Molecular Cloning and Characterization of an Intracellular Chloride Channel in the Proximal Tubule Cell Line, LLC-PK1
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m004840200
ISSN1083-351X
AutoresLara K. Dowland, Valérie A. Luyckx, Alissa H. Enck, Baudouin Leclercq, Alan S.L. Yu,
Tópico(s)Neuroscience and Neural Engineering
ResumoCLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate ≫ Cl− ≈ Br− > I− > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK a of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking. CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate ≫ Cl− ≈ Br− > I− > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK a of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking. polymerase chain reaction phosphate-buffered saline fluorescein isothiocyanate 2-(N-morpholino)ethanesulfonic acid kilobase pairs Functional evidence for chloride channels has been demonstrated in many intracellular organelles including the endoplasmic reticulum, Golgi, endosomes, lysosomes, and synaptic vesicles (1Al-Awqati Q. Curr. Opin. Cell Biol. 1995; 7: 504-508Crossref PubMed Scopus (96) Google Scholar). Although their molecular identity in most cases is unknown, members of several structurally distinct chloride channel gene families such as p64 (2Redhead C. Sullivan S.K. Koseki C. Fujiwara K. Edwards J.C. Mol. Biol. Cell. 1997; 8: 691-704Crossref PubMed Scopus (38) Google Scholar,3Tulk B.M. Edwards J.C. Am. J. Physiol. 1998; 274: F1140-F1149PubMed Google Scholar), cystic fibrosis transmembrane regulator (4Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar), and CLC6 (5Buyse G. Trouet D. Voets T. Missiaen L. Droogmans G. Nilius B. Eggermont J. Biochem. J. 1998; 330: 1015-1021Crossref PubMed Scopus (46) Google Scholar) have recently been found to be expressed intracellularly. Dent's disease is an X-linked inherited disorder characterized by hypercalciuria, nephrocalcinosis, and renal failure, associated with a Fanconi-like syndrome, low molecular weight proteinuria, and rickets (6Frymoyer P.A. Scheinman S.J. Dunham P.B. Jones D.B. Hueber P. Schroeder E.T. N. Engl. J. Med. 1991; 325: 681-686Crossref PubMed Scopus (135) Google Scholar, 7Bolino A. Devoto M. Enia G. Zoccali C. Weissenbach J. Romero G. Eur. J. Hum. Genet. 1993; 1: 269-279Crossref PubMed Scopus (61) Google Scholar, 8Wrong O.M. Norden A.G.W. Feest T.G. Q. J. Med. 1994; 87: 473-493Google Scholar), which is caused by inactivating mutations in the renal chloride channel, CLC5 (9Lloyd S.E. Pearce S.H.S. Fisher S.E. Steinmeyer K. Schwappach B. Scheinman S.J. Harding B. Bolino A. Devoto M. Goodyer P. Rigden S.P.A. Wrong O. Jentsch T.J. Craig I.W. Thakker R.V. Nature. 1996; 379: 445-449Crossref PubMed Scopus (620) Google Scholar). CLC5 has been shown to be expressed intracellularly in endosomes of the renal proximal tubule, where it colocalizes with the vacuolar H+-ATPase (10Günther W. Lüchow A. Cluzeaud F. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8075-8080Crossref PubMed Scopus (385) Google Scholar, 11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar, 12Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (251) Google Scholar, 13Sakamoto H. Sado Y. Naito I. Kwon T.H. Inoue S. Endo K. Kawasaki M. Uchida S. Nielsen S. Sasaki S. Marumo F. Am. J. Physiol. 1999; 277: F957-F965PubMed Google Scholar), but its physiological role is unknown. It has been postulated that CLC5 may provide an electrical shunt to dissipate the potential gradient across the endosomal membrane generated by electrogenic proton transport into the endosomal lumen. Thus, loss of CLC5 function would be predicted to impair endosomal acidification, which is normally required for endocytosis (14Gekle M. Mildenberger S. Freudinger R. Silbernagl S. Am. J. Physiol. 1995; 268: F899-F906PubMed Google Scholar), trafficking to lysosomes (15van Weert A.W.M. Dunn K.W. Geuze H.J. Maxfield F.R. Stoorvogel W. J. Cell Biol. 1995; 130: 821-834Crossref PubMed Scopus (299) Google Scholar), and recycling back to the surface (16Presley J.F. Mayor S. McGraw T.E. Dunn K.W. Maxfield F.R. J. Biol. Chem. 1997; 272: 13929-13936Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In this model, low molecular weight proteinuria in Dent's disease occurs because of failure of endocytosis from the proximal tubule lumen, which is the normal pathway for reabsorption of low molecular weight proteins (17Sumpio B.E. Hayslett J.P. Q. J. Med. 1985; 57: 611-635PubMed Google Scholar). The occurrence of a Fanconi-like syndrome may be explained by internalization of apical membrane sodium-coupled solute transporters into endosomal vesicles but failure to recycle these transporters back to the surface. The hypercalciuria in this disease appears to be absorptive in origin (18Luyckx V.A. Leclercq B. Dowland L. Yu A.S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12174-12179Crossref PubMed Scopus (67) Google Scholar) and may be due to abnormal regulation of proximal tubule 25-hydroxyvitamin D 1-hydroxylase activity. How this might be possible, given that the 1-hydroxylase is located on the inner membrane of mitochondria, remains unknown. To understand the cellular role of CLC5 and test these hypotheses, it will be critical to have a cell culture model that normally expresses CLC5 and physiologically resembles native proximal tubule epithelium. LLC-PK1 is a well characterized cell line that is an excellent model of the proximal tubule epithelium (19Hull R.N. Cherry W.R. Weaver G.W. In Vitro. 1976; 12: 670-677Crossref PubMed Scopus (492) Google Scholar). It shares many of the key properties of native proximal tubules that CLC5 might be predicted to regulate, including expression of apical transporters such as the sodium-hydrogen exchanger, NHE3 (20Shugrue C.A. Obermuller N. Bachmann S. Slayman C.W. Reilly R.F. J. Am. Soc. Nephrol. 1999; 10: 1649-1657Crossref PubMed Google Scholar), and the high affinity sodium-glucose cotransporter, SGLT1 (21Yet S.F. Kong C.T. Peng H. Lever J.E. J. Cell. Physiol. 1994; 158: 506-512Crossref PubMed Scopus (21) Google Scholar), endosomal expression of megalin (22Nielsen R. Birn H. Moestrup S.K. Nielsen M. Verroust P. Christensen E.I. J. Am. Soc. Nephrol. 1998; 9: 1767-1776PubMed Google Scholar), and the vacuolar H+-ATPase (23Rodman J.S. Stahl P.D. Gluck S. Exp. Cell. Res. 1991; 192: 445-452Crossref PubMed Scopus (15) Google Scholar), receptor-mediated endocytosis of proteins from the luminal surface (24Thakkar H. Lowe P.A. Price C.P. Newman D.J. Kidney Int. 1998; 54: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and 25-hydroxyvitamin D 1-hyroxylase activity (25Condamine L. Menaa C. Vrtovsnik F. Vztovsnik F. Friedlander G. Garabedian M. J. Clin. Invest. 1994; 94: 1673-1679Crossref PubMed Scopus (96) Google Scholar). We report here the cloning of a porcine CLC5 ortholog (pCLC5), its localization to Golgi and endosomal vesicles of LLC-PK1 cells, and its functional characterization by heterologous expression in the Xenopusoocyte system. LLC-PK1 cells (American Type Culture Collection, catalog number CL-101) were cultured to confluence on plastic plates in Dulbecco's modified Eagle's medium with 5% fetal bovine serum at 37 °C in 5% CO2. Transient transfections were performed on 70–80% confluent cells by lipofection using LipofectAMINE Plus Reagent (Life Technologies, Inc.), according to the manufacturer's instructions. Expression of enhanced green fluorescent protein by cotransfection of pEGFP-C2 (CLONTECH) was routinely used to assess transfection efficiency (typically 10–20% of cells) and identify positive cells. Northern blots under high stringency conditions (hybridization at 65 °C in the presence of 50% formamide, final washes at 65 °C in 0.1× SSCP) were performed on poly(A)+ RNA isolated from these cells, using digoxigenin-labeled antisense riboprobes of rat CLC5, CLC4, and CLC3 (11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar, 26Yu A.S.L. Hebert S.C. Lee S.-L. Brenner B.M. Lytton J. Am. J. Physiol. 1992; 263: F680-F685PubMed Google Scholar) and detected by chemiluminescence with CDP-Star and the Dig Genius System (Roche Molecular Biochemicals). To clone the pCLC5 cDNA, degenerate oligonucleotide primers (sense, ATHTCTGCNCAYTGGATGAC; antisense, TAYTTRTTRAARCARTGRCA) were designed to the conserved amino acid sequences of human (27Fisher S.E. van Bakel I. Lloyd S.E. Pearce S.H.S. Thakker R.V. Craig I.W. Genomics. 1995; 29: 598-606Crossref PubMed Scopus (137) Google Scholar), rat (28Steinmeyer K. Schwappach B. Bens M. Vanderwalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Crossref PubMed Scopus (250) Google Scholar), andXenopus (29Lindenthal S. Schmieder S. Ehrenfeld J. Wills N.K. Am. J. Physiol. 1997; 273: C1176-C1185Crossref PubMed Google Scholar) CLC5. By homology-based reverse transcription-polymerase chain reaction (PCR)1 amplification of LLC-PK1 cell mRNA, a 427-base pair cDNA was isolated that was highly homologous to CLC5 in other species. The remainder of the cDNA encompassing the coding sequence was cloned by a combination of primer walking and rapid amplification of cDNA ends and then sequenced in both directions by the dideoxynucleotide chain termination method. To exclude PCR misincorporation errors, each nucleotide sequence within the open reading frame was corroborated at least three times using overlapping clones amplified from independent PCRs. As a positive control, a rat CLC5 cDNA (rCLC5) containing the full coding region, identical to the published sequence (28Steinmeyer K. Schwappach B. Bens M. Vanderwalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Crossref PubMed Scopus (250) Google Scholar), was amplified by PCR and cloned in a similar fashion. Constructs encoding the rCLC5 coding region with a FLAG epitope tag (DYKDDDDK) at either the N or the C terminus and an optimized Kozak translation initiation sequence were generated by PCR-based oligonucleotide mutagenesis and verified by DNA sequencing. To generate mammalian expression constructs for transfection into LLC-PK1 cells, pCLC5 and rCLC5 cDNA were cloned into the plasmid vector, pcDNA3 (Invitrogen), under the control of a constitutive cytomegalovirus promoter. To generate vectors with convenient restriction sites suitable for expression in Xenopusoocytes, we adapted a pTLN II vector (30Lorenz C. Pusch M. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13362-13366Crossref PubMed Scopus (215) Google Scholar) in which the NcoI site and occult Kozak sequence had previously been deleted by mung bean exonuclease and replaced with an ApaI site (gifts of Dr. Peying Fong and Dr. Joseph Mindell). The polylinker was next removed with ApaI and BstEII, the ends made blunt with Klenow polymerase, and the polylinker of pcDNA3.1-zeo (Invitrogen) excised with PmeI and inserted into this site in both orientations to generate the vectors pOX(+) (oriented so that SP6 transcription proceeds from AflII to ApaI), and pOX(−). These vectors have the advantage that inserts cloned into the polylinker can be easily swapped as a restriction cassette into any of the pcDNA3.1 and pIND series of vectors for mammalian cell expression, which have the same polylinker, without the need to create new restriction sites. The coding sequences of pCLC5 and rCLC5 were cloned into the KpnI and XbaI sites of pOX(+). In vitro translation of pCLC5 and rCLC5 cDNA was performed in the presence or absence of canine microsomal membranes using the TNT-coupled reticulocyte lysate system (Promega), and the products were electrophoresed on a 7.5% denaturing SDS-polyacrylamide gel and electrotransferred onto polyvinylidene difluoride membrane. Calibration of molecular weights was achieved by electrophoresing in parallel a lane of unstained protein molecular weight standards. In the negative control sample, cDNA was omitted from the reaction. To detect all translated products, biotinylated lysine tRNA (Transcend tRNA, Promega) was included in the translation reaction, and the membrane was blotted with streptavidin-alkaline phosphatase followed by a chemiluminescent substrate, according to the manufacturer's instructions. To detect CLC5 immunoreactivity, Transcend tRNA was omitted, and the membranes were immunoblotted with the affinity-purified polyclonal antibody fractions, C1 and C2, exactly as described previously (11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar). To detect FLAG epitope-tagged proteins, immunoblots were performed with the M2 monoclonal antibody (Sigma) at a concentration of 10 μg/ml. To detect pCLC5 in LLC-PK1 membranes, confluent cultured cells were washed in phosphate-buffered saline (PBS), scraped off the plate, and suspended in sucrose/histidine buffer containing 0.25 msucrose, 30 mm histidine, 1 mm EDTA, 2× Complete protease inhibitor mixture (Roche Molecular Biochemicals), adjusted to pH 7.4. The cells were homogenized by 6 passes through a 25-gauge needle, centrifuged at 1000 × g for 5 min to sediment nuclei, and the post-nuclear supernatant centrifuged at 100,000 × g for 20 min to yield a microsomal membrane pellet. This was then resuspended in 2% SDS-containing gel-loading buffer for electrophoresis and immunoblotting as described above. Deglycosylation was performed by boiling the protein samples for 2 min in phosphate-buffered saline (PBS) containing 50 mmβ-mercaptoethanol, 10 mm EDTA, and 0.1% sodium dodecyl sulfate and then adding 0.5% Nonidet P-40 and 18 units/mlN-glycosidase F (Sigma) and incubating 12–16 h at 37 °C. Native or transfected LLC-PK1 cells grown on glass coverslips to 85% confluence were washed in PBS and fixed in methanol at −80 °C for 10 min. To entrap fluorescent markers in early endosomes of the apical endocytotic pathway, LLC-PK1 cells were incubated prior to fixation in Dulbecco's modified Eagle's medium containing fluorescein isothiocyanate (FITC)-labeled conjugates (all from Sigma) of dextran (1 mg/ml, average molecular mass 12 kDa), albumin (1 mg/ml), or Ricinus communis agglutinin (ricin, 0.1 mg/ml) for 15 min at 37 °C. Immunostaining of endogenous pCLC5 with the C1 antibody, using amplification by the direct tyramide signal amplification kit (PerkinElmer Life Sciences), was performed exactly as described previously (11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar). For double-staining studies with the Na+-K+-ATPase, a monoclonal antibody (gift of Dr. Kevin Bush) was used without dilution. For immunodetection of the FLAG epitope, the M2 anti-FLAG monoclonal antibody (Sigma) was used, diluted 1:100 into PBS containing 1% bovine serum albumin, 0.3% Triton X-100, and 0.2% skimmed milk. ARF1 rabbit polyclonal antibody (gift of Dr. Vladimir Marshansky), used as a marker of the Golgi complex (31Marshansky V. Bourgoin S. Londono I. Bendayan M. Vinay P. Electrophoresis. 1997; 18: 538-547Crossref PubMed Scopus (29) Google Scholar, 32Londono I. Marshansky V. Bourgoin S. Vinay P. Bendayan M. Kidney Int. 1999; 55: 1407-1416Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), was applied at a dilution of 1:250, also in the presence of 0.3% Triton X-100. Slides were visualized with a Bio-Rad MRC-1024 confocal krypton-argon laser scanning microscope. For double-labeled slides, images were acquired sequentially for each fluorophore in single label mode to minimize "bleed-through" between channels. Each pair of images was then imported into Adobe Photoshop 3.0, false color added, and merged to generate dual-color images. pOX(+) plasmid constructs encoding pCLC5 and rCLC5 cDNA were linearized with MluI, and capped cRNA was transcribed with SP6 polymerase. Stage V/VI Xenopus laevisoocytes were digested with collagenase, defolliculated, and injected with 50 nl of cRNA (1 μg/μl) or sterile water for negative controls and then incubated at 18 °C in ND96 (containing in mm: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, adjusted to pH 7.4) with 50 μg/ml gentamicin for 4–6 days. To detect CLC5 expression in the oocytes, total microsomal membranes were prepared. Oocytes were suspended in sucrose/histidine buffer, lysed by triturating 30 times through a 200-μl volume pipette tip, and then centrifuged at 1000 × g for 10 min to sediment cellular debris. The floating layer of yellow lipid was removed with a cotton-tipped applicator, and the supernatant was recovered and centrifuged at 100,000 × g for 20 min to yield a microsomal membrane pellet. To identify the subset of proteins expressed at the cell surface, a modification of a published surface biotinylation method (33Chillaron J. Estevez R. Samarzija I. Waldegger S. Testar X. Lang F. Zorzano A. Busch A. Palacin M. J. Biol. Chem. 1997; 272: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) was used. Between 40 and 80 oocytes were washed in OR2 (containing in mm: NaCl 82.5, KCl 2, MgCl2 1, Na phosphate 10, adjusted to pH 7.4) and then incubated in OR2 containing 4 mg/ml EZ-Link sulfo-NHS-biotin (Pierce) for 10 min at room temperature (22 °C). The reaction was then stopped by adding 0.5 mglycine, pH 7.4, and the oocytes were washed twice in glycine and twice in OR2. The oocytes were then suspended in lysis buffer containing 2% Nonidet P-40, 150 mm NaCl, 2 mmCaCl2, 20 mm Tris-HCl, 2× Complete protease inhibitor, adjusted to pH 7.4. The oocytes were lysed by trituration, centrifuged at 1000 × g for 10 min, the lipid layer removed, and the supernatant dialyzed overnight against SAV buffer containing 0.3% Nonidet P-40, 0.5 m NaCl, 1 mmCaCl2, 1 mm MgCl2, 10 mm Tris-HCl, adjusted to pH 8. The lysate was then centrifuged at 16,000 × g for 30 min to remove insoluble material. The supernatant was incubated with streptavidin-agarose beads overnight at 4 °C. The next day, the agarose was sedimented by brief centrifugation, and the supernatant, which contains non-biotinylated proteins, was recovered. The pellet was washed 4 times in SAV buffer, and the biotinylated proteins were recovered by solubilization in 1% SDS. The protein recovery of each fraction was determined by a detergent-compatible colorimetric assay (DC Protein Assay, Bio-Rad). Equal amounts of each protein sample (25 μg) were loaded in each lane of a denaturing 7.5% SDS-polyacrylamide gel for electrophoresis and immunoblotted with C1 antiserum. Two-microelectrode voltage clamp studies of Xenopus oocytes were performed at room temperature using a Clampator-1B (Dagan Instruments, Minneapolis, MN) controlled by pCLAMP 8.0 software (Axon Instruments, Foster City, CA). Microelectrodes were filled with 3 m KCl and had tip resistances of 0.5–2 mΩ. Oocytes were clamped at a holding potential of −50 mV, and voltage steps from −120 to +100 mV in 20 mV increment were applied. Data were acquired at 10 kHz and low pass-filtered at 2 kHz. For control studies, oocytes were superfused with ND96 modified to contain 98 mm NaCl and 0.3 mm CaCl2so as to minimize activation of the endogenous calcium-activated chloride conductance in oocytes. In anion substitution experiments, 80 mm chloride was substituted with an equimolar concentration of another anion (except where otherwise specified), and the bath Ag/AgCl electrode was protected by a KCl-agar bridge. In studies of the effect of lowering bath pH, MES was substituted for HEPES as the buffer and titrated to the desired pH with NaOH. Each experimental result reported was confirmed in at least 3 distinct batches of oocytes isolated from different frogs on separate days. By high stringency Northern blot analysis with a rat CLC5 probe, LLC-PK1 cells appear to express mRNA encoding a CLC5 homolog. A strong band of 9.5 kb was observed, similar to the size of human and rat CLC5 mRNA (28Steinmeyer K. Schwappach B. Bens M. Vanderwalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Crossref PubMed Scopus (250) Google Scholar, 34Fisher S.E. Black G.C.M. Lloyd S.E. Hatchwell E. Wrong O. Thakker R.V. Craig I.W. Hum. Mol. Genet. 1994; 3: 2053-2059PubMed Google Scholar), as well as a weaker band of approximately 3 kb, similar in size to the X. laevis CLC5 mRNA (29Lindenthal S. Schmieder S. Ehrenfeld J. Wills N.K. Am. J. Physiol. 1997; 273: C1176-C1185Crossref PubMed Google Scholar) (Fig.1). The pCLC5 cDNA was isolated and sequenced from multiple clones by homology-based reverse transcription-PCR of mRNA from LLC-PK1 cells. It contains a long open reading frame of over 2 kb. The first ATG codon after an in-frame upstream stop codon is not in the context of a suitable Kozak consensus sequence (35Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar), but the second ATG is, and the latter has therefore been assigned as the initiator codon (Fig. 2). The resultant open reading frame of 2238 base pairs predicts a protein of 746 amino acids with a molecular mass of 83 kDa (Fig.3 A). The protein sequence of pCLC5 is the same length as and highly homologous with rat CLC5 (rCLC5, 97% amino acid identity (28Steinmeyer K. Schwappach B. Bens M. Vanderwalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Crossref PubMed Scopus (250) Google Scholar)) and is also very similar to human CLC5 (98% amino acid identity in overlapping region (27Fisher S.E. van Bakel I. Lloyd S.E. Pearce S.H.S. Thakker R.V. Craig I.W. Genomics. 1995; 29: 598-606Crossref PubMed Scopus (137) Google Scholar)) except for the absence of a 20-residue stretch at the N-terminal of human CLC5. We conclude that pCLC5 is the porcine ortholog of CLC5.Figure 2Comparison of the upstream coding sequence and 5′-untranslated region of pCLC5 with that of rat and human CLC5 (27Fisher S.E. van Bakel I. Lloyd S.E. Pearce S.H.S. Thakker R.V. Craig I.W. Genomics. 1995; 29: 598-606Crossref PubMed Scopus (137) Google Scholar, 28Steinmeyer K. Schwappach B. Bens M. Vanderwalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Crossref PubMed Scopus (250) Google Scholar). Filled circles above the sequence denote nucleotides that are identical in all three. The predicted initiator codon is underlined, and the nearest upstream in-frame stop codon is double underlined. An alternative initiator codon in pCLC5, which is not in the context of a good Kozak consensus sequence, is indicated by an arrowhead.View Large Image Figure ViewerDownload (PPT)Figure 3Sequence analysis of pCLC5. A, complete deduced amino acid sequence of pCLC5, indicating the locations of the putative transmembrane domains, D1 to D10 (underlined), N-glycosylation site (asterisk), and protein kinase A phosphorylation sites (arrowheads). B, Kyte-Doolittle hydropathy plot using a window of 20 residues. The 13 hydrophobic domains are denoted by the boxes at the top, of which 10 (shaded) are predicted transmembrane domains.View Large Image Figure ViewerDownload (PPT) The hydropathy plot of pCLC5 is very similar to that of other CLC proteins and predicts 13 hydrophobic domains (Fig. 3 B). Based on glycosylation scanning and protease protection studies in CLC1 (36Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar), 10 of these domains are believed to span the membrane, with both N and C termini located on the cytosolic side of the membrane. pCLC5 has two potential N-glycosylation sites (Fig.3 A). One at residue 408 is predicted to be in the extracellular loop between the 7th and 8th transmembrane segments, is conserved in all CLC family members, has been shown to be used in vitro in CLC-K1, CLC-K2, and CLC1 (36Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar, 37Kieferle S. Fong P. Bens M. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6943-6947Crossref PubMed Scopus (242) Google Scholar), and is likely abona fide glycosylation site. The other site at residue 38 is predicted to be intracellular. There are two overlapping cAMP-dependent phosphorylation consensus sequences in the cytosolic loop between the 6th and 7th transmembrane domain and four potential protein kinase C phosphorylation sites that are predicted to be located on the cytosolic side (Fig. 3 A). We previously generated polyclonal antiserum against a fusion protein containing the C terminus of rCLC5 (11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar), and we affinity-purified from it two fractions, C1 (which reacts primarily with rCLC5 but also cross-reacts with CLC3 and CLC4), and C2 (which is specific for rCLC5). To determine if these sera could be used for immunodetection of pCLC5, we firstin vitro translated pCLC5. As shown by the streptavidin blot in Fig. 4 A, pCLC5 was successfully translated to yield a protein with an apparent molecular mass of 65 kDa, identical to the apparent size of in vitrotranslated rCLC5, and absent from the negative control lane. By Western blotting, the C1 antibody reacts strongly with the translated pCLC5 protein (Fig. 4 A), but C2 serum did not recognize pCLC5 at all (data not shown). Furthermore, C1 recognizes a band of the same apparent size in LLC-PK1 100,000 × g membranes (Fig.4 B). Because the apparent molecular mass of pCLC5 (and of rCLC5) on our denaturing gels (65 kDa) is lower than the 83-kDa size predicted from its amino acid sequence, and anomalous migration on protein gels has not been reported for any other CLC protein, the potential contribution of post-translational modification of pCLC5 and rCLC5 proteins by glycosylation and proteolysis was assessed. In vitro translated pCLC5 and rCLC5 polypeptides could both be glycosylated by canine microsomal membranes, yielding broad bands that migrate with an apparent molecular mass of ∼80–85 kDa (Fig.4 B), identical in size to the C1-immunoreactive band in rat kidney cortex (11Luyckx V.A. Goda F.O. Mount D.B. Nishio T. Hall A. Hebert S.C. Hammond T.G. Yu A.S.L. Am. J. Physiol. 1998; 275: F761-F769Crossref PubMed Google Scholar). This suggests that mature glycosylated CLC5 protein (as found in rat kidney cortex) migrates at 80–85 kDa, whereas the unglycosylated polypeptide (as found in LLC-PK1 cells) migrates at 65 kDa. Consistent with this, deglycosylation by N-glycosidase F collapsed the C1-immunoreactive band in rat kidney cortex from 80 to 85 kDa down to 65 kDa, but had no effect on the 65-kDa band in LLC-PK1 cells (Fig. 4 B). The finding of bands that migrate with a molecular weight that is consistently lower than predicted could also be explained by initiation of translation from an initiator codon downstream of the predicted start site, giving rise to a protein truncated at the N terminus or by proteolytic degradation. To address these possibilities, we generated epitope-tagged rCLC5 constructs that encode the FLAG octapeptide epitope fused directly to the N or C terminus. Both constructs could be translated in vitro and yielded bands that run only very slightly higher on the gel than those of the untagged proteins (Fig.4 A). Furthermore, both tagged polypeptides were immunoreactive with a monoclonal antibody directed against the FLAG epitope, demonstrating that neither the N nor the C terminus had been truncated. We conclude that the position of pCLC5 and rCLC5 bands on denaturing protein gels is the consequence of anomalous electrophoretic mobility of the n
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