Cell Swelling Stimulates Cytosol to Membrane Transposition of ICln
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m300374200
ISSN1083-351X
AutoresMarkus Ritter, Andrea Ravasio, Martin Jakab, Sabine Chwatal, Johannes Fürst, Andreas Laich, Martin Gschwentner, Sara Signorelli, Carmen M. Burtscher, Sonja Eichmüller, Markus Paulmichl,
Tópico(s)Pancreatic function and diabetes
ResumoICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane. ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane. The ability of volume regulation is a fundamental feature of cells. On the one hand, cells are persistently challenged by disturbances of the osmotic equilibrium between intra- and extracellular space due to changes in the ambient osmolarity, metabolism of osmotically active solutes or ion- and substrate transport. On the other hand, cells have to change their volume to execute specific functions such as cell division, migration, or secretion. Accordingly, cell volume regulatory mechanisms are involved in various cell functions, and disturbances of these mechanisms can lead to severe dysfunctions (1.Lang F. Busch G.L. Ritter M. Völkl H. Waldegger S. Gulbins E. Häussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar, 2.Ritter M. Fürst J. Wöll E. Chwatal S. Gschwentner M. Lang F. Deetjen P. Paulmichl M. Cell. Physiol. Biochem. 2001; 11: 1-18Crossref PubMed Scopus (63) Google Scholar). The ad hoc mechanism that enables cells to down-regulate their volume after swelling, a process called regulatory volume decrease (RVD), 1The abbreviations used are: RVDregulatory volume decreaseRVDCregulatory volume decrease currents/channelsCLICchloride intracellular channelECFPenhanced cyan fluorescent proteinEYFPenhanced yellow fluorescent proteinFRETfluorescence resonance energy transferIClnI = current, Cl = chloride, and n = nucleotide-sensitiveICln-CAAXfarnesylation sequence (K-ras-derived CAAX-motive, MSKDVKKKKKKSKTKCVIM) attached to IClnIClswellswelling-dependent chloride currentIClvolvolume-dependent chloride currentIGSSimmunogold silver stainingMDCK cellsMadin-Darby canine kidney cellsMEQ6-methoxy-N-ethyl-1,2-dihydroquinoline2-ME2-mercaptoethanolORFopen reading framePBSphosphate-buffered salinePDcell membrane potentialQRquenching rateROIregion of interestTBSTris-buffered salineCFP-IClnfusion protein of ICln attached to the C terminus of ECFPCFP-Mempalmitoylation sequence (N-terminal 20 amino acids of neuromodulin/GAP-43) attached to ECFPYFP-IClnfusion protein of ICln attached to the C terminus of EYFPYFP-Mempalmitoylation sequence attached to EYFP. is realized by the export of cellular ions and osmotically obliged water by the activation of ion channels. Besides K+ channels, most cells activate a distinct class of anion channels (termed IClvol, IClswell, volume-regulated anion channel, or volume-sensitive osmolyte and anion channel). Interestingly, these channels are also permeable to cations, organic osmolytes, and possibly ATP. Therefore, the (an)ion channels and the respective currents that are activated by cell swelling have recently been termed and are here referred to as regulatory volume decrease channels/currents (RVDC) (3.Fürst J. Gschwentner M. Ritter M. Botta G. Jakab M. Mayer M. Garavaglia L. Bazzini C. Rodighiero S. Meyer G. Eichmüller S. Wöll E. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2002; 444: 1-25Crossref PubMed Scopus (100) Google Scholar, 4.Jakab M. Fürst J. Gschwentner M. Botta G. Garavaglia M.L. Bazzini C. Rodighiero S. Meyer G. Eichmüller S. Wöll E. Chwatal S. Ritter M. Paulmichl M. Cell. Physiol. Biochem. 2002; 12: 235-258Crossref PubMed Scopus (119) Google Scholar). regulatory volume decrease regulatory volume decrease currents/channels chloride intracellular channel enhanced cyan fluorescent protein enhanced yellow fluorescent protein fluorescence resonance energy transfer I = current, Cl = chloride, and n = nucleotide-sensitive farnesylation sequence (K-ras-derived CAAX-motive, MSKDVKKKKKKSKTKCVIM) attached to ICln swelling-dependent chloride current volume-dependent chloride current immunogold silver staining Madin-Darby canine kidney cells 6-methoxy-N-ethyl-1,2-dihydroquinoline 2-mercaptoethanol open reading frame phosphate-buffered saline cell membrane potential quenching rate region of interest Tris-buffered saline fusion protein of ICln attached to the C terminus of ECFP palmitoylation sequence (N-terminal 20 amino acids of neuromodulin/GAP-43) attached to ECFP fusion protein of ICln attached to the C terminus of EYFP palmitoylation sequence attached to EYFP. The phenotypes of RVDC vary between cells but display some fingerprint features: they can be blocked by chloride channel blockers and extracellular nucleotides, are outwardly rectifying, inactivate at positive potentials, and are permeable to organic osmolytes and to both anions and cations in a manner depending on pH, calcium concentration, and lipid composition (3.Fürst J. Gschwentner M. Ritter M. Botta G. Jakab M. Mayer M. Garavaglia L. Bazzini C. Rodighiero S. Meyer G. Eichmüller S. Wöll E. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2002; 444: 1-25Crossref PubMed Scopus (100) Google Scholar). The molecular identity of RVDC is still elusive if we expect a one-molecule/one-function relation, i.e. that one molecule is responsible for generating all of the heterogeneous current phenotypes observed after cell swelling in different cells. However, several candidate-proteins can be envisioned to be involved in the formation of functional RVDC, such as ATP binding cassette transporters (P-glycoprotein, MDR1, CFTR), phospholemman, voltage-dependent anion channels, the CLC family of chloride channels (CLC2 and CLC3), and ICln (3.Fürst J. Gschwentner M. Ritter M. Botta G. Jakab M. Mayer M. Garavaglia L. Bazzini C. Rodighiero S. Meyer G. Eichmüller S. Wöll E. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2002; 444: 1-25Crossref PubMed Scopus (100) Google Scholar). ICln is a highly conserved and ubiquitously expressed protein that has been identified in all cells studied so far. The human gene for ICln is located on chromosome 11q13.5–14.1 and controlled by a constitutively highly active promoter (5.Scandella E. Nagl U.O. Oehl B. Bergmann F. Gschwentner M. Fürst J. Schmarda A. Ritter M. Waldegger S. Lang F. Deetjen P. Paulmichl M. J. Biol. Chem. 2000; 275: 15613-15620Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). The protein is essential for life, because all attempts to genetically knock out ICln in either mice, nematodes, or a cell line failed due to the lethality of the knockout (6.Pu W.T. Wickman K. Clapham D.E. J. Biol. Chem. 2000; 275: 12363-12366Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 2M. Ritter, A. Ravasio, M. Jakab, S. Chwatal, J. Fürst, A. Laich, M. Gschwentner, S. Signorelli, C. Burtscher, S. Eichmüller, and M. Paulmichl, unpublished observations. ICln is mainly found to be a water-soluble protein in the cytosol, but some portion is found within or in close association with the cell membrane. Different functions have been discussed for ICln. Overexpression of ICln in Xenopus laevis oocytes results in the occurrence of ion currents with features closely resembling RVDC. Accordingly it was assumed that ICln could form the ion conductive part of RVDC (7.Paulmichl M. Li Y. Wickman K. Ackerman M. Peralta E. Clapham D. Nature. 1992; 356: 238-241Crossref PubMed Scopus (310) Google Scholar). A knockdown of the ICln protein with ICln-specific antibodies (8.Krapivinsky G.B. Ackerman M.J. Gordon E.A. Krapivinsky L.D. Clapham D.E. Cell. 1994; 76: 439-448Abstract Full Text PDF PubMed Scopus (193) Google Scholar) or antisense oligodeoxynucleotides (9.Chen L. Wang L. Jacob T.J. Am. J. Physiol. 1999; 276: C182-C192Crossref PubMed Google Scholar, 10.Gschwentner M. Nagl U.O. Wöll E. Schmarda A. Ritter M. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 464-470Crossref PubMed Scopus (100) Google Scholar, 11.Hubert M.D. Levitan I. Hoffman M.M. Zraggen M. Hofreiter M.E. Garber S.S. Biochim. Biophys. 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ICln reconstituted in heart lipid is anion selective, in artificial lipids it is cation selective, with relative permeability sequences matching those of RVDC (14.Fürst J. Bazzini C. Jakab M. Meyer G. König M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2000; 440: 100-115Crossref PubMed Google Scholar, 15.Fürst J. Ritter M. Rudzki J. Danzl J. Gschwentner M. Scandella E. Jakab M. König M. Oehl B. Lang F. Deetjen P. Paulmichl M. J. Biol. Chem. 2002; 277: 4435-4445Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16.Garavaglia L. Rodighiero S. Bertocchi C. Manfredi R. Fürst J. Gschwentner M. Ritter M. Bazzini C. Botta G. Jakab M. Meyer G. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2002; 443: 748-753Crossref PubMed Scopus (17) Google Scholar, 17.Li 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). Analysis of the amino acid sequence of ICln predicts a transmembrane domain in the N-terminal half of the protein. This sequence shows a remarkable similarity to the pore-forming β-hairpin of α-hemolysin and leukocidin, which are water-soluble bacterial toxins that refold and build large pores upon insertion into cell membranes. Secondary structure and hydrophobicity pattern prediction suggests a transmembrane domain that is composed of a four-stranded β-sheet. To form a functional channel, the formation of an ICln dimer is required. The model of the ICln channel has been refined by site-directed mutagenesis experiments (7.Paulmichl M. Li Y. Wickman K. Ackerman M. Peralta E. Clapham D. Nature. 1992; 356: 238-241Crossref PubMed Scopus (310) Google Scholar, 14.Fürst J. Bazzini C. Jakab M. Meyer G. König M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2000; 440: 100-115Crossref PubMed Google Scholar, 16.Garavaglia L. Rodighiero S. Bertocchi C. Manfredi R. Fürst J. Gschwentner M. Ritter M. Bazzini C. Botta G. Jakab M. Meyer G. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2002; 443: 748-753Crossref PubMed Scopus (17) Google Scholar, 18.Fürst J. Jakab M. König M. Ritter M. Gschwentner M. Rudzki J. Danzl J. Mayer M. Burtscher C.M. Schirmer J. Maier B. Nairz M. Chwatal S. Paulmichl M. Cell. Physiol. Biochem. 2000; 10: 329-334Crossref PubMed Scopus (24) Google Scholar). All of these findings clearly link ICln functionally to RVD. It is however still a matter of debate, as to whether the protein itself is a part of the molecular RVDC complex or rather acts as a regulator thereof (4.Jakab M. Fürst J. Gschwentner M. Botta G. Garavaglia M.L. Bazzini C. Rodighiero S. Meyer G. Eichmüller S. Wöll E. Chwatal S. Ritter M. Paulmichl M. Cell. Physiol. Biochem. 2002; 12: 235-258Crossref PubMed Scopus (119) Google Scholar, 14.Fürst J. 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Kambach C. Fischer U. Curr. Biol. 2001; 11: 1990-1994Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), suggesting a multifunctional role for this protein. An intriguing feature of ICln is that it can appear in a water-soluble form, as well as in close association with the cell membrane. The mechanisms by which soluble ICln protein associates with and/or incorporates itself into the membrane are unknown. The present study has been performed to further investigate the intracellular distribution of ICln under normal conditions and during hypo-osmotic stress, as well as to correlate an enhanced presence of membrane-bound ICln with functional changes of the cell. All salts, chemicals, and drugs were of pro analysi grade and purchased from Sigma, Germany unless otherwise stated. Cell culture reagents were from Invitrogen, Germany. Optical filters were purchased from AHF Analysentechnik, Germany. Standard procedures (23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and procedures according to the manufacturers' protocols were used for DNA preparation, cloning, purification, and sequencing. Plasmids coding for fusion proteins of ICln attached to the C terminus of ECFP or EYFP (CFP-ICln and YFP-ICln) were prepared by cloning the ORF coding for the canine (MDCK) ICln inframe into the vectors pECFP-C1 and pEYFP-C1 (Clontech) using the XhoI and BamH1 restriction sites. For cell membrane labeling with fluorescent proteins (CFP-Mem and YFP-Mem) the vectors pECFPMem and pEYFPMem (Clontech) were used. To enhance targeting of ICln to the cell membrane, cells were transfected with the pCDNA3YFPCaaX vector (24.Fürst J. Haller T. Chwatal S. Wöll E. Dartsch P.C. Gschwentner M. Dienstl A. Zwierzina H. Lang F. Paulmichl M. Ritter M. Cell. Physiol. Biochem. 2002; 12: 19-30Crossref PubMed Scopus (12) Google Scholar, 25.van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), in which YFP was replaced by ICln (ICln-CAAX). The same vector lacking the ICln and YFP insert was used as a control vector. All constructs were sequenced using an automated sequencer (Gene ReadIR 4200, LiCor). The ORF of ICln was cloned in-frame into the pET3-His vector, which adds a histidine tag to the N terminus of ICln (14.Fürst J. Bazzini C. Jakab M. Meyer G. König M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2000; 440: 100-115Crossref PubMed Google Scholar). The protein was expressed in E. coli (BL21/DE3), purified using a nickel-nitrilotriacetic acid-agarose column (Qiagen) and stored at –74 °C in 50 mm K2HPO4, 200 mm imidazole, pH 8.00. NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells (strain II, low resistance) were grown in culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 μg/ml penicillin, and 100 units/ml streptomycin, at 37 °C and 5% CO2 and 95% air. Subcultures were routinely established every second to third day by trypsin/EDTA treatment (0.05%, pH 7.2). Cells were used for experiments at a degree of confluency of ∼40–70% unless otherwise stated. For transfection with the appropriate plasmids, NIH 3T3 fibroblasts were grown to 70% confluency and incubated for 6 h with 3 μg of plasmid DNA and 7 μl of LipofectAMINE 2000™ (Invitrogen). For double transfection with CFP-ICln and YFP-Mem a ratio of 2:1 yielded the best results. Thereafter the cells were subcultured and grown on uncoated glass cover slips for 24–48 h. The transfection efficacy was ∼15–20%. For all FRET experiments, ECFP served as a donor and EYFP as an acceptor (26.Gadella Jr., T.W. van der Krogt G.N. 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Visualization of ECFP- and/or EYFP-expressing cells and detection of FRET was performed on an Olympus IX70 inverted microscope equipped with a monochromator (Polychrome 4, TILL Photonics) and a cooled charge-coupled device camera (TILL Imago SVGA) controlled by the TILL Vision software (versions 3.3 and 4.0). Experiments were either performed by changing three separate Olympus BX cubes equipped with the appropriate filter combinations for ECFP, EYFP, and FRET measurements or by using a CFP/YFP dual-band polychroic mirror in combination with a real-time dual color imaging device (Dual-View™, Optical Insights) (Table I). All images were adjusted to pixel-by-pixel alignment and corrected for background, which was then clamped to zero (29.Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar).Table IFilters used for FRET experimentsExcitationExcitation filterBeamsplitter 1Beamsplitter 2Emission filternmSingle-cube setup ECFP436436/20455 DCLP480/40 EYFP515510/20530 DCLP560/40 FRET436436/20455 DCLP560/40Dual-View™ setup ECFP436CFP/YFP-dual-bandaPart no. F86-004.505 DCLP480/40 EYFP515CFP/YFP-dual-bandaPart no. F86-004.505 DCLP560/40 FRET436CFP/YFP-dual-bandaPart no. F86-004.505 DCLP560/40Acceptor photobleaching EYFP>515515 LPa Part no. F86-004. Open table in a new tab Light from a xenon lamp (XBO 75, Osram) was passed through a 515-nm cutoff filter toward the object. The FRET efficiency (FRETeff) was calculated from the increase in ECFP intensity after the bleaching of EYFP: FRETeff = 1 – (ICFP-bgDA/ICFP-bgD), where ICFP-bgDA and ICFP-bgD are the background-corrected fluorescence intensities of ECFP in the presence of both donor and acceptor and in the presence of the donor alone, respectively. As shown in Fig. 1 (B and C) YFP-Mem is bleached to 11 ± 2% (n = 6) within 10 min. This is paralleled by an increase of the CFP-ICln fluorescence to 114 ± 5% (n = 6). The calculated FRET efficiency after 10 min is 0.11 ± 0.04 (n = 6) and 0.20 ± 0.03 (n = 6) if calculated from the entire area of the cell and from selected regions close to the cells' outer margins, respectively. Fig. 1D shows the calculated FRET efficiency image of the cells shown in Fig. 1C. Corrected FRET images (NFRET) were calculated from the sensitized emission of EYFP (29.Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Each of the images was corrected for ECFP cross-talk (i.e. contribution of ECFP emission to the EYFP emission window) and EYFP cross-excitation (i.e. EYFP emission due to excitation at 436 nm), and this is referred to as, netFRET = IFRET-bg – (ICFP-bg × k1) – (IYFP-bg × k2), where IFRET-bg, ICFP-bg, and IYFP-bg are the background-corrected pixel gray values in the windows for FRET (ex, 436; em, 560 nm), ECFP (ex, 436; em, 480 nm), and EYFP (ex, 515; em, 560 nm), and k1 and k2 denote the relative contribution to the fluorescence intensity in the FRET window by ECFP cross-talk (k1) and EYFP cross-excitation (k2). For the excitation wavelengths and filter combinations provided in Table I, k1 and k2 were measured to be 0.3 and 0.04, respectively. These values did not change significantly under hypotonic conditions. The obtained netFRET values were normalized against protein expression levels and are referred to as, NFRET = netFRET × 100/(ICFP-bg × IYFP-bg)1/2 (29.Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). The integrated fluorescence density values of the images from total cells or from particular regions of interest therein were analyzed using TILL Vision and Microsoft Excel software. Because the fluorescence of green fluorescent protein and its variants is sensitive to pH (30.Llopis J. McCaffery J.M. Miyawaki A. Farquhar M.G. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6803-6808Crossref PubMed Scopus (930) Google Scholar), FRET could be affected by swelling-induced intracellular acidification (31.Ritter M. Paulmichl M. Lang F. Pfluegers Arch. Eur. J. Physiol. 1991; 418: 35-39Crossref PubMed Scopus (30) Google Scholar). During cell swelling the relative fluorescence of YFP-Mem decreased by 5.5 ± 2.3% (n = 17), and the measured FRET signals are therefore likely to be underestimated. Rat kidneys were removed after sacrifice, decapsulated, and cut into 2-mm sections from which the cortex, outer medulla, inner medulla, and papilla regions were separated (Fig. 2), washed in ice-cold PBS (pH 7.4), frozen and stored at –70 °C until further processing for Western analysis. For IGSS, the sections were fixed overnight (2.5% glutaraldehyde in PBS), washed in PBS, dehydrated in ethanol, incubated in xylol and paraplast, cut to 6-μm sections, and placed on poly-l-lysine-coated glass coverslips. Thereafter the paraplast was removed, the specimens were rehydrated, rinsed in distilled water and PBS (32.Huang W.M. Gibson S.J. Facer P. Gu J. Polak J.M. Histochemistry. 1983; 77: 275-279Crossref PubMed Scopus (290) Google Scholar), incubated in diluted (1:10) normal rabbit serum, then placed for 1 h in the primary polyclonal rabbit anti-ICln-antibody-containing solution (14.Fürst J. Bazzini C. Jakab M. Meyer G. König M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pfluegers Arch. Eur. J. Physiol. 2000; 440: 100-115Crossref PubMed Google Scholar), washed and incubated with the labeled (10-nm gold) secondary anti-rabbit IgG antibody. Then the samples were extensively washed, incubated in the dark for 5–10 min in a 1:1 mixture of hydrochinon and silver acetate (British Bio Cell International), washed extensively again in distilled water, subjected to iron hematoxilin staining and evaluated on a confocal laser-scanning microscope (LSM 410, Zeiss). The images obtained from the light reflections from the silver grains were overlaid with the conventional confocal transmission images. Subconfluent NIH 3T3 fibroblasts, LLC-PK1 cells, MDCK cells, or confluent LLC-PK1 cells were washed with PBS, detached by gentle treatment with trypsin, suspended in isotonic PBS, centrifuged for 10 min (700 × g, room temperature) and resuspended for 15 min in either isotonic or hypotonic buffer at 37 °C. The buffers were composed of (mm): phenylmethylsulfonyl fluoride 0.1, 2-ME 5 in PBS (pH 7.4; 310 mosm) and phenylmethylsulfonyl fluoride 0.1, 2-ME 5, Tris-HCl 20, in distilled water (pH 7.4; 110 mosm). Thereafter, the cells were homogenized by repeated freeze-thawing cycles (liquid nitrogen/37 °C) or by sonication (5–20 s, 4 °C) in lysis buffer (33.Stuart-Tilley A. Sardet C. Pouyssegur J. Schwartz M.A. Brown D. Alper S.L. Am. J. Physiol. 1994; 266: C559-C568Crossref PubMed Google Scholar). The cell lysates were centrifuged for 1 h at 100,000 × g at 4 °C. Supernatant (cytosolic fraction) and pellet (membrane fraction) were separated, and 2 μl of each supernatant was used to determine the protein concentration (34.Bradford M. Analyt. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Sample buffer (4× SDS-PAGE) was added to each sample containing supernatant. A volume of 1× SDS-PAGE sample buffer, equal to the volume of the corresponding supernatant sample, was added to the respective membrane fraction. Samples were then vortexed, heated to 100 °C for 5 min, centrifuged (19,000 × g, 5 min), and separated on a 13% SDS-PAGE according to the method of Laemmli (35.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). A volume containing 100 μg of cytosolic protein, and an equal volume from the corresponding membrane-sample was loaded onto the gel for electrophoretic protein separation. Thus the same relative amounts of the total cytosolic proteins and total membrane proteins, respectively, of a given cell preparation were separated. Proteins were then electroblotted (23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) onto a nitrocellulose membrane (Amersham Biosciences). Membranes were blocked in TBS containing 1% Tween and 5% nonfat dry milk for 1 h at room temperature, followed by incubation with primary anti-ICln polyclonal antibody for 1 h, washing, incubation with secondary antibody (1:2500) in TBST and antibody detection by enhanced chemiluminescence (ECL™-System, Amersham Biosciences). Significant immunoreactivity was detected only at an apparent molecular mass of 36 kDa. Densitometric quantification was performed for the 36-kDa bands (ImageQuant™, Amersham Biosciences or Eagle Eye™, Stratagene). Data from subconfluent and confluent LLC-PK1 cells were pooled. Whole Cell Patch Clamp and Perforated Patch Clamp Measurements—Cells were used 24–48 h after splitting. All experiments were performed at room temperature. For data acquisition and analysis, an EPC-9 (HEKA) or Axopatch 200A (Axon Instruments) amplifier, as well as Pulse/Pulsefit software (HEKA) were used. Current signals were filtered at 1 kHz (4-pole Bessel). The holding potential was 0 mV. The time courses of current activation were monitored by applying pulses (0.5 s) to +40 mV at intervals of 10 s. Current-voltage relations were registered by pulsing (0.5 s) from –120 mV to +100 mV with step increments of 20 mV or by applying a voltage ramp (–120 mV to +100 mV, 0.5 s). Bath and pipette solu
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