Use of Antibodies in the Analysis of Connexin 43 Turnover and Phosphorylation

METHODS 20, 129 –139 (2000) doi:10.1006/meth.1999.0931, available online at http://www.idealibrary.com on

Use of Antibodies in the Analysis of Connexin 43 Turnover and Phosphorylation Elliot L. Hertzberg,* ,† ,1 Juan C. Sa´ez,* ,2 Richard A. Corpina,* Christine Roy,* and John A. Kessler* ,‡ *Department of Neuroscience, †Department of Anatomy and Structural Biology, and ‡Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse– chase experiments in leptomeningeal cells with [ 35S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t 1/2 of 2.7 h. Pulse– chase labeling with [ 32P]P i indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t 1/2 of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system. © 2000 Academic Press Key Words: membrane proteins; cell– cell communication; membrane biogenesis; protein phosphorylation; membrane channels.

While it has been more than twenty years since the initial isolation of gap junctions and identification of 1 To whom correspondence should be sent. Fax: (718) 430-8944. E-mail: [email protected]. 2 Current address: Departmento de Ciencias Fisolo´gicas, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile.

1046-2023/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

the first connexins from liver (1, 2), the field achieved maturity only with preparation of antibodies to connexins (3, 4) and the cloning of gap junction cDNAs (5, 6). Antibodies to connexins have been used most extensively in analyzing their distribution in specific cell types by immunohistochemistry and Western blots. More recently, they have been used to address molecular issues of gap junction biology, including the pathways of biogenesis and degradation, as well as for immunoadsorption studies designed to identify the sites and function of connexin phosphorylation. In this report, we describe the preparation and characterization of a series of polyclonal antibodies specific for peptides corresponding to sequences of connexin 43 (Cx43), first identified as a major constituent of intercalated disks of heart muscle (7–9). Illustration of their application in techniques including immunohistochemistry, Western blots, and immunoprecipitation uses the leptomeningeal cell culture system (10) for analysis of Cx43 phosphorylation and turnover.

DESCRIPTION OF METHODS Preparation of Cell Cultures Leptomeningeal cultures were obtained by removing brains, with surrounding leptomeninges, from neonatal rats on Postnatal Day 1 or 2. The leptomeninges were carefully stripped from the cerebral hemispheres, incubated for 30 min in 0.25% trypsin at 37°C, and mechanically dissociated. The cells were plated into 100-mm tissue culture dishes (Falcon) in minimal essential medium (Gibco) containing 10% fetal calf serum (HyClone), streptomycin (50 ␮g/ml) (Gibco), and peni129

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cillin (50 units/ml) (Gibco). The cultures were maintained in 95% air/5% CO 2 (v/v) at 100% humidity. Cultures became confluent within 4 –5 days and were used for experiments on Days 5 through 21. These cultures consisted of greater than 98% of a single fibroblast-like cell type demonstrated by antifibronectin immunocytochemistry. Staining was absent with antibodies to glial fibrillary acidic protein (GFAP), galactocerebroside, and the monoclonal antibody A 2B 5. General Strategy for Preparation of Antibodies to Connexins Several considerations are necessary in deciding whether to prepare polyclonal or monoclonal antibodies as well as in selection of the material to serve as antigen (e.g., synthetic peptides, protein or connexin domains expressed using bacterial vector constructs). In general, greater success is likely in preparing polyclonal antibodies that have the requisite specificity and avidity. In addition, polyclonal antibodies, inasmuch as they comprise multiple antibody species, tend to be far more robust when applied to techniques including Western blots, immunohistochemistry, and immunoprecipitation. We have prepared many monoclonal antibodies to connexin peptides and, while it was relatively easy to obtain antibodies that worked satisfactorily by ELISA, relatively few would also recognize the protein in either or both Western blots and immunohistochemistry. Nonetheless, the rare hybridoma secreting antibody applicable to all of these techniques is a truly valuable resource and is critical for techniques such as double-label immunohistochemistry. While early generations of antibodies were prepared to purified connexins, this strategy has justifiably fallen out of favor due to the difficulty of gap junction isolation and ready availability of cDNA sequences for preparation of antipeptide or antidomain antibodies. Most investigators use synthetic peptides, the cost and purity of which are now quite reasonable. An alternative is to express a portion of a connexin, usually the cytoplasmically disposed C terminus, highly divergent among connexins, using an appropriate bacterial construct (e.g., the pET series of vectors). For both peptides and domain constructs it is useful to compare the sequence with those in a protein database to increase the likelihood of achieving the desired specificity. C-terminus domain constructs are particularly useful if one anticipates carrying out analysis and identification of phosphorylation sites since the same protein can be used for phosphorylation in vitro with specific protein kinases (11). Peptide Synthesis, Antibody Preparation, and Immunoassays A series of peptides corresponding to sequences of connexin 43 deduced from cDNA sequence analysis (8)

were synthesized using tBoc chemistry on an Applied Biosystems Model 430A Peptide Synthesizer. Peptide sequences not including a cysteine had one added at the amino or carboxy terminus (indicated by parentheses) to facilitate conjugation to protein for immunization, and the following peptides were synthesized: amino amino amino amino amino amino

acids acids acids acids acids acids

2–13 113–123 142–155 241–260 283–298 346 –360

(C)GDWSALGKLLDK (C)LKVAQTDGVNV HGKVKMRGGLLRTY(C) KGRSDPYHATTGPLSPSKDC PPGYKLVTGDRNNSSC (C)KVAAGHELQPLAIVD

Reduced peptide was conjugated to bovine serum albumin using succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) and used for immunization essentially as described earlier (12). To ensure immunization with peptide– bovine serum albumin (BSA) conjugate, an equal amount of peptide–BSA conjugate was prepared using glutaraldehyde-mediated coupling and mixed with the SMCC conjugate. Screening of antisera used ELISA, Western blot, and indirect immunofluorescence assays (12). Tissue or cell samples for Western blots were homogenized in 1 mM NaHCO 3 with phenylmethylsulfonyl fluoride (PMSF) added to 2 mM from a 0.2 M stock in 2-propanol immediately before use. Samples were then aliquoted and frozen at ⫺80°C for use. Fifty to seventy-five micrograms of homogenate protein was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blots. Prestained molecular weight markers (Rainbow Markers, Amersham) were used to estimate molecular weights. Antiserum was diluted 1:1000 for Western blots and indirect immunofluorescence experiments. No immunoreactivity was observed with preimmune serum in any of these assays. In other control experiments, a lack of immunoreactivity of these antibodies with liver gap junction proteins (connexin 32 and connexin 26) was also confirmed. Immunoelectron Microscopy Optimization of fixation and embedding conditions of cells and tissues for immunoelectron microscopy is similar to that followed for other proteins. In general, it is best to begin with indirect immunofluorescence of unfixed frozen sections of tissue expressing high levels of connexin, in this case heart ventricle for Cx43. Treatments of sections with acetone or ethanol can be optimized using this approach prior to similar studies of cultured cells. Similarly, levels of detergents that may be needed to ensure antibody penetration as well as tolerance of aldehyde fixatives and conditions used for electron microscopy can be more rapidly assessed by indirect immunofluorescence using tissue sections.

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Here, leptomeningeal cells grown on glass coverslips were fixed with 0.75% glutaraldehyde in 0.1 M NaP i, pH 7.4, for 1 h at 4°C, embedded in LR White (Ted Pella, Inc.), and polymerized under a UV light at 4°C. For the heart sections, rat hearts were first perfused briefly with phosphate-buffered saline (PBS) followed by 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The heart was then removed, the ventricle cut into small pieces, and fixation in glutaraldehyde continued for a total of 1 h. The tissue was embedded in LR White and sectioned. Ultrathin sections of rat ventricle and leptomeningeal cells were analyzed by immunogold labeling (13) with antiserum diluted 1:5000. Radiolabeling and Analysis of Connexin 43 Confluent monolayers of cultured leptomeningeal cells (above) were used for radiolabeling and immunoprecipitation of connexin 43. Cells were labeled with [ 35S]methionine (NEN, 686 Ci/mmol) or [ 32P]P i (Amersham PBS13A, carrier free) as indicated in the figure legends. Methionine-free medium (MEM, GIBCO) and

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dialyzed serum (Gibco) were used for labeling with [ 35S]methionine, and phosphate-free medium and dialyzed serum for labeling with [ 32P]P i. Immunoprecipitation of Cx43 Cells radiolabeled with [ 35S]methionine were scraped, collected, and washed twice in PBS and lysed in 100 ␮l of PBS containing 2% SDS, 100 U/ml Trasylol, 200 ␮g/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml ⑀-aminocaproic acid, and 2 mM fresh PMSF. The supernatant fraction was diluted with 1 ml of a buffer containing 190 mM NaCl, 50 mM Tris–HCl, 6 mM EDTA, 2.5% Triton X-100, 100 U/ml Trasylol, 200 ␮g/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml ⑀-aminocaproic acid, 2 mM fresh PMSF, pH 8.3, centrifuged briefly to remove insoluble material and 10 ␮l of immune or 10␮l of immune serum plus 1.5 ␮g antigenic peptide or 10␮l preimmune serum was added. Incubation with primary antibody was with continuous inversion for 3 h at 4°C. Sixty microliters of a 1:5 suspension of Pansorbin (Cal-

FIG. 1. Immunofluorescence localization of antibody binding on sections of heart ventricle. Eight-micrometer-thick cryostat sections of mouse ventricle were incubated with rabbit antibody followed by fluorescein-conjugated secondary antibody. The antibodies were raised to amino acids 113–123 (A), 241–260 (B), 283–298 (C), and 346 –360 (D) of the connexin 43 sequence. Bar ⫽ 50 ␮m.

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biochem), activated according to the supplier’s protocol, was added to adsorb immune complexes and incubation continued for 1 h at 4°C. Subsequent to centrifugation, the pellet was resuspended by sonication and washed twice with 150 mM NaCl, 10 mM Tris, 5 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, 100 U/ml Trasylol, 1 mg/ml BSA, pH 8.3. The pellets were then washed sequentially with 2 M NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40 (NP-40), 0.1 M Tris–HCl, pH 7.2; PBS containing

1% NP-40; and 0.05 M NaCl, 0.5% NP-40, 0.01 M Tris–HCl, pH 7.2, all containing 2 mM PMSF. The pellet was resuspended in SDS-containing buffer, heated at 100°C for 4 min, and processed for SDS– PAGE and fluorography using Enhance (DuPont). Immunoprecipitation of 32P-labeled protein used the same procedure as for other isotopes (above) with the addition of 20 mM sodium pyrophosphate and 100 mM NaF to inhibit potential endogenous phosphatase activity as described previously (14). Phosphoamino acid

FIG. 2. Immunogold labeling of heart gap junctions. Sections were incubated with rabbit preimmune antiserum (A) or anti-connexin 43 346 –360 (B) (1:5000)followed by incubation with goat anti-rabbit IgG adsorbed to 5 nm gold particles (BioCell). Bar ⫽ 0.2 ␮m.

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FIG. 3. Immunofluorescence on leptomeningeal cells. Cells grown on glass coverslips were treated with 70% ethanol for 20 min at ⫺20°C prior to incubation with primary antibody (anti-connexin 43 346 –360) and processing for indirect immunofluorescence (A) and phase-contrast (B) microscopy. Bar ⫽ 50 ␮m.

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FIG. 4. Immunogold labeling of gap junctions in cultured leptomeningeal cells. Sections were incubated with rabbit preimmune antiserum (A) or anti-connexin 43 346 –360 (B) (1:5000) followed by incubation with goat anti-rabbit IgG adsorbed to 5-nm gold particles (BioCell). Binding of antibodies was observed to gap junctions in the plasma membrane with the immune antiserum (B) and to annular gap junctions within the cell (D). No binding to gap junctions was observed with preimmune serum (A, C). Bar ⫽ 0.2 ␮m.

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analysis of immunoprecipitated protein hydrolysates was carried out as described previously (14, 15). Densitometric analysis of autoradiographs was carried out with a Molecular Dynamics 300A computing densitometer. Data analysis and plotting were carried out using Sigmaplot (Jandel Scientific). Special Considerations for Analysis and Immunoprecipitation of Connexins Analysis of connexins by Western blots, while straightforward, should avoid heating in SDScontaining buffers that can lead to their aggregation, as first shown for Cx32 (1), and loss of signal. More problematic is manipulation of immunoprecipitates prior to SDS–PAGE to release connexin from antibody complexes. While some antibodies will readily dissociate from antigen in the presence of SDS, others do not and require some heating in sample buffer. For such antibodies, a variety of heating protocols should be examined using Western blots to establish conditions for quantitative recovery of connexin. Special precautions are also necessary to ensure initial quantitative solubilization of connexin prior to incubation with antibody due to their limited solubility in many detergent-containing systems (16), a property initially used for gap junction isolation (1, 2, 17–19). In some cases, alternative protocols are needed for solubilization and immunoprecipitation (11) when Western blots, for example, indicate incomplete connexin solubilization in some cell samples after specific pharmacological or other manipulations.

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RESULTS Antibodies to peptides corresponding to several sequences of connexin 43 encoded by its cDNA and predicted to lie at the cytoplasmic surface of gap junctions (22, 23) were prepared in rabbits. These included antibodies to amino acids 113–123 (cytoplasmic loop connecting second and third transmembrane segments) and 241–260, 283–298, and 346 –360 (cytoplasmic carboxy-terminal “tail”). Such antibodies do not have access to the intercellular “gap” region of gap junctions because the size of this intercellular space, ca 1.5 nm, precludes penetration of antibodies used for immunocytological analyses. When applied to frozen sections of mouse heart ventricle, indirect immunofluorescence demonstrated that these antibodies bound to intercalated disks and some lateral membrane appositions (Fig. 1). Immunoelectron microscopy with antibodies to connexin 43 346 –360 demonstrated binding to gap junctions in LR White embedded rat ventricle sections (Fig. 2). Occasional immunoreactive annular gap junctions were observed. Similar images were obtained for antibodies to connexin 43 283–298 used for immunofluorescence in Fig. 1 (not shown). Indirect immunofluorescence of cultured leptomeningeal cells revealed antibody binding to discrete regions of appositional plasma membranes as well as

Phosphatase Treatment of Cells and Immunoprecipitated Protein Alkaline phosphatase from Escherichia coli (Sigma, Type III) or calf intestine (Boehringer-Mannheim) was diluted 1:5 in 10 mM Tris–HCl, 5 mM MgCl 2, 4 ␮M ZnCl 2, pH 9.7 and divided into equal aliquots. To minimize potential proteolytic activity in these enzyme preparations, one aliquot of each was heated at 100°C for 2 min (20). Assay of alkaline phosphatase activity in 20 mM Tris–HCl, pH 8.4, with p-nitrophenyl phosphate as substrate indicated that the E. coli enzyme retained 50 –75% of its activity after heating; the intestinal enzyme was found to be heat sensitive. The addition of up to 2% SDS to the assay mixture did not alter enzymatic activity in either case. Cultured leptomeningeal cells were scraped, centrifuged, and washed in PBS containing 2 mM fresh PMSF. The washed pellet was resuspended by gentle sonication in a minimal volume of H 2O and an equal volume of 2% SDS in 20 mM Tris–HCl, pH 8.4. Dilutions of alkaline phosphatase, as indicated in the figure legends, were then added to the solubilized sample and incubated at 37°C overnight. Samples were processed for SDS–PAGE and Western blots (21).

FIG. 5. Western blot analysis of anti-connexin 43 immunoreactivity in leptomeningeal cells, neonatal heart, and brain homogenates. Samples containing 75 ␮g of protein from leptomeningeal cells (A, a), neonatal heart (B, b), and brain homogenates (C, c) were resolved by electrophoresis on 10% SDS–polyacrylamide gels. One replicate was stained with Coomassie brilliant blue R (A, B, C) and a second electrophoretically transferred to nitrocellulose. Subsequent to incubation with primary antibody (anti-connexin 43 346 –360), blots were incubated with 125I-labeled protein A and antibody binding was visualized by autoradiography (a– c). The positions of the molecular weight markers as well as connexin 43 (43 kDa) and the 41-kDa protein visible particularly in the leptomeningeal and brain samples are indicated.

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punctate cytoplasmic fluorescence (Fig. 3). Immunoelectron microscopy demonstrated antibodies binding to gap junctions in the plasma membrane as well as to annular gap junctions within the cytoplasm of these cells (Fig. 4). The immunocytology of leptomeningeal cultures with these anti-connexin 43 antibodies is consistent with those obtained in another study using a different antibody (24). Characterization of the protein(s) responsible for antibody binding in immunocytological experiments was undertaken by Western blot analysis of tissue or cell homogenates. In the case of neonatal rat heart homogenates (Fig. 5, lane b), antibody binding was predominantly to a ca. 43-kDa protein, connexin 43. A very low level of antibody binding to a ca. 41-kDa protein in the heart sample was also observed. This pattern of antibody binding on Western blots is also seen for homogenates of adult rat heart (not shown). The binding of this antibody to these proteins was markedly reduced by carrying out incubations in the presence of 75 ng/ml peptide and no longer detectable at 750 ng/ml (not shown).

FIG. 6. Pulse– chase labeling of leptomeningeal cell connexin 43 with [ 35S]methionine. Confluent monolayers of leptomeningeal cells in 60-mm dishes were labeled for 20 min with 0.2 mCi [ 35S]methionine in methionine-free medium containing 10% dialyzed fetal bovine serum. Plates were then rinsed with complete medium and fresh medium added for chase periods as indicated. Immunoprecipitation with preimmune serum (“PRE”) was carried out in a duplicate of the zero-time sample. Cells were then scraped, collected by centrifugation, and lysed in a buffer containing 2% SDS. The solubilized mixture was diluted into a buffer containing NP-40 and deoxycholate with a cocktail of protease inhibitors and incubated with primary antibody (anti-connexin 43 346 –360) overnight at 4°C. Immune complexes were adsorbed using Pansorbin, washed, solubilized in SDS, and resolved by electrophoresis on 10% polyacrylamide gels. Gels were then stained with CBB, processed for fluorography, dried, and autoradiographed for 3 h.

In leptomeningeal cells (Fig. 5, lane a) and brain homogenates (Fig. 5, lane c), antibodies bound to bands with apparent molecular masses of 43 and 41 kDa. In other gels (e.g., Fig. 9, below), the 43-kDa band often resolved as a doublet. For the purposes of this study, the higher-molecular-mass band of the 43-kDa doublet is referred to as connexin 43-H or 43-kDa-H (for higher molecular mass), and the lower band of the doublet as connexin 43-L or 43 kDa-L (for lower molecular mass). Little difference in expression level of connexin 43 was observed in cultures maintained from 5 to 21 days. The addition of a cocktail of protease inhibitors during sample preparation for SDS–PAGE did not alter this pattern of antibody binding (not shown). Analysis of connexin 43 biogenesis was undertaken by pulse– chase experiments with [ 35 S]methionine (Fig. 6). When cultured leptomeningeal cells were pulse-labeled with [ 35 S]methionine for 20 min, followed by cell lysis, immunoprecipitation of connexin 43, and resolution of immunoprecipitated material by SDS–PAGE and fluorography, radiolabel was incorporated only into the 41-kDa protein. During a chase period with a 1000-fold excess of cold methionine, radiolabel began to appear by 1.5 h in a doublet at ca. 43 kDa. By 3 h, the radiolabel was concentrated in the higher band of this doublet (connexin 43-H), and by 4.5 h radiolabel was diminishing from the sample. By 12 h greater than 90% of the [ 35 S]methionine label was lost from the immunoprecipitate (not shown). In control experiments it was demonstrated that inclusion of 1.5 ␮g/ml peptide prevented immunoprecipitation of radiolabeled connexin 43, and no additional radiolabeled connexin 43 could be immunoprecipitated either by increasing

FIG. 7. Pulse– chase phosphorylation of connexin 43. Cells were labeled with 0.5 mCi of [ 32P]P i (see Fig. 6) except that phosphate-free medium was used prior to chase with complete medium. Immunoprecipitation was carried out as described for Fig. 6 and dried gels were analyzed by autoradiography. A fluorograph of immunoprecipiated connexin 43 radiolabeled for 2 h with [ 35S]methionine is included for comparison of [ 35S]methionine and [ 32P]P i labeling.

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the amount of primary antibody used or by a second round of immunoprecipitation (not shown). A parallel pulse– chase and immunoprecipitation experiment analyzed phosphorylation of connexin 43 (Fig. 7). In this experiment, [ 32P]P i was incorporated into the 43-kDa form of connexin 43 after 15–30 min of incubation, although resolution of bands in this experiment was not adequate to resolve the 43-kDa doublet. During the subsequent chase period with excess cold P i, radiolabel rapidly disappeared with no delay from the immunoprecipitate. Densitometric scans of autoradiographs of pulse– chase radiolabeled connexin 43 demonstrated exponential decay of both the [ 32P]P i and [ 35S]methionine radiolabels (Fig. 8). Turnover of the phosphoprotein had a t 1/ 2 of about 1.7 h, while that for the [ 35S]methionineradiolabeled mature form of connexin 43 was 2.7 h. In a separate experiment, immunoprecipitated 32Pradiolabeled connexin 43 resolved as a doublet, and bands were excised for phosphoamino acid analysis that indicated only serine was phosphorylated (Fig. 9). Several experiments were undertaken to determine the basis for the shift in apparent molecular weight of connexin 43 during the pulse– chase experiments. Addition of tunicamycin, which alters processing of N-linked oligosaccharides, had no influence on the relative amounts of the 41- and 43-kDa proteins on Western blots (not shown). No consensus sequence sites for N-linked glycosylation of connexin 43 exist in its predicted sequence based on cDNA sequence analysis (8). However, alkaline phosphatase treatment of SDSsolubilized leptomeningeal cells led to conversion of the 43-kDa doublet to the 41-kDa form (Fig 10A). In this Western blot experiment, incubation of the solubilized cell sample overnight at 37°C indicated no endogenous

FIG. 8. Densitometric analysis of turnover of [ 35S]methionine and 32 P-radiolabeled connexin 43. Autoradiographs of pulse– chase experiments were scanned and integrated values of absorbance normalized to 1000 for maximum values. The 32P data are indicated by filled boxes and 35S data by filled circles. Decay of the phosphorylated form of connexin 43 begins with the chase while that for total protein begins after a 2- to 4-h lag.

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degradation of immunoreactive proteins (compare lanes A and B). Treatment with boiled or unheated E. coli enzyme led to loss of the connexin 43 doublet and appearance of the 41-kDa form of the protein (lanes C–F). An additional intermediate phosphorylated form of the protein is indicated by the presence of immunoreactive material migrating faster than the 43-kDa doublet and slower than the 41-kDa form of the protein when a more dilute phosphatase treatment was used (lanes D and F). Intestinal phosphatase also led to conversion of the 43-kDa doublet to the 41-kDa form (lanes I and J) with no intermediate form detected. No loss of the connexin 43 doublet was observed with heat-treated intestinal phosphatase (lanes G and H). The loss of phosphate from the protein was confirmed by treatment of immunoprecipitated 32P-labeled connexin 43 with phosphatase (Fig. 10B, compare lanes A and B).

DISCUSSION Characterization of the antibodies raised to Cx43 included the demonstration of specificity by their

FIG. 9. Phosphoamino acid analysis of connexin 43. Leptomeningeal cells were labeled with 0.5 mCi [ 32P]P i for 1 h. (A) Connexin 43 was immunoprecipitated, resolved by SDS–PAGE and autoradiographed [compare immunoprecipitate with control using preimmmune (PRE) serum]. Connexin 43-H (B) and connexin 43-L (C) were excised and hydrolyzed, and phosphoamino acids were analyzed by thin-layer chromatography and autoradiography. The positions of unlabeled phosphoserine, phosphothreonine, and phosphotyrosine added as markers are indicated. Only serine was phosphorylated in these samples.

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binding to gap junctions by immunoelectron microscopy of heart ventricle (Fig. 2) and leptomeningeal cells (Fig. 4). In both cases, the pattern of binding reflects that observed at the level of light microscopy by indirect immunofluorescence (Figs. 1 and 3, respectively). The specificity is further demonstrated on Western blots (Fig. 5) and by immunoprecipitation (Figs. 6 and 7). That the multiple bands observed on Western blots arises as a result of phosphorylation was confirmed in experiments in which Cx43 was enzymatically dephosphorylated using alkaline phosphatase (Fig. 10), an observation consistent with earlier studies (25–29). The pulse– chase experiment with [ 35S]methionine (Fig. 6) indicates that the phosphorylation of Cx43 is postsynthetic, consistent with a similar analysis of cultured myocardial cells (29) and cell lines (16, 28). Nonetheless, the function(s) of phosphorylation is likely to be multiple and remains controversial, with numerous papers indicating a role in assembly, turnover, and regulation of the gap junction channel. Within this context, it should be noted that Cx26 is not phosphorylated (13, 15) and truncation mutants of Cx32 and Cx43 that are not phosphorylated are still capable of forming functional gap junctions (30 –32). That the phosphate radiolabel turnover in Cx43 is similar in rate to the turnover of the protein has also been observed in myocardial cells (29). The shortness of the half-life of Cx43, as well as of other connexins thus far analyzed (13, 33–36), is intriguing. Such rapid turnover of membrane proteins is usually found only for some ligand receptors. Clearly, a short half-life provides a mechanism for regulation of intercellular communication within a relatively short time frame. However, given the multitude of other mechanisms

available to cells for regulation of protein function, it is still not clear why (or whether) such a mechanism is used for regulation of gap junctional communication. While the results described and discussed above are relatively straightforward and generally consistent with similar analyses using other connexins and cell types, the possibility for unanticipated observations remains. For example, an antibody thought to be specific for the cytoplasmic loop of Cx26 cross-reacted with a protein of somewhat slower mobility on Western blots of astrocytes (37). It is likely that this other protein is Cx30 (38) based on the conservation of sequence with Cx26 in the region to which the antibody had been prepared. Perhaps the most intriguing finding using antibodies to connexins has been the finding of epitope masking of Cx43 in astrocytes as a result of insults such as kainate injection (39, 40), spinal cord compression (41), and focal ischemia (42). In these cases, an antibody to Cx43 346 –360 no longer recognizes Cx43 in gap junctions in situ by immunohistochemistry, although binding of a different antibody to Cx43 241–260 is retained and both antibodies continue to bind to Cx43 on Western blots. While these data might arise as a result of an alteration of Cx43 conformation, perhaps due to phosphorylation either within the epitope or elsewhere in the protein, it is also possible that these results are due to the binding of another protein to Cx43 that renders the affected epitope inaccessible to Cx43 346 –360 . In either case, the molecular consequences of this alteration of Cx43 are likely to lead to alteration of some aspect of the function of these gap junctions, a structure/function correlate that is the subject of ongoing studies.

FIG. 10. Enzymatic dephosphorylation of connexin 43. (A) Phosphatase treatment of connexin 43. Leptomeningeal cells solubilized in SDS were treated with phosphatase as indicated under Materials and Methods and analyzed by Western blots (lanes A–J) using anti-connexin 43 346 –360. Control, fresh cells (lane A) were compared with those incubated in the absence of phosphatase (lane B) to establish the absence of endogenous protease or phosphatase activity under the experimental conditions used. Treatment was with the E. coli phosphatase (lanes C–F) or intestinal enzyme (lanes G–J). Boiled phosphatase was used in lanes C, D, G, and H. Enzyme dilutions from stock were 1:5 (lanes C, E, H, and J), and 1:25 (lanes D, F, H, and J). (B) Phosphatase treatment of 32P-labeled connexin 43. Phosphatase treatment of immunoprecipitated connexin 43 radiolabeled with [ 32P]P i led to a loss of radiolabel from the sample (lane A, untreated control; lane B, treated).

TURNOVER AND PHOSPHORYLATION OF CONNEXIN 43

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health to E.L.H. (GM 30667), J.C.S. (DK41368), and J.A.K (NS34758), and a NIH Program Project grant (NS07512).

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