase-contrast images are shown. A and B: cortex stained with AP1058 and imaged with ×10 objective. Note glomerulus (top left) and intensely stained tubule (bottom right). C and D: kidney cortex stained with control antibody and imaged as in A and B. E and F: medulla stained with AP1058 and imaged with ×10 objective. G and H: medulla stained with control antibody and imaged as in E and F. I and J: high-power view (×40 objective) of glomerulus stained with AP1058. K and L: high-power view (×40 objective) of medulla stained with AP1058. Cells were photographed with conventional (i.e., nonconfocal) epifluorescence microscopy. Bar, 800 µm in A-H and 200 µm in I-L.

To determine which nephron segments were labeled with AP1058, sections were double-stained for H1 with a Cy3-conjugated secondary antibody and with FITC-conjugated, nephron segment-specific lectins. The lectin from Lotus tetragonobulus (LTA) specifically labels the proximal tubule in human kidney, whereas peanut agglutinin (PNA) labeling is restricted to the distal nephron, including the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the collecting duct (7). Cortical sections double-labeled with AP1058 and each of the lectins are shown in Fig. 10. For each pair of images in Fig. 10, the left panel is a single exposure showing the distribution of H1 (red), whereas the right panel is a double exposure showing the distributions of H1 (red) and lectin (green) superimposed. The top pair of images (Fig. 10, A and B) show double staining for H1 and the distal nephron marker PNA. Clearly, the lectin reacts with tubules distinct from those showing strong diffuse staining for H1. A higher power image of a similar section is shown in Fig. 10, C and D. This field contains cross sections of two tubules side by side. The tubule on the left shows a few punctate structures staining for H1, whereas the tubule on the right shows intense diffuse staining of the apical pole of the cells. Only the tubule showing vesicular staining is labeled with PNA. Figure 10, E and F, shows a section costained for H1 and the proximal tubule marker LTA. Again, two tubule fragments are shown, one with punctate H1 staining and one with the diffuse apical pattern of staining. The LTA reacts with the cells showing diffuse abundant expression of H1 and does not react with the cells showing more limited, vesicular staining. Furthermore, LTA is known to react most intensely with the apical domain and brush border of proximal tubule cells. The superimposition of AP1058 and LTA staining indicates that H1 is closely associated with the brush border of these cells.


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Fig. 10.   Colocalization of H1 with nephron segment-specific lectins in human kidney cortex. Single exposures (A, C, E) show staining with AP1058, and double exposures (B, D, F) show superimposed AP1058 (red) and lectin (green) staining. A and B: section stained with AP1058 and peanut agglutinin (PNA) at ×40 magnification. C and D: section stained as in A and B but at ×100 magnification. E and F: section stained with AP1058 and Lotus tetragonobulus (LTA; ×100 objective). Bar, 200 µm for A and B and 80 µm for C-F.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have reported the cDNA cloning of a novel human member of the p64 family of chloride channel proteins. Because of the very high homology of this new sequence with the previously reported rat cDNA H1, we conclude that this new human sequence is the human version of H1. Our data demonstrate the following. 1) huH1 is widely expressed in human tissues. 2) huH1, like p64 and NCC27, shows aberrantly low mobility on SDS-PAGE gels. 3) In Panc-1 cells, huH1 colocalizes with caveolin, a marker of caveolae and of the trans-Golgi network. It does not primarily reside in the ER. 4) In kidney, H1 is most highly expressed in proximal tubule cells, where it is present in a diffuse apical distribution. H1 is also expressed in a punctate vesicular pattern in glomeruli and in distal nephron.

The p64 gene family. Four distinct members of the p64 family have now been reported with versions of one of the family members described from two species. With the use of various methods, the appearance of novel chloride channel activity has been reported to be associated with expression of three of the four proteins (5, 6, 26). Clearly, the conserved portion of the molecule corresponds to the COOH-terminal half of p64. The two hydrophobic segments originally noted in p64 are particularly highly conserved throughout the family. Sites of maximum variability are 1) the NH2 terminus preceding the first hydrophobic segment, 2) a stretch of ~30 amino acids immediately after the first hydrophobic segment, 3) a segment of ~20 amino acids immediately preceding the second hydrophobic domain, and 4) the extreme COOH terminus. The pattern of conservation and divergence of sequence within the family suggests that the hydrophobic core of the molecule defines the family-specific function of these proteins (i.e., formation of a chloride-permeable pore), whereas the flanking regions confer version-specific patterns of subcellular distribution and perhaps regulation. It is attractive to speculate that this family of proteins may account for intracellular chloride channels with distinct patterns of targeting and regulation that could confer compartment-specific regulation of intravesicular pH. Despite the evidence for association of chloride channel activity with p64 family members, a second possibility is that these proteins are not channels themselves but could function as regulators or accessory subunits of other proteins that in fact provide the pore-forming function. Whether H1 forms multi-subunit complexes with other proteins has not yet been addressed.

Despite the pattern of conservation and the presence of hydrophobic amino acid segments typical of transmembrane domains, it seems that standard models of membrane insertion will not explain the biochemical behavior of p64 family proteins. In this study, we demonstrate that a sizable fraction of huH1 protein behaves as a soluble cytosolic protein in a standard cell fractionation, while the remainder of the protein partitions with membranes where it is resistant to alkaline extraction, the characteristic behavior of an integral membrane protein. A similar behavior has been demonstrated for NCC27 (25), and expression of p64 in HeLa cells leads to the presence of p64 in both soluble and membrane fractions (6). Thus these proteins must be able to assume both a conformation that is soluble in aqueous solution as well as one that can reside in phospholipid membranes. There are a few proteins (e.g., bacterial porins, diphtheria toxin, and, perhaps, pICln) that are known to shuttle between soluble and membrane-inserted forms where they have been proposed to function as pores (23). The structural features that confer this behavior are poorly understood. It is not apparent that p64 shares any structural similarities with these other proteins, which can be either soluble or membrane inserted.

Subcellular distribution of H1. We have addressed the subcellular distribution of H1 in Panc-1 cells with double-fluorescence staining and confocal microscopy. Our results clearly demonstrate that the distribution of H1 is distinct from markers of ER, fluid phase endocytosis, and the transferrin-recycling compartment. H1 does colocalize with caveolin in these cells. Caveolin is known to be present in trans-Golgi network vesicles and is also the major protein of cell surface invaginations known as caveolae. The central small vesicular structures that costain for H1 and caveolin are likely to represent trans-Golgi network vesicles. Trans-Golgi network vesicles are more acidic than the cytoplasm, and the intravesicular pH is an important determinant of trans-Golgi network compartment-specific functions such as sialylation. H1 could function as the chloride conductance, allowing and perhaps regulating acidification of this compartment. Whether the peripheral structures that costain for H1 and caveolin are in fact caveolae remains to be determined. There is considerable debate surrounding the function of caveolae (18). A role for a chloride conductance in these structures is uncertain. Panc-1 cells are derived from a human pancreatic cancer. Whether the distribution of H1 in these cells, particularly in relation to caveolin, is typical of more normal cells remains to be determined.

In our experiments, the distribution of H1 is clearly distinct from a well-characterized marker of ER. However, H1 was previously reported to colocalize with a lipid marker of the ER (5). This lipid marker is extracted from cells if they are permeabilized with detergent. Thus, in the previous report, the cells were labeled with the lipid ER stain, then fixed and reacted with antibodies against H1 without permeabilization of the cells. In our experience, attempts to stain Panc-1 cells with the AP1058 antisera without prior permeabilization resulted in no signal.

H1 in kidney. H1 is expressed in two distinct patterns in kidney cells. In proximal tubule, we find a diffuse apical-subapical staining pattern. At the level of resolution of light microscopy, it is uncertain whether H1 is present in the plasma membrane or in subapical vesicles. In distal nephron and in glomeruli, we find scattered intracellular punctate staining consistent with intracellular vesicles. This pattern is of interest when compared and contrasted with the pattern of NCC27 staining in kidney (25). Like H1, NCC27 is highly expressed in the apical domain of proximal tubule cells, but NCC27 is not present in the punctate staining pattern that H1 demonstrates in distal tubule cells. Both NCC27 and H1 are present in glomeruli, but the H1 staining is more punctate and discrete, whereas the NCC27 staining is more diffuse. Finally, NCC27 antibodies were found to stain vascular smooth muscle in kidney intensely, whereas H1 was not detected. Thus the distributions of H1 and NCC27 overlap in proximal tubule but are distinct in other cells of the kidney. It will be of interest to determine whether the two proteins are present in the same vesicles in proximal tubule or whether they are each in distinct membranes that are interspersed.

Proposed new nomenclature. With the appearance of p64 family members in the literature, the nomenclature has become confusing. Heiss and Poustka (11) proposed the name CLIC, derived from Cl intracellular channel. They proposed that NCC27 be named CLIC1 and the new sequence they identified be named CLIC2. I suggest these names be adopted and extended to the other family members. In this scheme, the original isolate p64 is named CLIC3, and H1 is CLIC4. This nomenclature has been included in parentheses in Fig. 3.


    ACKNOWLEDGEMENTS

I thank Joyce Yee, Amy Driskell, and Shefalee Kapadia for excellent technical assistance.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R29-DK-46212 and by a grant from the Barnes-Jewish Hospital Foundation.

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. §1734 solely to indicate this fact.

Address for reprint requests: J. C. Edwards, Renal Div., Barnes-Jewish Hospital North Campus, 216 S. Kingshighway, St. Louis, MO 63110.

Received 5 August 1998; accepted in final form 7 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-Awqati, Q. Chloride channels of intracellular organelles. Curr. Opin. Cell Biol. 7: 504-508, 1995[Medline].

2.   Bae, H.-R., and A. S. Verkman. Protein kinase A regulates chloride conductance in endocytotic vesicles from proximal tubule. Nature 348: 637-639, 1990[Medline].

3.   Barasch, J., M. D. Gershon, E. A. Nunez, H. Tamir, and Q. Al-Awqati. Thyrotropin induces acidification of the secretory granules of parafollicular cells by increasing the conductance of the granular membrane. J. Cell Biol. 107: 2137-2147, 1988[Abstract].

4.   Bole, D. G., R. Dowin, M. Doriaux, and J. D. Jamieson. Immunocytochemical localization of BiP to rough endoplasmic reticulum: evidence for protein sorting by selective retention. J. Histochem. Cytochem. 37: 1817-1823, 1989[Abstract].

5.   Duncan, R. R., P. K. Westwood, A. Boyd, and R. H. Ashley. Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum. J. Biol. Chem. 272: 23880-23886, 1997[Abstract/Free Full Text].

6.   Edwards, J. C., B. M. Tulk, and P. H. Schlesinger. Functional expression of p64, and intracellular chloride channel protein. J. Membr. Biol. 163: 119-127, 1998[Medline].

7.   Farraggiana, T., F. Malchiodi, A. Prado, and J. Churg. Lectin-peroxidase conjugate reactivity in normal human kidney. J. Histochem. Cytochem. 30: 451-458, 1986[Abstract].

8.   Glenney, J. R., and D. Soppet. Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts. Proc. Natl. Acad. Sci. USA 89: 10517-10521, 1992[Abstract].

9.   Guan, K., and J. E. Dixon. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione-S-transferase. Anal. Biochem. 192: 262-267, 1991[Medline].

10.   Harlow, E., and D. Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988.

11.   Heiss, N. S., and A. Pustka. Genomic structure of a novel chloride channel gene, CLIC2, in Xq28. Genomics 45: 224-228, 1997[Medline].

12.   Kozak, M. An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol. 115: 887-903, 1991[Abstract].

13.   Kurzchalia, T. V., P. Dupree, R. G. Parton, R. Kellner, H. Virta, M. Lehnert, and K. Simons. VIP21, a 21-kD membrane protein, is an integral component of trans-Golgi-network-derived transport vesicles. J. Cell Biol. 118: 1003-1014, 1992[Abstract].

14.   Landry, D. W., M. H. Akabas, C. R. Redhead, A. Edelman, E. J. Cragoe, Jr., and Q. Al-Awqati. Purification and reconstitution of chloride channels from kidney and trachea. Science 244: 1469-1472, 1989[Medline].

15.   Landry, D. W., S. Sullivan, M. Nicolaides, C. Redhead, A. Edelman, M. Field, Q. Al-Awqati, and J. Edwards. Molecular cloning and characterization of p64, a chloride channel protein from kidney microsomes. J. Biol. Chem. 268: 14948-14955, 1993[Abstract/Free Full Text].

16.   Mellman, I., R. Fuchs, and A. Helenius. Acidification of the endocytic and exocytotic pathway. J. Biol. Chem. 55: 663-700, 1986.

17.   Mulberg, A. E., B. M. Tulk, and M. Forgac. Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A phosphorylation. J. Biol. Chem. 266: 20590-20593, 1991[Abstract/Free Full Text].

18.   Parton, R. G. Caveolae and caveolins. Curr. Opin. Cell Biol. 8: 542-548, 1996[Medline].

19.   Redhead, C. R., A. E. Edelman, D. Brown, D. W. Landry, and Q. Al-Awqati. An ubiquitous 64-kDa protein is a component of a chloride channel of plasma and intracellular membranes. Proc. Natl. Acad. Sci. USA 89: 3716-3720, 1992[Abstract].

20.   Redhead, C., S. K. Sullivan, C. Koseki, K. Fujiwara, and J. C. Edwards. Subcellular distribution and targeting of the intracellular chloride channel p64. Mol. Biol. Cell 8: 691-704, 1997[Abstract].

21.   Rothberg, K., J. E. Heuser, W. C. Donzell, Y.-S. Ying, J. R. Glenney, and R. G. W. Anderson. Caveolin, a protein component of caveolae membrane coats. Cell 68: 673-682, 1992[Medline].

22.   Schlesinger, P. M., H. C. Blair, S. L. Teitelbaum, and J. C. Edwards. Characterization of the chloride channel of osteoclast ruffled membrane and its role in bone resorption. J. Biol. Chem. 272: 18636-18643, 1997[Abstract/Free Full Text].

23.   Strange, K., F. Emma, and P. S. Jackson. Cellular and molecular physiology of volume-sensitive anion channels. Am. J. Physiol. 270 (Cell Physiol. 39): C711-C730, 1996[Abstract/Free Full Text].

24.   Tamir, H., I. Piscopo, K. Liu, S. Hsiung, M. Adlersberg, M. Nicolaides, Q. Al-Awqati, and M. Gershon. Secretagogue-induced gating of chloride channels in the secretory vesicles of parafollicular cells. Endocrinology 135: 2045-2057, 1994[Abstract].

25.   Tulk, B. M., and J. C. Edwards. NCC27, a homolog of intracellular C1 channel, p64, is expressed in brush border of renal proximal tubule. Am. J. Physiol. 274 (Renal Physiol. 43): F1140-F1149, 1998[Abstract/Free Full Text].

26.   Valenzuaela, S. M., D. K. Martin, S. B. Por, J. M. Robbins, K. Warton, M. R. Bootcov, P. R. Schofield, T. J. Campbell, and S. N. Breit. Molecular cloning and expression of a chloride ion channel of cell nuclei. J. Biol. Chem. 272: 12575-12582, 1997[Abstract/Free Full Text].

27.   Yamashiro, D. J., B. Tycko, S. R. Fluss, and F. R. Maxfield. Segregation of transferrin to a mildly acidic (pH 6.5) paraGolgi compartment in the recycling pathway. Cell 37: 789-800, 1984[Medline].


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