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Heteromeric connexons formed by the lens connexins, connexin43 and connexin56
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European Journal of Cell Biology 80, 11 ± 19 (2001, January) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
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Heteromeric connexons formed by the lens connexins, connexin43 and connexin56 Viviana M. Berthoud1)a, Elisabeth A. Montegnaa, Namita Atalb, Naga H. Aithala, Peter R. Brinkc, Eric C. Beyera a b c
Department of Pediatrics, Section of Hematology/Oncology University of Chicago, Chicago, IL/USA Department of Medicine, Division of Infectious Diseases, Washington University Medical School, St. Louis, MO/USA Department of Physiology and Biophysics, State University of New York, Stony Brook, NY/USA
Received August 23, 2000 Received in revised version September 11, 2000 Accepted September 11, 2000
Heteromeric connexons ± lens connexins ± sucrose gradient sedimentation ± double whole-cell patch-clamp ± gap junctions In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.
Dr. Viviana M. Berthoud, Section of Pediatric Hematology/Oncology, University of Chicago, 5841 S. Maryland Ave., MC 4060, Chicago, IL 60637, e-mail: [email protected], Fax: 773 702 9881.
1)
Abbreviations. Cx Connexin. ± Cy3 Indocarbocyanine. ± ECL Enhanced chemiluminescence. ± FITC Fluorescein isothiocyanate. ± ginitial Initial conductance. ± Gj Normalized junctional conductance. ± gsteady-state Steadystate junctional conductance. ± Gmin Voltage-insensitive component of normalized gsteady-state. ± HEPES N-2-Hydroxyethylpiperazine-N'-2ethanesulfonic acid. ± Ij Junctional current. ± PAGE Polyacrylamide gel electrophoresis. ± PBS Phosphate-buffered saline. ± PMSF Phenylmethylsulfonyl fluoride. ± SDS Sodium dodecyl sulfate. ± TBS Tris-buffered saline. ± Vh Holding potential. ± Vj Transjunctional voltage. ± Vm Membrane potential. ± Vo Voltage at which the decrease in steady-state conductance is half maximal.
Introduction Gap junctions are membrane specializations that contain aggregates of intercellular channels that allow intercellular passage of ions and small molecules of up to 1000 Da. These channels are oligomeric assemblies of members of a family of related proteins called connexins (Cx) (Goodenough et al., 1996). Six connexin monomers assemble to form a hemi-gap junctional channel or connexon, which, in turn, may form a complete gap junction channel by docking with a connexon from the adjacent cell. The diversity of connexins and the multisubunit structure of the gap junctional channel allow for the formation of molecularly diverse channels. A homomeric hemichannel contains all subunits of a single connexin type. A homotypic gap junctional channel is formed when one homomeric connexon docks with another homomeric connexon in the adjacent cell containing the same connexin type; a heterotypic gap junctional channel is formed when one homomeric connexon docks with a homomeric connexon made of a different connexin type. A heteromeric connexon contains subunits of more than one connexin type. Docking of heteromeric connexons would lead to the greatest diversity of channel types. The lens is an avascular organ formed by an anterior epithelial cell layer and fiber cells which form the bulk of the
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V. M. Berthoud, E. A. Montegna et al.
organ. Gap junctions have been morphologically identified by electron microscopy between epithelial cells and between fiber cells (Goodenough, 1992). Recently published data have demonstrated that gap junctions are critical for lens function (Gong et al., 1997; Shiels et al., 1998; Steele et al., 1998; White et al., 1998; Mackay et al., 1999). Lens gap junctions contain at least three different connexins with somewhat overlapping cellular distributions. Lens epithelial cells express Cx43 (Beyer et al., 1989; Musil et al., 1990a) and fiber cells express lensspecific connexins such as rat Cx46 (Paul et al., 1991), mouse Cx50 (White et al., 1992), chicken Cx56 (Rup et al., 1993; Berthoud et al., 1994) and Cx45.6 (Jiang et al., 1994), bovine Cx44 (Gupta et al., 1994), ovine Cx49 (Yang and Louis, 1996), human Cx50 (Church et al., 1995) and Cx46 (Mackay et al., 1999). During development, epithelial cells in the bow region differentiate into fiber cells; a process that continues throughout the life-span of the individual. This process is characterized by the loss of organelles (including the nuclei) and changes in connexin expression. The differences in pH sensitivity of chicken lens gap junctions observed during development (Schuetze and Goodenough, 1982) have been correlated with changes in gap junction morphology (Schuetze and Goodenough, 1982) and connexin expression (Jiang et al., 1995), although the developmental switch in pH sensitivity cannot be explained on the basis of connexin type alone (Jiang et al., 1995). Impedance measurements in the lens suggest a gradient of junctional conductance from the poles to the equator (Mathias et al., 1979, 1991; Baldo and Mathias, 1992). Immunofluorescence studies on the distribution of Cx43 and of the lens-specific connexins suggest that the bow region/differentiating fiber cells co-express Cx43 with lens-specific connexins (Musil et al., 1990a; Evans et al., 1993; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995). Recent studies also suggest the presence of chicken Cx56 and Cx45.6 or its mammalian homolog, Cx50, in epithelial cells (Jiang et al., 1995; Dahm et al., 1999). Formation of gap junctional channels containing both bovine Cx46 and Cx50 (Konig and Zampighi, 1995), and of heteromeric connexons containing chicken Cx45.6 and Cx56 (Jiang and Goodenough, 1996) have been demonstrated. However, the existence of heteromeric connexons between Cx43 and either one of the lens-specific connexins has not been studied, nor have the functional properties of channels made of such heteromeric connexons been reported. In this study, we report the formation of heteromeric connexons between Cx43 and Cx56 in NRK cells transfected with Cx56 and in the chicken lens. These transfectants have been characterized for the expression, modification, targeting, and assembly of the transfected connexin into gap junctional channels, and the properties of Cx56 in these cells have been compared to those of Cx56 in the lens.
Materials and methods Chemicals
All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless specified otherwise.
Animals
Fertilized White Leghorn chicken eggs were obtained from SPAFAS (Norwich, CT, USA).
Cell culture
Parental normal rat kidney cells (NRK-52E, ATCC CRL 1571) were obtained from ATCC and grown in DMEM medium with 5% fetal bovine serum, 100 units/ml penicillin G and 100 mg/ml streptomycin sulfate. Cx56 in pSFFV-neo (Rup et al., 1993) was transfected into NRK cells using lipofectin, and stably transfected clones were selected by their resistance to 0.25 mg/ml active G418 in the medium.
RNA blotting
Total cellular RNA was prepared from cell cultures or 18E lenses according to Chomczynski and Sacchi (1987). RNA was separated on formaldehyde/agarose gels, and transferred to Hybond N nylon membranes (Amersham, Arlington Heights, IL) as previously described (Beyer et al., 1987). Hybridization was performed using a specific 32P-labeled DNA probe, containing base pairs 911 ± 1530 of Cx56, prepared using random hexanucleotide primers and the Klenow fragment of DNA polymerase I (Feinberg and Vogelstein, 1983).
Immunofluorescence
Untransfected NRK cells or NRK cells transfected with Cx56 were plated on 4-well slides (LAB TEK, Nalge Nunc International, Naperville, IL, USA) and allowed to reach 80 ± 90% confluence. Cells were then rinsed with phosphate-buffered saline, pH 7.4 (PBS), fixed in methanol : acetone (1 : 1) for 2 minutes at room temperature, permeabilized in 1% Triton X-100 in PBS for 15 min at room temperature and blocked in 2% normal goat serum, 1% Triton X-100 in PBS for 10 min at room temperature as previously described (Steinberg et al., 1994). Fixed cells were then incubated in mouse monoclonal anti-Cx43 antibody (Chemicon International, Temecula, CA, USA) and rabbit polyclonal anti-Cx56 antibodies (Berthoud et al., 1994) overnight at 4 8C. Cells were rinsed 4 times with PBS and then sequentially incubated in secondary antibody, Cy3-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA, USA). After 1.5 h incubation at room temperature, cells were rinsed 4 times in PBS, and coverslips were mounted with 2% n-propylgallate in PBS : glycerol (1 : 1). Confocal images were obtained using a Zeiss LSM 410 confocal microscope equipped with an argon/krypton laser.
Modification of Cx56 by phosphorylation
Cultured cells were harvested in PBS and centrifuged for 7 min at 14 000g. Cell pellets or 18E lenses were homogenized by sonication in 1 mM NaHCO3, 2 mM phenylmethylsulfonyl fluoride (PMSF). Homogenate aliquots containing 100 mg of protein were treated with alkaline phosphatase as previously described (Berthoud et al., 1994). Then, the proteins were resolved in sodium dodecyl sulfate (SDS)-containing gels and the pattern of bands analyzed after immunoblotting.
Sedimentation through sucrose density gradients
The procedure was performed as described by Koval et al. (1997) with minor modifications. Briefly, cultured cells were harvested in incubation buffer (PBS containing 0.8 mM MgSO4, 2.7 mM CaCl2, 1 mM Na2V2O5, 10 mM NaF, 10 mM N-ethylmaleimide, 20 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 3 mg/ml antipain, 3 mg/ml phosphoramidon, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 20 mg/ml Pefabloc; protease inhibitors were purchased from Roche Molecular Biochemicals, Indianapolis, IN, USA) and centrifuged at 150g for 7 min. Pelleted cells or chicken lenses were homogenized in incubation buffer. Triton X-100 was added to aliquots containing 300 mg of protein to a final concentration of 1% and incubated on ice for 30 min. The aliquots were centrifuged at 100 000gave for 30 min and the supernatant was layered on top of a sucrose density gradient (4.8 ml 5 ± 20% linear sucrose gradient with a 0.2-ml 25% sucrose cushion). The gradients were centrifuged at 100 000g for 19 h and 250-ml fractions were collected. In some cases, oligomers were dissociated by incubation of the Triton X-100-soluble material in 0.2% SDS for 1 h at 4 8C prior to layering it on top of the sucrose gradient.
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The percentage concentration of sucrose in each fraction was determined using a hand held Brix refractometer (0 ± 328; Fisher Scientific, Pittsburgh, PA, USA). Fractions were analyzed by immunoblotting. Horseradish peroxidase and catalase were subjected to sedimentation through sucrose densities as standards for connexin monomers (5S) and connexons (9S), respectively. The 5S standard, horseradish peroxidase, was detected between 6.6 ± 9% sucrose with its peak centered at 7.8% sucrose, the 9S standard, catalase, was detected between 10.2 ± 12.7% sucrose with its peak centered at 11.6% sucrose (not shown).
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filtered at 1 kHz. All recordings shown are currents observed in the non-stepped cell held at membrane potential (Vm) of 0 mV. These recordings represent Ij only.
Results Expression of Cx56 in NRK cells
Immunoblotting
Alkaline phosphatase experiments. 100 mg of protein from whole cell or lens homogenates treated with alkaline phosphatase or with buffer only (controls) were resolved on 9% SDS-containing polyacrylamide gels and subjected to immunoblot analysis using rabbit polyclonal anti-Cx56 or anti-Cx43 antibodies. After overnight incubation in primary antibodies at 4 8C, the membranes were rinsed in Tris-buffered saline (TBS) and incubated in peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. After this period of time, the membranes were rinsed in TBS and detection of secondary antibody binding was performed using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL, USA). Sucrose density gradient fractions. Equal volumes of the sucrose density gradient fractions were loaded in each lane and resolved on 9% SDS-containing polyacrylamide gels; the proteins were subjected to immunoblotting as described above using either rabbit polyclonal antiCx56 or anti-Cx43 antibodies. After reaction with ECL, membranes were stripped of primary antibody by incubation in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM b-mercaptoethanol for 30 min at 50 8C. Then, membranes were reacted with the other primary antibody.
Co-precipitation of Cx43 and Cx56
Cultured NRK-Cx56 cells or embryonic chicken lenses were sedimented through sucrose gradients as described above. Sucrose gradient fractions with sucrose densities between 8.6% and 15.2% were pooled and immunoprecipitated using anti-Cx56 antibodies as previously described (Berthoud et al., 1997). Immunoprecipitated proteins were resolved on SDS-containing 4 ± 15% polyacrylamide gradient gels and subjected to immunoblotting as described above using a mouse monoclonal anti-Cx43 antibody (Chemicon International, Temecula, CA, USA).
Protein determination
To study the functional properties of gap junctional channels formed by heteromeric Cx43/Cx56 connexons, we generated clones from a cell line stably transfected with Cx56. We chose NRK-52E, a normal rat kidney cell line that endogenously expresses Cx43 and that has been extensively used to study the solubility properties of the different phosphorylated forms of Cx43 and its assembly into oligomeric forms (Musil and Goodenough, 1993). The expression of the transfected Cx56 RNA in the clones was demonstrated by RNA blotting. A single band of the appropriate size for the transfected Cx56 was observed in total cellular RNA (Fig. 1a). The stably transfected clones were analyzed for the presence of Cx56 protein by immunoblotting. Multiple bands of Cx56 were detected in whole cell homogenates of stably transfected NRK-Cx56 (Fig. 1b, lane 3). The immunoblot pattern of Cx56 in NRK-Cx56 was similar to that obtained in an 18E whole lens homogenate (Fig. 1b, lane 1). Because Cx56 is a phosphoprotein (Berthoud et al., 1997), we studied the possibility that the observed complexity in the immunoblot pattern was due to phosphorylation. Similar to results obtained from lens homogenates (Berthoud et al., 1994, and Fig. 1b, lane 2), when NRK-Cx56 whole cell homogenates were incubated in the presence of alkaline phosphatase before resolution of the proteins by SDSPAGE, the multiple Cx56 bands collapsed to a doublet (Fig. 1b, lane 4). These results suggest that, similar to the lens, Cx56 is modified by phosphorylation in NRK-Cx56 transfectants. The distribution of Cx43 and Cx56 in untransfected NRK and NRK-Cx56 was studied by immunofluorescence. In agreement with previous results (Musil and Goodenough, 1993), immunopositive staining for Cx43 was localized to a perinuclear region and to puncta at appositional membranes in NRK-52E cells (Fig. 2a). A similar Cx43 staining pattern was
Proteins were determined using the BioRad Protein Assay (BioRad, Hercules, CA, USA) based on the Bradford dye-binding procedure (Bradford, 1976).
Electrophysiology
Experiments were carried out on cell pairs using the double whole-cell patch-clamp technique (Brink et al., 1997). During the experiments, the cells were bathed in a solution containing 180 mM CsCl or KCl, 1 mM CaCl2, 1.8 mM MgCl2, and 10 mM HEPES, pH 7.1 ± 7.3. For the wholecell recordings, the pipette solution contained 180 mM CsCl, 1 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 mM CaCl2, 1.8 mM MgCl2 and 10 mM HEPES, pH 7.0. Flow of intercellular junctional current (Ij) was induced by different voltage protocols. For the recording of the macroscopic current-voltage relationship, both cells were held at a holding potential of 0 mV. From this holding potential, the voltage of one cell was stepped to varying voltages (transjunctional voltages of 10 ± 150 mV or 120 mV, in 20 mV increments). For all experiments shown, the first voltage step was negative. After holding the potential for 250 or 400 ms, the voltage was flipped to the equal magnitude but opposite polarity for the same time. Current recording data analyses were performed as previously described (Brink et al., 1997). All macroscopic records were
Fig. 1. Expression and modification of Cx56 in NRK cells transfected with Cx56. (a) Ten mg of total RNA from NRK (lane 1) and NRK-Cx56 (lane 2) cells were resolved on formaldehyde-containing 1% agarose gels and transferred to Hybond membranes. The membranes were hybridized with a 32P-labeled Cx56 probe and exposed to X-ray film. The positions of the 18S and 28S rRNAs are indicated. (b) Immunoblot pattern of Cx56 in homogenates of 18-day embryonic lens (lanes 1 and 2) and NRK-Cx56 cells (lanes 3 and 4). The homogenates were left untreated (lanes 1 and 3) or they were incubated with alkaline phosphatase (lanes 2 and 4) prior to resolution of the bands by SDSPAGE. Note that several bands of Cx56 are detected in NRK-Cx56 cell homogenates. Similar to the results obtained using a lens homogenate, these bands collapse to a doublet after alkaline phosphatase treatment.
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Fig. 2. Localization of Cx43 and Cx56 in NRK and NRK-Cx56 cells. Confocal images from double immunofluorescence staining of NRK (a ± c) and NRK-Cx56 (d ± f) cells using mouse monoclonal anti-Cx43 (a, d) and rabbit polyclonal anti-Cx56 (b, e) antibodies followed by
Cy3-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies. Panels c and f show superposition of panels a and b and d and e, respectively. Bar represents 10 mm.
observed in NRK-Cx56 cells (Fig. 2d). As expected, no immunopositive staining was observed in NRK-52E with anti-Cx56 antibodies (Fig. 2b). In NRK-Cx56 cells, anti-Cx56 antibodies showed punctate immunopositive staining at appositional membranes and diffuse staining in the perinuclear region (Fig. 2e). When the confocal images were superimposed, the staining appeared red for the NRK-52E cells (Fig. 2c) because of the presence of Cx43 alone and yellow for the NRK-Cx56 cells (Fig. 2f), suggesting co-localization of Cx43 and Cx56 in the transfected cells.
monomeric and oligomeric forms by sedimentation through sucrose density gradients (Musil and Goodenough, 1993). We used a similar approach to study the assembly of oligomeric forms in NRK-Cx56 cells. When we applied this technique to the Triton X-100-soluble fraction of NRK-52E cells (used as a control), we obtained two peaks of immunoreactive Cx43 (Fig. 3, top panel), in agreement with the data previously published (Musil and Goodenough, 1993); the peaks were centered at 8.6 and 10.8% sucrose. In the detergent-soluble fraction of NRK-Cx56 cells, two major peaks of immunoreactive Cx43 were detected in all experiments after sedimentation through sucrose gradients (n 4). One of these peaks was centered at 7.8 ± 9.2% sucrose (Fig. 3, middle panel), similar to the sucrose percentage at which the Cx43 monomer peak was detected in NRK-52E cells. The second main peak of Cx43 immunoreactivity was centered at 12.2 ± 13.6% sucrose, a
Separation of monomeric and oligomeric forms of Cx43 and Cx56 by sedimentation through sucrose density gradients
It has been previously reported that the Triton X-100-soluble fraction of Cx43 from NRK-52E cells can be separated into
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reacted with anti-Cx56 antibodies, two peaks of immunoreactive Cx56 centered at 7.8 ± 9.4 and 12.7 ± 13.6% sucrose were observed in NRK-Cx56 cells (Fig. 3, middle panel); the fractions containing the apparent middle peak of immunoreactive Cx43 were almost devoid of anti-Cx56 immunoreactivity (Fig. 3, middle panel). When SDS was added to the Triton X-100-soluble fraction of NRK-Cx56 cells prior to layering the sample onto the gradient, only one peak, cosedimenting with the monomer peak, was observed with either anti-Cx43 or anti-Cx56 antibodies, confirming that the connexin immunoreactivity detected between 10.6 and 13.6% sucrose represented connexin oligomers (Fig. 3, bottom panel).
Heteromeric connexons in NRK-Cx56 cells
The presence of an oligomer peak of Cx43 which co-migrated with Cx56 oligomers suggested that in NRK-Cx56, Cx43 was forming heteromeric connexons with Cx56. Formation of heteromeric connexons can be demonstrated by immunoprecipitation using a specific antibody to one of the connexins involved and immunoblotting of the immunoprecipitated protein with an antibody to the other connexin. To test the hypothesis that Cx43 formed heteromeric connexons with Cx56, Cx56 was immunoprecipitated from connexin-containing sucrose gradient fractions of Triton X-100 extracts of NRKCx56 using anti-Cx56 antibodies; then, the immunoprecipitate was resolved on SDS-containing gels and analyzed by immunoblotting with anti-Cx43 antibody. Several bands of Cx43 were detected by immunoblotting in these samples (Fig. 4, lane 1); these likely correspond to the non-phosphorylated and phosphorylated forms of Cx43 (Musil et al., 1990b).
Do Cx43/Cx56 heteromeric connexons exist in the lens?
Studies on the distribution of lens connexins in several species suggest that Cx43 is co-expressed with the lens-specific
Fig. 3. Analysis of oligomeric association of Cx43 and Cx56 in NRK and NRK-Cx56 by sucrose gradient centrifugation. Graphs depict the densitometric values obtained after quantitation of Cx43 (*; top, middle and bottom panels) or Cx56 (*; middle and bottom panels) immunoblots of sucrose gradient fractions from Triton X-100-soluble material from NRK (top panel) or NRK-Cx56 (middle and bottom panels) cells. For NRK-Cx56, the Triton X-100-soluble material was untreated (middle panel) or treated (bottom panel) with 0.2% SDS for 1 h at 4 8C before layering it on top of the sucrose gradient.
sucrose percentage higher than that at which the Cx43 oligomer peak from NRK-52E cells was detected (Fig. 3, middle panel). In two out of four experiments, Cx43 immunoreactivity seemed to resolve in three peaks, with an additional apparent peak centered at 10.6% sucrose (Fig. 3, middle panel), a sucrose percentage similar to that containing Cx43 oligomers in NRK-52E cells. When the membranes were
Fig. 4. Co-precipitation of Cx43 and Cx56. Sucrose gradient fractions from NRK-Cx56 cells (lane 1) or embryonic chicken lens (lane 2) were immunoprecipitated using affinity-purified rabbit polyclonal anti-Cx56 antibodies. The immunoprecipitated proteins were resolved on SDScontaining gradient gels, and Cx43 was detected by immunoblotting using a mouse monoclonal anti-Cx43 antibody. The positions of the Cx43 bands in lane 1 are indicated (Ð, ). The positions of the Cx43 non-phosphorylated (Cx43-NP) and Cx43 phosphorylated (Cx43-P1 and Cx43-P2) forms are indicated for lane 2. Cx43 phosphoforms were not distinctly resolved in lane 1 ( ).
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connexins in epithelial cells and bow region/differentiating fiber cells (Musil et al., 1990a; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995; Jiang et al., 1995; Dahm et al., 1999). This raised the possibility that Cx43 formed heteromeric connexons with lens-specific connexins in the chicken lens. A similar approach to that taken for NRK-Cx56 cells was used for connexin-containing sucrose density gradient fractions from embryonic lenses. When Cx56 was immunoprecipitated using anti-Cx56 antibodies, three Cx43 bands corresponding to Cx43-NP, Cx43-P1 and Cx43-P2 were detected after immunoblotting with a monoclonal anti-Cx43 antibody (Fig. 4, lane 2). These results demonstrate the association of Cx43 with Cx56 in oligomeric forms in the lens.
Junctional currents and voltage-dependence of gap junction channels in NRK and NRK-Cx56 cell pairs
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magnitude but different polarity, a junctional current was elicited that decayed less steeply. A similar paradigm applied to NRK-Cx56 cells elicited junctional currents which behaved differently (Fig. 6a). Application of a voltage step elicited a junctional current which did not show a pronounced decay, even when the voltage step was prolonged to 400 ms (Fig. 6a). Application of a second voltage pulse of equal magnitude but opposite polarity, elicited non-decaying junctional currents of about the same magnitude (Fig. 6a). The difference observed in the junctional currents elicited in NRK or NRK-Cx56 cells was not due to a loss of cell impedance in the NRK-Cx56 cell pairs; the cell impedance remained relatively constant throughout the range of voltage pulses used (Fig. 6c). The difference in behavior of gap junctional channels in NRK and NRK-Cx56 cells can be best observed by plotting the steadystate junctional conductance (gsteady-state) vs. the transjunctional voltage (Vj) (Figs. 5b and 6b). While the voltage-dependence
Because the functional properties of heteromeric connexons might differ from those of homomeric connexons, gap junctional channels in NRK and NRK-Cx56 cells were analyzed by the dual whole-cell patch-clamp technique. Representative examples of the families of currents obtained are shown (Figs. 5a and 6a). In NRK cells, the initial junctional current elicited by a voltage pulse > 50 mV decayed to a steady-state value; the higher the voltage pulse, the steeper the decay. On application of a second voltage pulse of equal
Fig. 5. Junctional currents and voltage-dependence of gap junctional conductance in a pair of NRK cells. (a) Junctional currents (Ij) obtained from parental NRK cells generated in the non-stepped cell of a pair by stepping the other cell of the pair to a positive or negative voltage for 250 ms followed immediately by a step of equal magnitude but opposite polarity for 250 ms. The holding potential was kept at 0 mV; steps of either polarity were applied in increments of 20 mV from 10 to 150 mV. (b) Steady-state junctional conductance vs. transjunctional voltage (Vj) relationship. Vo 70 mV, gmin 1 nS, ratio of gsteady-state/ginitial at Vj /ÿ 150 mV is 0.2.
Fig. 6. Gap junctional currents and voltage-dependence of steadystate junctional conductance in a pair of NRK-Cx56 cells. (a) Junctional currents (Ij) generated in the non-stepped cell of a pair of NRK-Cx56 cells by stepping the other cell of the pair to a positive or a negative voltage for 400 ms followed immediately by a step of equal magnitude but opposite polarity for 400 ms. The holding potential was 0 mV; steps were applied in 20 mV increments from 10 to 150 mV. Junctional conductance was 4 nS. (b) Steady-state (400 ms) junctional conductance vs. transjunctional voltage (Vj) relationship. (c) Steady-state whole cell current vs. holding potential (Vh) curves for both cells from which junctional currents were obtained. Whole-cell slope conductance 20 nS.
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Fig. 7. Voltage-dependence relationship of junctional conductance between NRK or NRK-Cx56 cells. Normalized steady-state junctional conductance (mean SEM) obtained for several parental NRK (n 3; ±^±) and NRK-Cx56 (n 8; ±&±) cell pairs after applying the voltage pulse paradigm described in Figs. 5 and 6 vs. transjunctional voltage (Vj). Standard error bars are shown at 150 mV, 110 mV and 70 mV. The data on NRK-Cx56 differ significantly from the NRK data (p 0.05).
of gap junction channels observed in NRK cells was similar to that described for Cx43 in many cell types, little voltagedependence was observed for gap junctional channels in NRKCx56 cells. In these cells, the voltage-dependence typical of homotypic gap junction channels made of either Cx43 or Cx56 was absent and the apparent Vo was broadened such that Gmin was significantly higher than either homotypic form. Some variability in the voltage-dependence of gap junctional channels was observed in NRK-Cx56 cell pairs; the average data for several NRK and NRK-Cx56 cell pairs are shown in Figure 7.
Discussion Transfection of a variety of cell lines with wild-type and mutant connexins has been extensively used to study the biochemical properties of connexins. By using a similar approach, we have demonstrated in the present paper that stable transfection of NRK cells with Cx56 resulted in expression of the exogenous Cx56 RNA and its translation to levels detectable by immunoblotting. The immunoblot pattern of translated Cx56 was similar to that observed in the native tissue (i. e. the chicken lens), suggesting that Cx56 was modified as in the lens in vivo. Further support for this conclusion was obtained when the pattern of Cx56 bands collapsed to a doublet after treatment of NRK-Cx56 cell homogenates with alkaline phosphatase, as occurs for lens homogenates (Berthoud et al., 1994). Moreover, expressed Cx56 was detected in hexamer-containing fractions after velocity sedimentation through sucrose density gradients, and at appositional membranes by immunofluorescence. These results suggest that the exogenous Cx56 followed similar life cycle steps to endogenously expressed connexins. Expression of more than one connexin in a cell, as has been reported for several cell types (Nicholson et al., 1987; Traub et al., 1989; Kanter et al., 1993), allows the possible formation of heteromeric connexons (i. e. connexin hexamers containing more than one connexin type). Formation of heteromeric
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connexons has been inferred from biochemical data in which the two connexins involved co-purify (Stauffer, 1995) or coprecipitate (Jiang and Goodenough, 1996; He et al., 1999). The difference in Mr of the two connexins studied in this paper presented some advantages for the analysis of connexin mixing. In agreement with previously reported data (Musil and Goodenough, 1993), we found the endogenously expressed Cx43 in NRK cells in a monomer peak and a hexamer peak. A Cx56 monomer peak and a Cx56 hexamer peak were also detected in the Triton X-100-soluble fraction from NRKCx56 cells. Interestingly, the main oligomeric peak of Cx43 detected in NRK-Cx56 cells sedimented at a higher sucrose percentage than the Cx43 oligomer peak in NRK cells and comigrated with the peak of oligomeric forms of Cx56 in the NRK-Cx56 cells. Because we transfected cells expressing Cx43 (Mr, 42 ± 46 kDa) with Cx56 (Mr, 67 ± 90 kDa), hexameric forms containing Cx56 would be expected to sediment into denser sucrose gradient fractions than hexameric forms containing only Cx43. The presence of a denser peak of Cx43 hexamers co-sedimenting with the Cx56 hexamer peak suggests that Cx43 and Cx56 form heteromeric connexons. Furthermore, the co-precipitation of Cx43 after immunoprecipitation of Cx56 from sucrose gradient fractions from NRKCx56 cells, also suggests the formation of heteromeric connexons containing both connexins. The presence of an apparent Cx43 oligomer peak of intermediate density in NRK-Cx56 cells suggests that some Cx43 hexamers contained either no Cx56 or very little Cx56. The converse situation is probably also true, i. e. some Cx56 hexamers contained few or no Cx43 molecules. The functional properties of gap junctional channels in cells expressing two (or more) connexins are distinct from those of homomeric channels containing only one of the two connexins (Brink et al., 1997; Bevans et al., 1998; He et al., 1999). Similarly, our electrophysiological characterization of gap junctional channels from NRK-Cx56 cells revealed a Gj vs. Vj relationship that was less voltage-dependent and had a higher Gmin than that from NRK cells, or that expected based on studies in other systems expressing Cx43 (Moreno et al., 1992; White et al., 1994; present data) or Cx56 (Ebihara et al., 1995) alone. These data further suggest the formation of heteromeric connexons made of Cx56 and Cx43 in NRK-Cx56 cells. Because our biochemical data imply that these cells may contain both homomeric and heteromeric connexons, it is possible that the macroscopic channel data obtained in NRKCx56 cells reflects a mixture of heteromeric/heterotypic gap junction channels with homomeric/heterotypic and homomeric/homotypic gap junction channels. The electrophysiological data obtained from NRK-Cx56 might be of physiological relevance in the lens. All three identified lens connexins are found in the bow region/ differentiating fiber cells by immunofluorescence staining (Musil et al., 1990a; Evans et al., 1993; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995). In addition to Cx43, bovine Cx50 and chicken Cx45.6 and Cx56 have been found in lens epithelial cells (Jiang et al., 1995; Dahm et al., 1999). Coexpression of these connexins in the same cells suggests that ªfiber cell connexinsº, including Cx56, might form heteromeric connexons with Cx43 in the lens. In the present paper, we demonstrated co-precipitation of Cx43 after immunoprecipitation of Cx56 from embryonic chicken lens samples. These results suggest the existence of interactions between Cx43 and Cx56 in connexin oligomers; thus, it is likely that Cx43 and
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Cx56 form heteromeric connexons in the lens in vivo. Formation of heteromeric connexons by lens connexins might explain the observed different properties of gap junctions in different lens regions. Studies on NRK-Cx56 cells might help elucidate the contribution of heteromeric connexons formed by Cx56/Cx43 to these properties. Acknowledgements. This research was supported by NIH grants EY08368 (to E. C. Beyer), HL59199 (to E. C. Beyer) and GM55263 (to P. R. Brink). The authors would like to thank Dr. Steven Bassnett for his assistance in obtaining and analyzing the confocal images.
References Baldo, G. J., Mathias, R. T. (1992): Spatial variations in membrane properties in the intact rat lens. Biophys. J. 63, 518 ± 529. Berthoud, V. M., Cook, A. J., Beyer, E. C. (1994): Characterization of the gap junction protein connexin56 in the chicken lens by immunofluorescence and immunoblotting. Invest. Ophthalmol. Vis. Sci. 35, 4109 ± 4117. Berthoud, V. M., Beyer, E. C., Kurata, W. E., Lau, A. F., Lampe, P. D. (1997): The gap junction protein connexin56 is phosphorylated in both the intracellular loop and the carboxy terminal regions. Eur. J. Biochem. 244, 89 ± 97. Bevans, C. G., Kordel, M., Rhee, S. K., Harris, A. L. (1998): Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules. J. Biol. Chem. 273, 2808 ± 2816. Beyer, E. C., Kistler, J., Paul, D. L., Goodenough, D. A. (1989): Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108, 595 ± 605. Beyer, E. C., Paul, D. L., Goodenough, D. A. (1987): Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105, 2621 ± 2629. Bradford, M. M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 ± 254. Brink, P. R., Cronin, K., Banach, K., Peterson, E., Westphale, E. M., Seul, K. H., Ramanan, S. V., Beyer, E. C. (1997): Evidence for heteromeric gap junction channels formed from rat connexin43 and human Cx37. Am. J. Physiol. 273, C1386 ± C1396. Chomczynski, P., Sacchi, N. (1987): Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156 ± 159. Church, R. L., Wang, J. H., Steele, E. (1995): The human lens intrinsic membrane protein mp70 (cx50) gene: clonal analysis and chromosome mapping. Curr. Eye Research 14, 215 ± 221. Dahm, R., van Marle, J., Prescott, A. R., Quinlan, R. A. (1999): Gap junctions containing a8-connexin (MP70) in the adult mammalian lens epithelium suggests a re-evaluation of its role in the lens. Exp. Eye Res. 69, 45 ± 56. Donaldson, P. J., Dong, Y., Roos, M., Green, C., Goodenough, D. A., Kistler, J. (1995): Changes in lens connexin expression lead to increased gap junctional voltage dependence and conductance. Am. J. Physiol. 269, C590 ± C600. Ebihara, L., Berthoud, V. M., Beyer, E. C. (1995): Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys. J. 68, 1796 ± 1803. Evans, C. W., Eastwood, S., Rains, J., Gruitjers, W. T., Bullivant, S., Kistler J. (1993): Gap junction formation during development of the mouse lens. Eur. J. Cell Biol. 60, 243 ± 249. Feinberg, A. P., Vogelstein, B. (1983): A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 ± 13.
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Gong, X., Li, E., Klier, G., Huang, Q., Wu, Y., Lei, H., Kumar, N. M., Horwitz, J., Gilula N. B. (1997): Disruption of a3 connexin gene leads to proteolysis and cataractogenesis in mice. Cell 91, 833 ± 843. Goodenough, D. A. (1992): The crystalline lens. A system networked by gap junctional intercellular communication. Semin. Cell Biol. 3, 49 ± 58. Goodenough, D. A., Goliger, J. A., Paul, D. L. (1996): Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475 ± 502. Gupta, V. K., Berthoud, V. M., Atal, N., Jarillo, J. A., Barrio, L. C., Beyer, E. C. (1994): Bovine connexin44, a lens gap junction protein: molecular cloning, immunological characterization, and functional expression. Invest. Ophthalmol. Vis. Sci. 35, 3747 ± 3758. He, D. S., Jiang, J. X., Taffet, S. M., Burt, J. M. (1999): Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells. Proc. Natl. Acad. Sci. USA 96, 6495 ± 6500. Jiang, J. X., White, T. W., Goodenough, D. A. (1995): Changes in connexin expression and distribution during chick lens development. Dev. Biol. 168, 649 ± 661. Jiang, J. X., White, T. W., Goodenough, D. A., Paul, D. L. (1994): Molecular cloning and functional characterization of chick lens fiber connexin45.6. Mol. Biol. Cell 5, 363 ± 373. Jiang, J. X., Goodenough, D. A. (1996): Heteromeric connexons in lens gap junction channels. Proc. Natl. Acad. Sci. USA 93, 1287 ± 1291. Kanter, H. L., Laing. J. G., Beyer, E. C., Green, K. G., Saffitz, J. E. (1993): Multiple connexins colocalize in canine ventricular myocyte gap junctions. Circ. Res. 73, 344 ± 350. Konig, N., Zampighi, G. A. (1995): Purification of bovine lens cell-tocell channels composed of connexin44 and connexin50. J. Cell Sci. 108, 3091 ± 3098. Koval, M., Harley, J. E., Hick, E., Steinberg, T. H. (1997): Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells. J. Cell Biol. 137, 847 ± 857. Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A., Bhattacharya, S. (1999): Connexin46 mutations in autosomal dominant congenital cataract. Am. J. Hum. Genet. 64, 1357 ± 1364. Mathias, R. T., Rae, J. L., Eisenberg, R. S. (1979): Electrical properties of structural components of the crystalline lens. Biophys. J. 25, 181 ± 201. Mathias, R. T., Riquelme, G., Rae. J. L. (1991): Cell to cell communication and pH in the frog lens. J. Gen. Physiol. 98, 1085 ± 1103. Moreno, A. P., Fishman, G. I., Spray, D. C. (1992): Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys. J. 62, 51 ± 53. Musil, L. S., Beyer, E. C., Goodenough, D. A. (1990a): Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membr. Biol. 116, 163 ± 175. Musil, L. S., Goodenough, D. A. (1993): Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74, 1065 ± 1077. Musil, L. S., Cunningham, B. A., Edelman, G. M., Goodenough, D. A. (1990b): Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell Biol. 111, 2077 ± 2088. Nicholson, B. J., Dermietzel, R., Teplow, D., Traub, O., Willecke, K., Revel, J. P. (1987): Two homologous protein components of hepatic gap junctions. Nature 329, 732 ± 734. Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I., Goodenough, D. A. (1991): Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115, 1077 ± 1089. Rup, D. M., Veenstra, R. D., Wang, H. Z., Brink, P. R., Beyer, E. C. (1993): Chick connexin56, a novel lens gap junction protein. J. Biol. Chem. 268, 706 ± 712. Schuetze, S. M., Goodenough, D. A. (1982): Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development. J. Cell Biol. 92, 694 ± 705.
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Shiels, A., Mackay, D., Irisawa, H., Berry, V., Moore, A., Bhattacharya, S. (1998): A mis-sense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant ªZonular Pulverulentº cataract on chromosome 1q. Am. J. Hum. Genet. 62, 526 ± 532. Stauffer, K. A. (1995): The gap junction proteins b1-connexin (connexin-32) and b2-connexin (connexin-26) can form heteromeric hemichannels. J. Biol. Chem. 270, 6768 ± 6772. Steele, E. C. Jr., Lyon, M. F. Glenister, P. H., Guillot, P. V., Church, R. L. (1998): Identification of a mutation in the connexin50 (Cx50) gene of the No2 cataractous mouse mutant. In: R. Werner (ed.): Gap Junctions, IOS Press, Amsterdam, pp. 289 ± 293. Steinberg, T. H., Civitelli, R., Geist, S. T., Robertson, A. J., Hick, E., Veenstra, R. D., Wang, H. Z., Warlow, P. M., Westphale, E. M., Laing, J. G., Beyer, E. C. (1994): Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J. 13, 744 ± 750 . Traub, O., Look, J., Dermietzel, R., Brümmer, F., Hülser, D., Willecke, K. (1989): Comparative characterization of the 21-kD and 26-kD
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gap junction proteins in murine liver and cultured hepatocytes. J. Cell Biol. 108, 1039 ± 1051. White, T. W., Bruzzone, R., Goodenough, D. A., Paul, D. L. (1992): Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol. Biol. Cell 3, 711 ± 720. White, T. W., Bruzzone, R., Wolfram, S., Paul, D. L., Goodenough, D. A. (1994): Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J. Cell Biol. 125, 879 ± 892. White, T. W., Goodenough, D. A., Paul, D. L. (1998): Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts. J. Cell Biol. 143, 815 ± 825. Yang, D.-I., Louis, C. F. (1996): Molecular cloning of sheep connexin49 and its identity with MP70. Curr. Eye Res. 15, 307 ± 314.
European Journal of Cell Biology 80, 11 ± 19 (2001, January) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
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Heteromeric connexons formed by the lens connexins, connexin43 and connexin56 Viviana M. Berthoud1)a, Elisabeth A. Montegnaa, Namita Atalb, Naga H. Aithala, Peter R. Brinkc, Eric C. Beyera a b c
Department of Pediatrics, Section of Hematology/Oncology University of Chicago, Chicago, IL/USA Department of Medicine, Division of Infectious Diseases, Washington University Medical School, St. Louis, MO/USA Department of Physiology and Biophysics, State University of New York, Stony Brook, NY/USA
Received August 23, 2000 Received in revised version September 11, 2000 Accepted September 11, 2000
Heteromeric connexons ± lens connexins ± sucrose gradient sedimentation ± double whole-cell patch-clamp ± gap junctions In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.
Dr. Viviana M. Berthoud, Section of Pediatric Hematology/Oncology, University of Chicago, 5841 S. Maryland Ave., MC 4060, Chicago, IL 60637, e-mail: [email protected], Fax: 773 702 9881.
1)
Abbreviations. Cx Connexin. ± Cy3 Indocarbocyanine. ± ECL Enhanced chemiluminescence. ± FITC Fluorescein isothiocyanate. ± ginitial Initial conductance. ± Gj Normalized junctional conductance. ± gsteady-state Steadystate junctional conductance. ± Gmin Voltage-insensitive component of normalized gsteady-state. ± HEPES N-2-Hydroxyethylpiperazine-N'-2ethanesulfonic acid. ± Ij Junctional current. ± PAGE Polyacrylamide gel electrophoresis. ± PBS Phosphate-buffered saline. ± PMSF Phenylmethylsulfonyl fluoride. ± SDS Sodium dodecyl sulfate. ± TBS Tris-buffered saline. ± Vh Holding potential. ± Vj Transjunctional voltage. ± Vm Membrane potential. ± Vo Voltage at which the decrease in steady-state conductance is half maximal.
Introduction Gap junctions are membrane specializations that contain aggregates of intercellular channels that allow intercellular passage of ions and small molecules of up to 1000 Da. These channels are oligomeric assemblies of members of a family of related proteins called connexins (Cx) (Goodenough et al., 1996). Six connexin monomers assemble to form a hemi-gap junctional channel or connexon, which, in turn, may form a complete gap junction channel by docking with a connexon from the adjacent cell. The diversity of connexins and the multisubunit structure of the gap junctional channel allow for the formation of molecularly diverse channels. A homomeric hemichannel contains all subunits of a single connexin type. A homotypic gap junctional channel is formed when one homomeric connexon docks with another homomeric connexon in the adjacent cell containing the same connexin type; a heterotypic gap junctional channel is formed when one homomeric connexon docks with a homomeric connexon made of a different connexin type. A heteromeric connexon contains subunits of more than one connexin type. Docking of heteromeric connexons would lead to the greatest diversity of channel types. The lens is an avascular organ formed by an anterior epithelial cell layer and fiber cells which form the bulk of the
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organ. Gap junctions have been morphologically identified by electron microscopy between epithelial cells and between fiber cells (Goodenough, 1992). Recently published data have demonstrated that gap junctions are critical for lens function (Gong et al., 1997; Shiels et al., 1998; Steele et al., 1998; White et al., 1998; Mackay et al., 1999). Lens gap junctions contain at least three different connexins with somewhat overlapping cellular distributions. Lens epithelial cells express Cx43 (Beyer et al., 1989; Musil et al., 1990a) and fiber cells express lensspecific connexins such as rat Cx46 (Paul et al., 1991), mouse Cx50 (White et al., 1992), chicken Cx56 (Rup et al., 1993; Berthoud et al., 1994) and Cx45.6 (Jiang et al., 1994), bovine Cx44 (Gupta et al., 1994), ovine Cx49 (Yang and Louis, 1996), human Cx50 (Church et al., 1995) and Cx46 (Mackay et al., 1999). During development, epithelial cells in the bow region differentiate into fiber cells; a process that continues throughout the life-span of the individual. This process is characterized by the loss of organelles (including the nuclei) and changes in connexin expression. The differences in pH sensitivity of chicken lens gap junctions observed during development (Schuetze and Goodenough, 1982) have been correlated with changes in gap junction morphology (Schuetze and Goodenough, 1982) and connexin expression (Jiang et al., 1995), although the developmental switch in pH sensitivity cannot be explained on the basis of connexin type alone (Jiang et al., 1995). Impedance measurements in the lens suggest a gradient of junctional conductance from the poles to the equator (Mathias et al., 1979, 1991; Baldo and Mathias, 1992). Immunofluorescence studies on the distribution of Cx43 and of the lens-specific connexins suggest that the bow region/differentiating fiber cells co-express Cx43 with lens-specific connexins (Musil et al., 1990a; Evans et al., 1993; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995). Recent studies also suggest the presence of chicken Cx56 and Cx45.6 or its mammalian homolog, Cx50, in epithelial cells (Jiang et al., 1995; Dahm et al., 1999). Formation of gap junctional channels containing both bovine Cx46 and Cx50 (Konig and Zampighi, 1995), and of heteromeric connexons containing chicken Cx45.6 and Cx56 (Jiang and Goodenough, 1996) have been demonstrated. However, the existence of heteromeric connexons between Cx43 and either one of the lens-specific connexins has not been studied, nor have the functional properties of channels made of such heteromeric connexons been reported. In this study, we report the formation of heteromeric connexons between Cx43 and Cx56 in NRK cells transfected with Cx56 and in the chicken lens. These transfectants have been characterized for the expression, modification, targeting, and assembly of the transfected connexin into gap junctional channels, and the properties of Cx56 in these cells have been compared to those of Cx56 in the lens.
Materials and methods Chemicals
All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless specified otherwise.
Animals
Fertilized White Leghorn chicken eggs were obtained from SPAFAS (Norwich, CT, USA).
Cell culture
Parental normal rat kidney cells (NRK-52E, ATCC CRL 1571) were obtained from ATCC and grown in DMEM medium with 5% fetal bovine serum, 100 units/ml penicillin G and 100 mg/ml streptomycin sulfate. Cx56 in pSFFV-neo (Rup et al., 1993) was transfected into NRK cells using lipofectin, and stably transfected clones were selected by their resistance to 0.25 mg/ml active G418 in the medium.
RNA blotting
Total cellular RNA was prepared from cell cultures or 18E lenses according to Chomczynski and Sacchi (1987). RNA was separated on formaldehyde/agarose gels, and transferred to Hybond N nylon membranes (Amersham, Arlington Heights, IL) as previously described (Beyer et al., 1987). Hybridization was performed using a specific 32P-labeled DNA probe, containing base pairs 911 ± 1530 of Cx56, prepared using random hexanucleotide primers and the Klenow fragment of DNA polymerase I (Feinberg and Vogelstein, 1983).
Immunofluorescence
Untransfected NRK cells or NRK cells transfected with Cx56 were plated on 4-well slides (LAB TEK, Nalge Nunc International, Naperville, IL, USA) and allowed to reach 80 ± 90% confluence. Cells were then rinsed with phosphate-buffered saline, pH 7.4 (PBS), fixed in methanol : acetone (1 : 1) for 2 minutes at room temperature, permeabilized in 1% Triton X-100 in PBS for 15 min at room temperature and blocked in 2% normal goat serum, 1% Triton X-100 in PBS for 10 min at room temperature as previously described (Steinberg et al., 1994). Fixed cells were then incubated in mouse monoclonal anti-Cx43 antibody (Chemicon International, Temecula, CA, USA) and rabbit polyclonal anti-Cx56 antibodies (Berthoud et al., 1994) overnight at 4 8C. Cells were rinsed 4 times with PBS and then sequentially incubated in secondary antibody, Cy3-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA, USA). After 1.5 h incubation at room temperature, cells were rinsed 4 times in PBS, and coverslips were mounted with 2% n-propylgallate in PBS : glycerol (1 : 1). Confocal images were obtained using a Zeiss LSM 410 confocal microscope equipped with an argon/krypton laser.
Modification of Cx56 by phosphorylation
Cultured cells were harvested in PBS and centrifuged for 7 min at 14 000g. Cell pellets or 18E lenses were homogenized by sonication in 1 mM NaHCO3, 2 mM phenylmethylsulfonyl fluoride (PMSF). Homogenate aliquots containing 100 mg of protein were treated with alkaline phosphatase as previously described (Berthoud et al., 1994). Then, the proteins were resolved in sodium dodecyl sulfate (SDS)-containing gels and the pattern of bands analyzed after immunoblotting.
Sedimentation through sucrose density gradients
The procedure was performed as described by Koval et al. (1997) with minor modifications. Briefly, cultured cells were harvested in incubation buffer (PBS containing 0.8 mM MgSO4, 2.7 mM CaCl2, 1 mM Na2V2O5, 10 mM NaF, 10 mM N-ethylmaleimide, 20 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 3 mg/ml antipain, 3 mg/ml phosphoramidon, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 20 mg/ml Pefabloc; protease inhibitors were purchased from Roche Molecular Biochemicals, Indianapolis, IN, USA) and centrifuged at 150g for 7 min. Pelleted cells or chicken lenses were homogenized in incubation buffer. Triton X-100 was added to aliquots containing 300 mg of protein to a final concentration of 1% and incubated on ice for 30 min. The aliquots were centrifuged at 100 000gave for 30 min and the supernatant was layered on top of a sucrose density gradient (4.8 ml 5 ± 20% linear sucrose gradient with a 0.2-ml 25% sucrose cushion). The gradients were centrifuged at 100 000g for 19 h and 250-ml fractions were collected. In some cases, oligomers were dissociated by incubation of the Triton X-100-soluble material in 0.2% SDS for 1 h at 4 8C prior to layering it on top of the sucrose gradient.
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The percentage concentration of sucrose in each fraction was determined using a hand held Brix refractometer (0 ± 328; Fisher Scientific, Pittsburgh, PA, USA). Fractions were analyzed by immunoblotting. Horseradish peroxidase and catalase were subjected to sedimentation through sucrose densities as standards for connexin monomers (5S) and connexons (9S), respectively. The 5S standard, horseradish peroxidase, was detected between 6.6 ± 9% sucrose with its peak centered at 7.8% sucrose, the 9S standard, catalase, was detected between 10.2 ± 12.7% sucrose with its peak centered at 11.6% sucrose (not shown).
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filtered at 1 kHz. All recordings shown are currents observed in the non-stepped cell held at membrane potential (Vm) of 0 mV. These recordings represent Ij only.
Results Expression of Cx56 in NRK cells
Immunoblotting
Alkaline phosphatase experiments. 100 mg of protein from whole cell or lens homogenates treated with alkaline phosphatase or with buffer only (controls) were resolved on 9% SDS-containing polyacrylamide gels and subjected to immunoblot analysis using rabbit polyclonal anti-Cx56 or anti-Cx43 antibodies. After overnight incubation in primary antibodies at 4 8C, the membranes were rinsed in Tris-buffered saline (TBS) and incubated in peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. After this period of time, the membranes were rinsed in TBS and detection of secondary antibody binding was performed using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL, USA). Sucrose density gradient fractions. Equal volumes of the sucrose density gradient fractions were loaded in each lane and resolved on 9% SDS-containing polyacrylamide gels; the proteins were subjected to immunoblotting as described above using either rabbit polyclonal antiCx56 or anti-Cx43 antibodies. After reaction with ECL, membranes were stripped of primary antibody by incubation in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM b-mercaptoethanol for 30 min at 50 8C. Then, membranes were reacted with the other primary antibody.
Co-precipitation of Cx43 and Cx56
Cultured NRK-Cx56 cells or embryonic chicken lenses were sedimented through sucrose gradients as described above. Sucrose gradient fractions with sucrose densities between 8.6% and 15.2% were pooled and immunoprecipitated using anti-Cx56 antibodies as previously described (Berthoud et al., 1997). Immunoprecipitated proteins were resolved on SDS-containing 4 ± 15% polyacrylamide gradient gels and subjected to immunoblotting as described above using a mouse monoclonal anti-Cx43 antibody (Chemicon International, Temecula, CA, USA).
Protein determination
To study the functional properties of gap junctional channels formed by heteromeric Cx43/Cx56 connexons, we generated clones from a cell line stably transfected with Cx56. We chose NRK-52E, a normal rat kidney cell line that endogenously expresses Cx43 and that has been extensively used to study the solubility properties of the different phosphorylated forms of Cx43 and its assembly into oligomeric forms (Musil and Goodenough, 1993). The expression of the transfected Cx56 RNA in the clones was demonstrated by RNA blotting. A single band of the appropriate size for the transfected Cx56 was observed in total cellular RNA (Fig. 1a). The stably transfected clones were analyzed for the presence of Cx56 protein by immunoblotting. Multiple bands of Cx56 were detected in whole cell homogenates of stably transfected NRK-Cx56 (Fig. 1b, lane 3). The immunoblot pattern of Cx56 in NRK-Cx56 was similar to that obtained in an 18E whole lens homogenate (Fig. 1b, lane 1). Because Cx56 is a phosphoprotein (Berthoud et al., 1997), we studied the possibility that the observed complexity in the immunoblot pattern was due to phosphorylation. Similar to results obtained from lens homogenates (Berthoud et al., 1994, and Fig. 1b, lane 2), when NRK-Cx56 whole cell homogenates were incubated in the presence of alkaline phosphatase before resolution of the proteins by SDSPAGE, the multiple Cx56 bands collapsed to a doublet (Fig. 1b, lane 4). These results suggest that, similar to the lens, Cx56 is modified by phosphorylation in NRK-Cx56 transfectants. The distribution of Cx43 and Cx56 in untransfected NRK and NRK-Cx56 was studied by immunofluorescence. In agreement with previous results (Musil and Goodenough, 1993), immunopositive staining for Cx43 was localized to a perinuclear region and to puncta at appositional membranes in NRK-52E cells (Fig. 2a). A similar Cx43 staining pattern was
Proteins were determined using the BioRad Protein Assay (BioRad, Hercules, CA, USA) based on the Bradford dye-binding procedure (Bradford, 1976).
Electrophysiology
Experiments were carried out on cell pairs using the double whole-cell patch-clamp technique (Brink et al., 1997). During the experiments, the cells were bathed in a solution containing 180 mM CsCl or KCl, 1 mM CaCl2, 1.8 mM MgCl2, and 10 mM HEPES, pH 7.1 ± 7.3. For the wholecell recordings, the pipette solution contained 180 mM CsCl, 1 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 mM CaCl2, 1.8 mM MgCl2 and 10 mM HEPES, pH 7.0. Flow of intercellular junctional current (Ij) was induced by different voltage protocols. For the recording of the macroscopic current-voltage relationship, both cells were held at a holding potential of 0 mV. From this holding potential, the voltage of one cell was stepped to varying voltages (transjunctional voltages of 10 ± 150 mV or 120 mV, in 20 mV increments). For all experiments shown, the first voltage step was negative. After holding the potential for 250 or 400 ms, the voltage was flipped to the equal magnitude but opposite polarity for the same time. Current recording data analyses were performed as previously described (Brink et al., 1997). All macroscopic records were
Fig. 1. Expression and modification of Cx56 in NRK cells transfected with Cx56. (a) Ten mg of total RNA from NRK (lane 1) and NRK-Cx56 (lane 2) cells were resolved on formaldehyde-containing 1% agarose gels and transferred to Hybond membranes. The membranes were hybridized with a 32P-labeled Cx56 probe and exposed to X-ray film. The positions of the 18S and 28S rRNAs are indicated. (b) Immunoblot pattern of Cx56 in homogenates of 18-day embryonic lens (lanes 1 and 2) and NRK-Cx56 cells (lanes 3 and 4). The homogenates were left untreated (lanes 1 and 3) or they were incubated with alkaline phosphatase (lanes 2 and 4) prior to resolution of the bands by SDSPAGE. Note that several bands of Cx56 are detected in NRK-Cx56 cell homogenates. Similar to the results obtained using a lens homogenate, these bands collapse to a doublet after alkaline phosphatase treatment.
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Fig. 2. Localization of Cx43 and Cx56 in NRK and NRK-Cx56 cells. Confocal images from double immunofluorescence staining of NRK (a ± c) and NRK-Cx56 (d ± f) cells using mouse monoclonal anti-Cx43 (a, d) and rabbit polyclonal anti-Cx56 (b, e) antibodies followed by
Cy3-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies. Panels c and f show superposition of panels a and b and d and e, respectively. Bar represents 10 mm.
observed in NRK-Cx56 cells (Fig. 2d). As expected, no immunopositive staining was observed in NRK-52E with anti-Cx56 antibodies (Fig. 2b). In NRK-Cx56 cells, anti-Cx56 antibodies showed punctate immunopositive staining at appositional membranes and diffuse staining in the perinuclear region (Fig. 2e). When the confocal images were superimposed, the staining appeared red for the NRK-52E cells (Fig. 2c) because of the presence of Cx43 alone and yellow for the NRK-Cx56 cells (Fig. 2f), suggesting co-localization of Cx43 and Cx56 in the transfected cells.
monomeric and oligomeric forms by sedimentation through sucrose density gradients (Musil and Goodenough, 1993). We used a similar approach to study the assembly of oligomeric forms in NRK-Cx56 cells. When we applied this technique to the Triton X-100-soluble fraction of NRK-52E cells (used as a control), we obtained two peaks of immunoreactive Cx43 (Fig. 3, top panel), in agreement with the data previously published (Musil and Goodenough, 1993); the peaks were centered at 8.6 and 10.8% sucrose. In the detergent-soluble fraction of NRK-Cx56 cells, two major peaks of immunoreactive Cx43 were detected in all experiments after sedimentation through sucrose gradients (n 4). One of these peaks was centered at 7.8 ± 9.2% sucrose (Fig. 3, middle panel), similar to the sucrose percentage at which the Cx43 monomer peak was detected in NRK-52E cells. The second main peak of Cx43 immunoreactivity was centered at 12.2 ± 13.6% sucrose, a
Separation of monomeric and oligomeric forms of Cx43 and Cx56 by sedimentation through sucrose density gradients
It has been previously reported that the Triton X-100-soluble fraction of Cx43 from NRK-52E cells can be separated into
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Lens Cx43 and Cx56 form heteromeric connexons
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reacted with anti-Cx56 antibodies, two peaks of immunoreactive Cx56 centered at 7.8 ± 9.4 and 12.7 ± 13.6% sucrose were observed in NRK-Cx56 cells (Fig. 3, middle panel); the fractions containing the apparent middle peak of immunoreactive Cx43 were almost devoid of anti-Cx56 immunoreactivity (Fig. 3, middle panel). When SDS was added to the Triton X-100-soluble fraction of NRK-Cx56 cells prior to layering the sample onto the gradient, only one peak, cosedimenting with the monomer peak, was observed with either anti-Cx43 or anti-Cx56 antibodies, confirming that the connexin immunoreactivity detected between 10.6 and 13.6% sucrose represented connexin oligomers (Fig. 3, bottom panel).
Heteromeric connexons in NRK-Cx56 cells
The presence of an oligomer peak of Cx43 which co-migrated with Cx56 oligomers suggested that in NRK-Cx56, Cx43 was forming heteromeric connexons with Cx56. Formation of heteromeric connexons can be demonstrated by immunoprecipitation using a specific antibody to one of the connexins involved and immunoblotting of the immunoprecipitated protein with an antibody to the other connexin. To test the hypothesis that Cx43 formed heteromeric connexons with Cx56, Cx56 was immunoprecipitated from connexin-containing sucrose gradient fractions of Triton X-100 extracts of NRKCx56 using anti-Cx56 antibodies; then, the immunoprecipitate was resolved on SDS-containing gels and analyzed by immunoblotting with anti-Cx43 antibody. Several bands of Cx43 were detected by immunoblotting in these samples (Fig. 4, lane 1); these likely correspond to the non-phosphorylated and phosphorylated forms of Cx43 (Musil et al., 1990b).
Do Cx43/Cx56 heteromeric connexons exist in the lens?
Studies on the distribution of lens connexins in several species suggest that Cx43 is co-expressed with the lens-specific
Fig. 3. Analysis of oligomeric association of Cx43 and Cx56 in NRK and NRK-Cx56 by sucrose gradient centrifugation. Graphs depict the densitometric values obtained after quantitation of Cx43 (*; top, middle and bottom panels) or Cx56 (*; middle and bottom panels) immunoblots of sucrose gradient fractions from Triton X-100-soluble material from NRK (top panel) or NRK-Cx56 (middle and bottom panels) cells. For NRK-Cx56, the Triton X-100-soluble material was untreated (middle panel) or treated (bottom panel) with 0.2% SDS for 1 h at 4 8C before layering it on top of the sucrose gradient.
sucrose percentage higher than that at which the Cx43 oligomer peak from NRK-52E cells was detected (Fig. 3, middle panel). In two out of four experiments, Cx43 immunoreactivity seemed to resolve in three peaks, with an additional apparent peak centered at 10.6% sucrose (Fig. 3, middle panel), a sucrose percentage similar to that containing Cx43 oligomers in NRK-52E cells. When the membranes were
Fig. 4. Co-precipitation of Cx43 and Cx56. Sucrose gradient fractions from NRK-Cx56 cells (lane 1) or embryonic chicken lens (lane 2) were immunoprecipitated using affinity-purified rabbit polyclonal anti-Cx56 antibodies. The immunoprecipitated proteins were resolved on SDScontaining gradient gels, and Cx43 was detected by immunoblotting using a mouse monoclonal anti-Cx43 antibody. The positions of the Cx43 bands in lane 1 are indicated (Ð, ). The positions of the Cx43 non-phosphorylated (Cx43-NP) and Cx43 phosphorylated (Cx43-P1 and Cx43-P2) forms are indicated for lane 2. Cx43 phosphoforms were not distinctly resolved in lane 1 ( ).
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V. M. Berthoud, E. A. Montegna et al.
connexins in epithelial cells and bow region/differentiating fiber cells (Musil et al., 1990a; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995; Jiang et al., 1995; Dahm et al., 1999). This raised the possibility that Cx43 formed heteromeric connexons with lens-specific connexins in the chicken lens. A similar approach to that taken for NRK-Cx56 cells was used for connexin-containing sucrose density gradient fractions from embryonic lenses. When Cx56 was immunoprecipitated using anti-Cx56 antibodies, three Cx43 bands corresponding to Cx43-NP, Cx43-P1 and Cx43-P2 were detected after immunoblotting with a monoclonal anti-Cx43 antibody (Fig. 4, lane 2). These results demonstrate the association of Cx43 with Cx56 in oligomeric forms in the lens.
Junctional currents and voltage-dependence of gap junction channels in NRK and NRK-Cx56 cell pairs
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magnitude but different polarity, a junctional current was elicited that decayed less steeply. A similar paradigm applied to NRK-Cx56 cells elicited junctional currents which behaved differently (Fig. 6a). Application of a voltage step elicited a junctional current which did not show a pronounced decay, even when the voltage step was prolonged to 400 ms (Fig. 6a). Application of a second voltage pulse of equal magnitude but opposite polarity, elicited non-decaying junctional currents of about the same magnitude (Fig. 6a). The difference observed in the junctional currents elicited in NRK or NRK-Cx56 cells was not due to a loss of cell impedance in the NRK-Cx56 cell pairs; the cell impedance remained relatively constant throughout the range of voltage pulses used (Fig. 6c). The difference in behavior of gap junctional channels in NRK and NRK-Cx56 cells can be best observed by plotting the steadystate junctional conductance (gsteady-state) vs. the transjunctional voltage (Vj) (Figs. 5b and 6b). While the voltage-dependence
Because the functional properties of heteromeric connexons might differ from those of homomeric connexons, gap junctional channels in NRK and NRK-Cx56 cells were analyzed by the dual whole-cell patch-clamp technique. Representative examples of the families of currents obtained are shown (Figs. 5a and 6a). In NRK cells, the initial junctional current elicited by a voltage pulse > 50 mV decayed to a steady-state value; the higher the voltage pulse, the steeper the decay. On application of a second voltage pulse of equal
Fig. 5. Junctional currents and voltage-dependence of gap junctional conductance in a pair of NRK cells. (a) Junctional currents (Ij) obtained from parental NRK cells generated in the non-stepped cell of a pair by stepping the other cell of the pair to a positive or negative voltage for 250 ms followed immediately by a step of equal magnitude but opposite polarity for 250 ms. The holding potential was kept at 0 mV; steps of either polarity were applied in increments of 20 mV from 10 to 150 mV. (b) Steady-state junctional conductance vs. transjunctional voltage (Vj) relationship. Vo 70 mV, gmin 1 nS, ratio of gsteady-state/ginitial at Vj /ÿ 150 mV is 0.2.
Fig. 6. Gap junctional currents and voltage-dependence of steadystate junctional conductance in a pair of NRK-Cx56 cells. (a) Junctional currents (Ij) generated in the non-stepped cell of a pair of NRK-Cx56 cells by stepping the other cell of the pair to a positive or a negative voltage for 400 ms followed immediately by a step of equal magnitude but opposite polarity for 400 ms. The holding potential was 0 mV; steps were applied in 20 mV increments from 10 to 150 mV. Junctional conductance was 4 nS. (b) Steady-state (400 ms) junctional conductance vs. transjunctional voltage (Vj) relationship. (c) Steady-state whole cell current vs. holding potential (Vh) curves for both cells from which junctional currents were obtained. Whole-cell slope conductance 20 nS.
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Fig. 7. Voltage-dependence relationship of junctional conductance between NRK or NRK-Cx56 cells. Normalized steady-state junctional conductance (mean SEM) obtained for several parental NRK (n 3; ±^±) and NRK-Cx56 (n 8; ±&±) cell pairs after applying the voltage pulse paradigm described in Figs. 5 and 6 vs. transjunctional voltage (Vj). Standard error bars are shown at 150 mV, 110 mV and 70 mV. The data on NRK-Cx56 differ significantly from the NRK data (p 0.05).
of gap junction channels observed in NRK cells was similar to that described for Cx43 in many cell types, little voltagedependence was observed for gap junctional channels in NRKCx56 cells. In these cells, the voltage-dependence typical of homotypic gap junction channels made of either Cx43 or Cx56 was absent and the apparent Vo was broadened such that Gmin was significantly higher than either homotypic form. Some variability in the voltage-dependence of gap junctional channels was observed in NRK-Cx56 cell pairs; the average data for several NRK and NRK-Cx56 cell pairs are shown in Figure 7.
Discussion Transfection of a variety of cell lines with wild-type and mutant connexins has been extensively used to study the biochemical properties of connexins. By using a similar approach, we have demonstrated in the present paper that stable transfection of NRK cells with Cx56 resulted in expression of the exogenous Cx56 RNA and its translation to levels detectable by immunoblotting. The immunoblot pattern of translated Cx56 was similar to that observed in the native tissue (i. e. the chicken lens), suggesting that Cx56 was modified as in the lens in vivo. Further support for this conclusion was obtained when the pattern of Cx56 bands collapsed to a doublet after treatment of NRK-Cx56 cell homogenates with alkaline phosphatase, as occurs for lens homogenates (Berthoud et al., 1994). Moreover, expressed Cx56 was detected in hexamer-containing fractions after velocity sedimentation through sucrose density gradients, and at appositional membranes by immunofluorescence. These results suggest that the exogenous Cx56 followed similar life cycle steps to endogenously expressed connexins. Expression of more than one connexin in a cell, as has been reported for several cell types (Nicholson et al., 1987; Traub et al., 1989; Kanter et al., 1993), allows the possible formation of heteromeric connexons (i. e. connexin hexamers containing more than one connexin type). Formation of heteromeric
Lens Cx43 and Cx56 form heteromeric connexons
17
connexons has been inferred from biochemical data in which the two connexins involved co-purify (Stauffer, 1995) or coprecipitate (Jiang and Goodenough, 1996; He et al., 1999). The difference in Mr of the two connexins studied in this paper presented some advantages for the analysis of connexin mixing. In agreement with previously reported data (Musil and Goodenough, 1993), we found the endogenously expressed Cx43 in NRK cells in a monomer peak and a hexamer peak. A Cx56 monomer peak and a Cx56 hexamer peak were also detected in the Triton X-100-soluble fraction from NRKCx56 cells. Interestingly, the main oligomeric peak of Cx43 detected in NRK-Cx56 cells sedimented at a higher sucrose percentage than the Cx43 oligomer peak in NRK cells and comigrated with the peak of oligomeric forms of Cx56 in the NRK-Cx56 cells. Because we transfected cells expressing Cx43 (Mr, 42 ± 46 kDa) with Cx56 (Mr, 67 ± 90 kDa), hexameric forms containing Cx56 would be expected to sediment into denser sucrose gradient fractions than hexameric forms containing only Cx43. The presence of a denser peak of Cx43 hexamers co-sedimenting with the Cx56 hexamer peak suggests that Cx43 and Cx56 form heteromeric connexons. Furthermore, the co-precipitation of Cx43 after immunoprecipitation of Cx56 from sucrose gradient fractions from NRKCx56 cells, also suggests the formation of heteromeric connexons containing both connexins. The presence of an apparent Cx43 oligomer peak of intermediate density in NRK-Cx56 cells suggests that some Cx43 hexamers contained either no Cx56 or very little Cx56. The converse situation is probably also true, i. e. some Cx56 hexamers contained few or no Cx43 molecules. The functional properties of gap junctional channels in cells expressing two (or more) connexins are distinct from those of homomeric channels containing only one of the two connexins (Brink et al., 1997; Bevans et al., 1998; He et al., 1999). Similarly, our electrophysiological characterization of gap junctional channels from NRK-Cx56 cells revealed a Gj vs. Vj relationship that was less voltage-dependent and had a higher Gmin than that from NRK cells, or that expected based on studies in other systems expressing Cx43 (Moreno et al., 1992; White et al., 1994; present data) or Cx56 (Ebihara et al., 1995) alone. These data further suggest the formation of heteromeric connexons made of Cx56 and Cx43 in NRK-Cx56 cells. Because our biochemical data imply that these cells may contain both homomeric and heteromeric connexons, it is possible that the macroscopic channel data obtained in NRKCx56 cells reflects a mixture of heteromeric/heterotypic gap junction channels with homomeric/heterotypic and homomeric/homotypic gap junction channels. The electrophysiological data obtained from NRK-Cx56 might be of physiological relevance in the lens. All three identified lens connexins are found in the bow region/ differentiating fiber cells by immunofluorescence staining (Musil et al., 1990a; Evans et al., 1993; Berthoud et al., 1994; Jiang et al., 1994; Donaldson et al., 1995). In addition to Cx43, bovine Cx50 and chicken Cx45.6 and Cx56 have been found in lens epithelial cells (Jiang et al., 1995; Dahm et al., 1999). Coexpression of these connexins in the same cells suggests that ªfiber cell connexinsº, including Cx56, might form heteromeric connexons with Cx43 in the lens. In the present paper, we demonstrated co-precipitation of Cx43 after immunoprecipitation of Cx56 from embryonic chicken lens samples. These results suggest the existence of interactions between Cx43 and Cx56 in connexin oligomers; thus, it is likely that Cx43 and
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Cx56 form heteromeric connexons in the lens in vivo. Formation of heteromeric connexons by lens connexins might explain the observed different properties of gap junctions in different lens regions. Studies on NRK-Cx56 cells might help elucidate the contribution of heteromeric connexons formed by Cx56/Cx43 to these properties. Acknowledgements. This research was supported by NIH grants EY08368 (to E. C. Beyer), HL59199 (to E. C. Beyer) and GM55263 (to P. R. Brink). The authors would like to thank Dr. Steven Bassnett for his assistance in obtaining and analyzing the confocal images.
References Baldo, G. J., Mathias, R. T. (1992): Spatial variations in membrane properties in the intact rat lens. Biophys. J. 63, 518 ± 529. Berthoud, V. M., Cook, A. J., Beyer, E. C. (1994): Characterization of the gap junction protein connexin56 in the chicken lens by immunofluorescence and immunoblotting. Invest. Ophthalmol. Vis. Sci. 35, 4109 ± 4117. Berthoud, V. M., Beyer, E. C., Kurata, W. E., Lau, A. F., Lampe, P. D. (1997): The gap junction protein connexin56 is phosphorylated in both the intracellular loop and the carboxy terminal regions. Eur. J. Biochem. 244, 89 ± 97. Bevans, C. G., Kordel, M., Rhee, S. K., Harris, A. L. (1998): Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules. J. Biol. Chem. 273, 2808 ± 2816. Beyer, E. C., Kistler, J., Paul, D. L., Goodenough, D. A. (1989): Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108, 595 ± 605. Beyer, E. C., Paul, D. L., Goodenough, D. A. (1987): Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105, 2621 ± 2629. Bradford, M. M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 ± 254. Brink, P. R., Cronin, K., Banach, K., Peterson, E., Westphale, E. M., Seul, K. H., Ramanan, S. V., Beyer, E. C. (1997): Evidence for heteromeric gap junction channels formed from rat connexin43 and human Cx37. Am. J. Physiol. 273, C1386 ± C1396. Chomczynski, P., Sacchi, N. (1987): Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156 ± 159. Church, R. L., Wang, J. H., Steele, E. (1995): The human lens intrinsic membrane protein mp70 (cx50) gene: clonal analysis and chromosome mapping. Curr. Eye Research 14, 215 ± 221. Dahm, R., van Marle, J., Prescott, A. R., Quinlan, R. A. (1999): Gap junctions containing a8-connexin (MP70) in the adult mammalian lens epithelium suggests a re-evaluation of its role in the lens. Exp. Eye Res. 69, 45 ± 56. Donaldson, P. J., Dong, Y., Roos, M., Green, C., Goodenough, D. A., Kistler, J. (1995): Changes in lens connexin expression lead to increased gap junctional voltage dependence and conductance. Am. J. Physiol. 269, C590 ± C600. Ebihara, L., Berthoud, V. M., Beyer, E. C. (1995): Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys. J. 68, 1796 ± 1803. Evans, C. W., Eastwood, S., Rains, J., Gruitjers, W. T., Bullivant, S., Kistler J. (1993): Gap junction formation during development of the mouse lens. Eur. J. Cell Biol. 60, 243 ± 249. Feinberg, A. P., Vogelstein, B. (1983): A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 ± 13.
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Gong, X., Li, E., Klier, G., Huang, Q., Wu, Y., Lei, H., Kumar, N. M., Horwitz, J., Gilula N. B. (1997): Disruption of a3 connexin gene leads to proteolysis and cataractogenesis in mice. Cell 91, 833 ± 843. Goodenough, D. A. (1992): The crystalline lens. A system networked by gap junctional intercellular communication. Semin. Cell Biol. 3, 49 ± 58. Goodenough, D. A., Goliger, J. A., Paul, D. L. (1996): Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475 ± 502. Gupta, V. K., Berthoud, V. M., Atal, N., Jarillo, J. A., Barrio, L. C., Beyer, E. C. (1994): Bovine connexin44, a lens gap junction protein: molecular cloning, immunological characterization, and functional expression. Invest. Ophthalmol. Vis. Sci. 35, 3747 ± 3758. He, D. S., Jiang, J. X., Taffet, S. M., Burt, J. M. (1999): Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells. Proc. Natl. Acad. Sci. USA 96, 6495 ± 6500. Jiang, J. X., White, T. W., Goodenough, D. A. (1995): Changes in connexin expression and distribution during chick lens development. Dev. Biol. 168, 649 ± 661. Jiang, J. X., White, T. W., Goodenough, D. A., Paul, D. L. (1994): Molecular cloning and functional characterization of chick lens fiber connexin45.6. Mol. Biol. Cell 5, 363 ± 373. Jiang, J. X., Goodenough, D. A. (1996): Heteromeric connexons in lens gap junction channels. Proc. Natl. Acad. Sci. USA 93, 1287 ± 1291. Kanter, H. L., Laing. J. G., Beyer, E. C., Green, K. G., Saffitz, J. E. (1993): Multiple connexins colocalize in canine ventricular myocyte gap junctions. Circ. Res. 73, 344 ± 350. Konig, N., Zampighi, G. A. (1995): Purification of bovine lens cell-tocell channels composed of connexin44 and connexin50. J. Cell Sci. 108, 3091 ± 3098. Koval, M., Harley, J. E., Hick, E., Steinberg, T. H. (1997): Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells. J. Cell Biol. 137, 847 ± 857. Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A., Bhattacharya, S. (1999): Connexin46 mutations in autosomal dominant congenital cataract. Am. J. Hum. Genet. 64, 1357 ± 1364. Mathias, R. T., Rae, J. L., Eisenberg, R. S. (1979): Electrical properties of structural components of the crystalline lens. Biophys. J. 25, 181 ± 201. Mathias, R. T., Riquelme, G., Rae. J. L. (1991): Cell to cell communication and pH in the frog lens. J. Gen. Physiol. 98, 1085 ± 1103. Moreno, A. P., Fishman, G. I., Spray, D. C. (1992): Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys. J. 62, 51 ± 53. Musil, L. S., Beyer, E. C., Goodenough, D. A. (1990a): Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membr. Biol. 116, 163 ± 175. Musil, L. S., Goodenough, D. A. (1993): Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74, 1065 ± 1077. Musil, L. S., Cunningham, B. A., Edelman, G. M., Goodenough, D. A. (1990b): Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell Biol. 111, 2077 ± 2088. Nicholson, B. J., Dermietzel, R., Teplow, D., Traub, O., Willecke, K., Revel, J. P. (1987): Two homologous protein components of hepatic gap junctions. Nature 329, 732 ± 734. Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I., Goodenough, D. A. (1991): Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115, 1077 ± 1089. Rup, D. M., Veenstra, R. D., Wang, H. Z., Brink, P. R., Beyer, E. C. (1993): Chick connexin56, a novel lens gap junction protein. J. Biol. Chem. 268, 706 ± 712. Schuetze, S. M., Goodenough, D. A. (1982): Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development. J. Cell Biol. 92, 694 ± 705.
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Shiels, A., Mackay, D., Irisawa, H., Berry, V., Moore, A., Bhattacharya, S. (1998): A mis-sense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant ªZonular Pulverulentº cataract on chromosome 1q. Am. J. Hum. Genet. 62, 526 ± 532. Stauffer, K. A. (1995): The gap junction proteins b1-connexin (connexin-32) and b2-connexin (connexin-26) can form heteromeric hemichannels. J. Biol. Chem. 270, 6768 ± 6772. Steele, E. C. Jr., Lyon, M. F. Glenister, P. H., Guillot, P. V., Church, R. L. (1998): Identification of a mutation in the connexin50 (Cx50) gene of the No2 cataractous mouse mutant. In: R. Werner (ed.): Gap Junctions, IOS Press, Amsterdam, pp. 289 ± 293. Steinberg, T. H., Civitelli, R., Geist, S. T., Robertson, A. J., Hick, E., Veenstra, R. D., Wang, H. Z., Warlow, P. M., Westphale, E. M., Laing, J. G., Beyer, E. C. (1994): Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J. 13, 744 ± 750 . Traub, O., Look, J., Dermietzel, R., Brümmer, F., Hülser, D., Willecke, K. (1989): Comparative characterization of the 21-kD and 26-kD
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gap junction proteins in murine liver and cultured hepatocytes. J. Cell Biol. 108, 1039 ± 1051. White, T. W., Bruzzone, R., Goodenough, D. A., Paul, D. L. (1992): Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol. Biol. Cell 3, 711 ± 720. White, T. W., Bruzzone, R., Wolfram, S., Paul, D. L., Goodenough, D. A. (1994): Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J. Cell Biol. 125, 879 ± 892. White, T. W., Goodenough, D. A., Paul, D. L. (1998): Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts. J. Cell Biol. 143, 815 ± 825. Yang, D.-I., Louis, C. F. (1996): Molecular cloning of sheep connexin49 and its identity with MP70. Curr. Eye Res. 15, 307 ± 314.