ClC-3 Is a Fundamental Molecular Component of Volume-sensitive Outwardly Rectifying Cl− Channels and Volume Regulation in HeLa Cells and Xenopus laevis Oocytes
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m205132200
ISSN1083-351X
AutoresMarcela A. Hermoso, Christina Satterwhite, Yaniré N. Andrade, Jorge Hidalgo, Sean M. Wilson, Burton Horowitz, Joseph R. Hume,
Tópico(s)Magnesium in Health and Disease
ResumoVolume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells andX. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes. Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells andX. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes. regulatory volume decrease volume-sensitive osmolyte and anion channel antisense oligonucleotide mismatched oligonucleotide antibody X. laevis oocyte ClC-3 fluorescein isothiocyanate 5-(and -6)-carboxytetramethylrhodamine nucleotide(s) analysis of variance N-methyl-d-glucamine cystic fibrosis transmembrane conductance regulator A basic homeostatic function of all cells is to regulate their cell volume when exposed to either intracellular or extracellular anisosmotic conditions. Most vertebrate cells respond to hypoosmotically induced cell swelling by actively decreasing their volume, a process known as regulatory volume decrease (RVD)1 (1Hoffmann E.K. Dunham P.B. Int. Rev. Cytol. 1995; 161: 173-262Crossref PubMed Scopus (443) Google Scholar). The RVD response occurs by efflux of K+, Cl−, and organic osmolytes accompanied by osmotically obliged water loss. Approximately 70% of the osmolyte loss during the RVD response is accounted for by loss of KCl via separate conductive pathways (2Lang F. Busch G.L. Volkl H. Cell. Physiol. Biochem. 1998; 8: 1-45Crossref PubMed Scopus (293) Google Scholar, 3Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar). Cell volume regulatory mechanisms are fundamentally important in physiological processes such as cell differentiation, cell growth, apoptosis, and cellular metabolism (4Waldegger S. Steuer S. Risler T. Heidland A. Capasso G. Massry S. Lang F. Nephrol. Dial. Transplant. 1998; 13: 867-874Crossref PubMed Scopus (43) Google Scholar). Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Blockade of VSOACs were found to suppress RVD in a variety of cell types (5Okada Y. Am. J. Physiol. 1997; 273: C755-C789Crossref PubMed Google Scholar). Macroscopic outwardly rectifying VSOAC currents are characterized by activation after cell volume increase (6Diaz M. Valverde M.A. Higgins C.F. Rucareanu C. Sepulveda F.V. Pflugers Arch. 1993; 422: 347-353Crossref PubMed Scopus (121) Google Scholar), a SCN > I > Br > Cl > F > gluconate permeability sequence (6Diaz M. Valverde M.A. Higgins C.F. Rucareanu C. Sepulveda F.V. Pflugers Arch. 1993; 422: 347-353Crossref PubMed Scopus (121) Google Scholar), time-dependent inactivation at positive potentials (7Worrell R.T. Butt A.G. Cliff W.H. Frizzell R.A. Am. J. Physiol. 1989; 256: C1111-C1119Crossref PubMed Google Scholar), inhibition by tamoxifen, 1,9-dideoxyforskolin, and stilbene derivatives (8Valverde M.A. Mintenig G.M. Sepulveda F.V. Pflugers Arch. 1993; 425: 552-554Crossref PubMed Scopus (86) Google Scholar), dependence on intracellular ATP (6Diaz M. Valverde M.A. Higgins C.F. Rucareanu C. Sepulveda F.V. Pflugers Arch. 1993; 422: 347-353Crossref PubMed Scopus (121) Google Scholar), and a single channel conductance in the range of 20–40 pS (9Strange K. Emma F. Jackson P.S. Am. J. Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar). These biophysical and pharmacological characteristics are considered to represent classic characteristics of VSOACs. However, variations in the pharmacological characteristics, voltage dependence, and signaling mechanisms responsible for channel activation have been described in different cell types (5Okada Y. Am. J. Physiol. 1997; 273: C755-C789Crossref PubMed Google Scholar, 10Nilius B. Eggermont J. Voets T. Buyse G. Manolopoulos V. Droogmans G. Prog. Biophys. Mol. Biol. 1997; 68: 69-119Crossref PubMed Scopus (322) Google Scholar). These findings have led to the current debate as to whether a group of channel proteins rather than just one protein may comprise the overall biophysical and pharmacological characteristics of VSOACs described in different cell types (11Stutzin A. Torres R. Oporto M. Pacheco P. Eguiguren A.L. Cid L.P. Sepulveda F.V. Am. J. Physiol. 1999; 277: C392-C402Crossref PubMed Google Scholar, 12Valverde M.A. Curr. Opin. Cell Biol. 1999; 11: 509-516Crossref PubMed Scopus (40) Google Scholar). The protein or proteins conclusively responsible for the native VSOAC current have yet to be identified. Several proteins have been implicated as candidates for VSOAC, including P-glycoprotein (13Valverde M.A. Diaz M. Sepulveda F.V. Gill D.R. Hyde S.C. Higgins C.F. Nature. 1992; 355: 830-833Crossref PubMed Scopus (524) Google Scholar), pICln (14Paulmichl M., Li, Y. Wickman K. Ackerman M. Peralta E. Clapham D. Nature. 1992; 356: 238-241Crossref PubMed Scopus (310) Google Scholar), ClC-2 (15Thiemann A. Grunder S. Pusch M. Jentsch T.J. Nature. 1992; 356: 57-60Crossref PubMed Scopus (510) Google Scholar), and ClC-3 (16Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar, 17Wang L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (114) Google Scholar). P-glycoprotein is now thought to be a regulator of this channel (18Valverde M.A. Bond T.D. Hardy S.P. Taylor J.C. Higgins C.F. Altamirano J. Alvarez-Leefmans F.J. EMBO J. 1996; 15: 4460-4468Crossref PubMed Scopus (89) Google Scholar, 19Bond T.D. Higgins C.F. Valverde M.A. Methods Enzymol. 1998; 292: 359-370Crossref PubMed Scopus (25) Google Scholar) and pICln is also thought to possibly regulate VSOAC; however, its precise role is still under investigation (20Furst J. Bazzini C. Jakab M. Meyer G. Konig M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pflugers Arch. 2000; 440: 100-115Crossref PubMed Google Scholar, 21Li C. Breton S. Morrison R. Cannon C.L. Emma F. Sanchez-Olea R. Bear C. Strange K. J. Gen. Physiol. 1998; 112: 727-736Crossref PubMed Scopus (40) Google Scholar). ClC-2, when expressed, has been shown to display biophysical and pharmacological characteristics that differ from native VSOACs and has also been shown not to contribute to RVD (15Thiemann A. Grunder S. Pusch M. Jentsch T.J. Nature. 1992; 356: 57-60Crossref PubMed Scopus (510) Google Scholar, 22Bond T.D. Ambikapathy S. Mohammad S. Valverde M.A. J. Physiol. (Lond.). 1998; 511: 45-54Crossref Scopus (61) Google Scholar). ClC-3 was originally cloned from rat kidney by Kawasakiet al. (23Sasaki S. Uchida S. Kawasaki M. Adachi S. Marumo F. Jpn. J. Physiol. 1994; 44 Suppl. 2: S3-S8PubMed Google Scholar, 24Uchida S. Kawasaki M. Sasaki S. Marumo F. Jpn. J. Physiol. 1994; 44 Suppl. 2: S55-S62PubMed Google Scholar), and, when expressed, currents showed basal activation and inhibition by phorbol esters and Ca2+ (25Kawasaki M. Uchida S. Monkawa T. Miyawaki A. Mikoshiba K. Marumo F. Sasaki S. Neuron. 1994; 12: 597-604Abstract Full Text PDF PubMed Scopus (218) Google Scholar,26Kawasaki M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar). ClC-3 cloned from guinea pig ventricular myocytes was proposed to be the molecular candidate responsible for cardiac VSOACs (16Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). When expressed in NIH3T3 cells ClC-3 gave rise to a basally active chloride conductance that was modulated by cell volume, strongly inhibited by protein kinase C, displayed outward rectification with a unitary slope conductance of 40 pS, an anion selectivity sequence of I > Cl > Asp, voltage-dependent inactivation at positive potentials, and similar pharmacological characteristics as native VSOACs (27Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar). Furthermore, Wang et al. (17Wang L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (114) Google Scholar) showed in bovine epithelial cells that ClC-3 antisense treatment delayed the rate of activation of native VSOAC and reduced its amplitude by up to 60% in a dose-dependent manner. Also, a polyclonal anti-ClC-3 antibody (Ab) was shown to functionally inhibit native VSOACs in guinea pig cardiac cells, canine pulmonary arterial smooth muscle cells, andXenopus laevis oocytes (28Duan D. Zhong J. Hermoso M. Satterwhite C.M. Rossow C.F. Hatton W.J. Yamboliev I. Horowitz B. Hume J.R. J. Physiol. 2001; 531: 437-444Crossref PubMed Scopus (82) Google Scholar). Although the ClC-3 hypothesis has received additional experimental support from other laboratories, (29Coca-Prados M. Sanchez-Torres J. Peterson-Yantorno K. Civan M.M. J. Membr. Biol. 1996; 150: 197-208Crossref PubMed Scopus (74) Google Scholar, 30Schmid A. Blum R. Krause E. J. Physiol. (Lond.). 1998; 513: 453-465Crossref Scopus (22) Google Scholar, 31von Weikersthal S.F. Barrand M.A. Hladky S.B. J. Physiol. (Lond.). 1999; 516: 75-84Crossref Scopus (74) Google Scholar, 32Higgins C.F. Weylandt K.H. Nastrucci C. Sardini A. Linton K. Diaz M. Valverde M.A. Kozlowski R. Chloride Channels. Isis Medical Media Ltd., Oxford1999: 35-36Google Scholar) its role as a volume sensitive chloride channel has recently become controversial (33Shimada K., Li, X., Xu, G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar, 34Li X. Shimada K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; 275: 35994-35998Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 35Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 36Weylandt K.H. Valverde M.A. Nobles M. Raguz S. Amey J.S. Diaz M. Nastrucci C. Higgins C.F. Sardini A. J. Biol. Chem. 2001; 276: 17461-17467Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The purpose of the present study was to investigate the role of endogenous ClC-3 in native VSOAC activation and the RVD mechanism intrinsic to HeLa cells and X. laevis oocytes. HeLa cells or X. laevis oocytes were transiently treated with an antisense oligonucleotide or injected with antisense cRNA against ClC-3, respectively, to abolish endogenous protein expression. Our results demonstrate that antisense treatment abolished both mRNA and protein expression of ClC-3 in HeLa cells andX. laevis oocytes, significantly reduced native VSOAC current density, and significantly diminished the ability of cells to undergo RVD. All experiments on HeLa cells were performed at the Instituto de Ciencias Biomédicas, Facultad de Medicina Universidad de Chile, and all oocyte experiments were conducted at the University of Nevada, Reno, School of Medicine. HeLa cells were grown to ∼70% confluence at 37 °C in an 5%/95%: CO2/air atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 80,000 IU/liter penicillin, and 50 μg/liter streptomycin. Oocytes were collected as previously described (28Duan D. Zhong J. Hermoso M. Satterwhite C.M. Rossow C.F. Hatton W.J. Yamboliev I. Horowitz B. Hume J.R. J. Physiol. 2001; 531: 437-444Crossref PubMed Scopus (82) Google Scholar). Oocytes were removed and incubated in ND96 solution containing, in mm: 96 NaCl, 2 KCl, 1.8 CaCl2, 2.8 MgCl2, 5 Hepes, 2.5 sodium pyruvate, and 1 mg/ml gentamicin, pH 7.5. Follicle-enclosed oocytes were used in all experiments. 733-bp in situhybridization probes were obtained from HeLa cells cDNA, complementary to the human ClC-3 mRNA initiation codon region (37Borsani G. Rugarli E.I. Taglialatela M. Wong C. Ballabio A. Genomics. 1995; 27: 131-141Crossref PubMed Scopus (77) Google Scholar). An antisense probe was obtained using conventional procedures. HeLa cells were grown up to 70% confluence and fixed in 4% paraformaldehyde at 4 °C for 30 min. Excess fixative was washed with cold phosphate-buffered saline containing 0.1% Triton X-100. Fixed HeLa cells were pre-hybridized with Denhardt's solution for 30 min and hybridized overnight with 10 ng/μl ClC-3 antisense or sense RNA probes at 65 °C. After washing in 2× SSC/0.1% Triton X-100 and 0.2× SSC/0.1% Triton X-100, cells were incubated overnight with a rabbit anti-digoxigenin (alkaline phosphatase-conjugated) Ab at 4 °C. After excess Ab was washed away, cells were incubated with NTP/5-bromo-4-chloro-3-indolyl phosphate substrates, mounted with Permount (Fisher Scientific, NJ) and examined under a light microscope. The antisense (ASO) or mismatched (MMO) oligonucleotides used for HeLa cell transfection were second generation chimeras high-performance liquid chromatography-purified (Oligos Etc. Inc., Wilsonville, OR). The ASO and MMO sequence was 5′-CGTCCCTCTTTAACTGGTT-3′ and 5′-CTGCCTCCATTTGTCATTG-3′, respectively. The ASO sequence was complementary to the human ClC-3 mRNA initiation codon region (37Borsani G. Rugarli E.I. Taglialatela M. Wong C. Ballabio A. Genomics. 1995; 27: 131-141Crossref PubMed Scopus (77) Google Scholar), starting from the second base before the initiation codon (ATG corresponding to the third open reading frame). For electrophysiological and volume measurement experiments, the transfection efficiency was assessed with a fluorescein isothiocyanate (FITC)- or a rhodamine derivative (TAMRA)-labeled oligonucleotides at bases 1 and 19, or 1 and 2, respectively. Confluent HeLa cell cultures were acutely transfected with 1 μm ASO- or MMO-ClC-3 in the presence of LipofectAMINE Plus reagent (Invitrogen, Paisley, UK). Cells were then grown for another 24, 48, or 72 h until electrophysiological recordings, cell volume measurements, and ClC-3 mRNA and protein content determinations were conducted. Antisense cRNA was used to abolish ClC-3 mRNA transcription in X. laevis oocytes. X. laevis oocyte ClC-3 (xClC-3) cDNA, corresponding to a 211-bp fragment (15–225 bp, GenBankTM accession numberY09941) in either the sense or antisense direction, was subcloned into a TA cloning vector PCR 2.1 (Invitrogen). The sequence includes the start codon of xClC-3 mRNA and is not homologous to other known ClC channels. The constructs were linearized by digestion with the restriction enzyme BamHI and complementary RNA (cRNA) was synthesized by the bacteriophage RNA polymerase T7 using the mMessage mMachine in vitro transcription kit (Ambion). Injection pipettes with tips of ∼1 μm in diameter were baked at >150 °C overnight to destroy RNases and were mounted in a Drummound Nanoject autoinjector (Drummound Scientific, Broomall, PA). Oocytes were injected with 9 ng of either sense or antisense cRNA in 50 nl of diethyl pyrocarbonate water. Total RNA was isolated from either HeLa cells transfected with ASO- or MMO-ClC-3 orX. laevis oocytes injected with sense or antisense ClC-3 cRNA by the use of a TRIzol® total RNA isolation reagent (Invitrogen, Gaithersburg, MD) following the manufacturer's instructions. The total RNA was quantified by absorbance at 260 nm in a spectrophotometer, and the 260/280 absorbance ratio was determined for purity. Total RNA was incubated with RNase-free DNase (Promega, Madison, WI) for 20 min at 25 °C followed by heat inactivation at 65 °C. cDNA was synthesized using 1 μg of RNA reverse-transcribed with 200 units of Superscript IITMreverse transcriptase (Invitrogen) in a 20-μl reaction containing 25 ng of oligo(dT)12–18 primer (HeLa cells) or random hexamer primer (oocytes), 500 μm each of dNTP, 50 mmTris-HCl, pH 8.3, 75 mm KCl, 3 mmMgCl2, and 10 mm dithiothreitol. For ClC-3 transcript content determination in transfected HeLa cells, a 570-bp product, including the 5′ ClC3 coding region was amplified by PCR. The specific primers used to quantitate ClC-3 expression in HeLa cells were as follows: ClC-3 (GenBankTM accession number X78520), forward 5′-CATGTCAATGGGGAGG-3′ (sense, nt 633–680) and reverse 5′-GCAAGAAAGGCAAAACT-3′ (antisense, nt 1233–1216). For ClC-3 semiquantification, a 208-bp product was amplified from the housekeeping gene glutaraldehyde-3-phosphate dehydrogenase and used as an internal standard. The specific primers used to amplify glutaraldehyde-3-phosphate dehydrogenase expression in HeLa cells were as follows: (GenBankTM accession number U51572), forward 5′-GCCCACCAGAACATCATCC-3′ (sense, nt 207–227) and reverse 5′-GCCATCCCTGTCAGCTTC-3′ (antisense, nt 478–460). PCR reactions were carried out in Taq polymerase buffer (Invitrogen) with 2.5 mm MgCl2, 0.4 mm dNTP, and 10 pmol of each primer. Amplification was performed in a DNA thermal cycler (PerkinElmer Life Sciences, model 2400) with 2 units ofTaq polymerase/tube (50-μl final volume). The thermal profile used was 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 60 s with a total of 30 cycles. The quantification of X. laevis oocyte ClC-3 mRNA content was done using real-time PCR. The specific primers used to quantitate xClC-3 expression in X. laevisoocytes were as follows: xClC-3 (GenBankTM accession numberY09941), forward 5′-CCCAATGGATATCTCTTCAGATC-3′ (sense, nt 87–109) and reverse 5′-CAATAGGTGAGTCGTGCTGT-3′ (antisense, nt 225–206), amplicon 138 bp; 18 S rRNA (GenBankTMaccession number K01373), forward 5′-ACAGTGAAACTGCGAATGGCT-3′ (sense, nt 77–97) and reverse 5′-GCTCGTCGGCATGTATTAGCT-3′ (antisense, nt 183–163), amplicon 107 bp. The amplification profile for these primer pairs was as follows: 95 °C for 10 min to activate the AmpliTaq polymerase, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min, performed in a GeneAmp 2400 thermal cycler (PE Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed with the use of SYBR® Green chemistry on an ABI 5700 sequence detector (PE Applied Biosystems, Foster City, CA). Standard curves were generated for xClC-3 and the constitutively expressed 18 S rRNA from regression analysis of the mean values of RT-PCR values for the log10 diluted cDNA. Unknown quantities relative to the standard curve for the xClC-3 primers were calculated, yielding transcriptional quantitation of xClC-3 cDNA relative to the endogenous standard (18 S rRNA). Each cDNA sample was tested in triplicate, and cDNA was obtained from at least five different frogs. The reproducibility of the assay was tested by analysis of variance (ANOVA) comparing repeat runs of samples, and mean values generated at individual time points were compared by Student's t test. The PCR products were resolved in 1.5 and 2.5% agarose/ethidium bromide gels, for the HeLa and X. laevis ClC-3 amplification products, respectively. Bands were observed under an UV transilluminator and analyzed with Molecular Analyst software (Bio-Rad, Richmond, CA). Net intensity values were obtained for each band resolved in the agarose gels. Crude and membrane protein from HeLa cells and X. laevis oocytes were extracted as described previously (28Duan D. Zhong J. Hermoso M. Satterwhite C.M. Rossow C.F. Hatton W.J. Yamboliev I. Horowitz B. Hume J.R. J. Physiol. 2001; 531: 437-444Crossref PubMed Scopus (82) Google Scholar). Protein content in HeLa cells and X. laevis oocytes was determined by the Bradford method (38Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar) or the bicinchoninic acid method (39Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18709) Google Scholar), respectively. Total protein was dissolved in sample buffer (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar) and subjected to electrophoresis in parallel to prestained low range molecular weight standards on 8% SDS-PAGE gels. Gels were then blotted to nitrocellulose, and proteins were electrically transferred (41Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44938) Google Scholar). Nitrocellulose nonspecific binding was blocked with 5% dried nonfat milk, 0.05 m Tris-buffered saline (TBS), pH 7.2. Blots were then initially incubated with the commercially available anti-ClC-3 Ab (Alomone Laboratories, Jerusalem, Israel) raised against a C terminus epitope (amino acids 592–661) at a final concentration of 7.5 μg. Due to recent concerns (35Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar) regarding the specificity of this Ab, some blots were incubated with a new rabbit polyclonal affinity-purified anti-ClC-3 Ab (42Hatton W.J. Yamboliev I. Hume J.R. Biophys. J. 2002; 82: 242aGoogle Scholar) raised against a different C terminus epitope (amino acids 670–687 of the mouse sequence) at a final concentration of 25 μg. The peptide used to generate the C670–687 Ab is similar to the ClC-3 epitope previously used to generate a specific C terminus anti-ClC-3 Ab, which exhibited no cross-reactivity with human ClC-1, rat ClC-2, or human ClC-4 (43Huang P. Liu J., Di, A. Robinson N.C. Musch M.W. Kaetzel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Ab excess was removed with 0.1% Tween 20-TBS, and specific ClC-3 reactivity was determined with a goat-anti rabbit IgG conjugated to peroxidase or alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA). The peroxidase activity was detected by the ECL chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA) and revealed with Kodak films. Immunoreactive bands present in the films were observed under a visible light transilluminator and digitized with a Kodak DC40/DC120 camera. Images were analyzed with the Kodak digital science 1D program (KdS 1d, Kodak). Phosphatase activity was detected with Nitro BT/5-bromo-4-chloro-3-indolyl phosphate tablets. Net intensity values for each band were analyzed with Molecular Analyst software (Bio-Rad, Richmond, CA). Solutions were chosen to facilitate Cl− currents in HeLa cells and in X. laevis oocytes. The isotonic solution for HeLa cells current recordings contained, in mm: 140N-methyl-d-glucamine chloride, 1.2 CaCl2, 0.5 MgCl2, 10 HEPES, 1 EGTA, 70 mannitol, pH 7.4. All whole cell recordings were in either isotonic (300 mosm) or hypotonic (220 mosm, by exclusion of mannitol) bath solutions. The pipette solution contained (in mm) 140 N-methyl-d-glucamine chloride, 1.2 MgCl2, 10 HEPES, 1 EGTA, and 2 ATP, pH 7.3 (285 mosm). The same isotonic and hypotonic bath solutions were used in cell volume experiments. The isotonic solution (ND72) for X. laevis oocyte current recordings contained, in mm: 72 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, and 55 mannitol, pH 7.5. 100 μm niflumic acid was added to all solutions to eliminate contamination by endogenous ICl.Ca. All whole cell recordings were in either isotonic (220 mosm) or hypotonic (165 mosm, by exclusion of mannitol) bath solutions. For cell volume measurements, NaCl was replaced byN-methyl-d-glucamine chloride at a concentration of 100 mm adjusting the solution to be 50% hypotonic. All solutions were tested with a freezing point depression osmometer. HeLa cells transfected with FITC-ASO-ClC-3 or FITC-MMO-ClC-3 were plated on 25-mm round coverslips and placed in a microchamber mounted on a fluorescent inverted microscope. The bath was grounded via an agar bridge. Standard whole-cell patch-clamp recordings were performed using the Axopatch 200 B (Axon Instruments, Foster City, CA) amplifier. Patch-clamp pipettes were made from thin borosilicate (hard) glass capillary tubing with an outside diameter of 1.5 or 1.7 mm (Clark Electromedical, Edenbridge, UK) using a BB-CH puller (Mecanex, Geneva, Switzerland) and had resistances of 3–5 MΩ. Voltage and current signals from the amplifier were recorded on a digital tape recorder (DTR-1204, Biologic, France), digitized using a computer equipped with a Digidata 1200 (Axon Instruments) analog-to-digital/digital-to-analog interface and analyzed with Axon software. Changes in liquid junction potentials, which occurred as a result of bath solution changes during an experiment, were calculated (44Barry P.H. J. Neurosci. Methods. 1994; 51: 107-116Crossref PubMed Scopus (550) Google Scholar), and current-voltage relations were corrected accordingly. HeLa cells were clamped at a 0-mV holding potential, and voltage steps were applied from −80 to +120 mV for 500 ms in 40-mV increments unless otherwise stated. Membrane currents from X. laevis oocytes 0–3 days post surgery were recorded using a two-microelectrode voltage clamp system (GeneClamp 500B, Axon Instruments). Double-blind recordings were performed in all experiments. Microelectrodes, filled with 3 m KCl, had resistances of 0.5–3 MΩ. Voltages are reported with reference to the bath. Membrane currents were filtered at 1.0 kHz, digitized on-line, and stored on a computer. Data acquisition and analysis were performed using pClamp 6 software (Axon Instruments). Oocytes were clamped at a −30mV holding potential and voltage steps were applied from −100 to +120 mV for 400 ms in +20-mV increments unless otherwise stated. Cell water volume was assessed in single HeLa cells by measuring changes in concentration of an intracellularly trapped fluorescent dye (45Alvarez-Leefmans F.J. Altamirano J. Crowe W.E. Methods Neurosci. 1995; 27: 361-391Crossref Scopus (55) Google Scholar). HeLa cells transfected with TAMRA-ASO-ClC-3 or TAMRA-MMO-ClC-3 were loaded with 5 μm calcein-AM/10% pluronic acid for 5 min at 22 °C, and volume measurements were performed using a confocal laser imaging system (LSM, Carl Zeiss). Excitation light was 488 and 523 nm, for calcein and TAMRA, respectively, and emitted light was measured at wavelengths longer than 515 nm. Images of transfected HeLa cells were obtained at 10-s intervals, and fluorescence of a ∼10-μm2 area in the cell center was measured and subsequent correction for fluorescence decay was depicted. The data are presented as V t/V 0, the ratio between the cell water volume in isoosmotic solution at time = 0 and time = t, calculated fromF 0/F t (F= fluorescence intensity) as described (45Alvarez-Leefmans F.J. Altamirano J. Crowe W.E. Methods Neurosci. 1995; 27: 361-391Crossref Scopus (55) Google Scholar). Cell volume was estimated in oocytes by diameter measurements (46Furukawa T. Ogura T. Katayama Y. Hiraoka M. Am. J. Physiol. 1998; 274: C500-C512Crossref PubMed Google Scholar). Oocyte diameters were measured using a digital video line measurement system (Ionoptix, Milton, MA) with contrast analysis of digitized image data. The measurement system was mounted to a stereo-optic microscope (Cambridge Instruments). Oocytes were illuminated with focused broad-spectrum light emitted from a halogen lamp. Specifically, the left and right edges of the oocyte were measured independently using separate video lines placed at the peripheral edges of the oocyte. These lines were stripped out of the image, and intensity was displayed following online smoothing with digital filters to improve the signal-to-noise ratio. A
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