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• W2146581722 abstract "Indirect evidence suggests that the permeability of connexin 43 (Cx43) gap-junctional channels (connexons) to small organic molecules (Mr < 1,000) is decreased by protein kinase C (PKC)-mediated phosphorylation of Ser-368. However, it is currently unknown whether this effect is produced directly by phosphorylation of this residue or whether cytoplasmic regulatory factors are required for the decrease in Cx43 gap-junctional channel permeability. Here we studied the effects of PKC-mediated phosphorylation on purified recombinant wild-type Cx43 and a PKC-unresponsive mutant (S368A). Our studies show that (a) PKC phosphorylates Ser-368, (b) the phosphorylation by PKC of purified and reconstituted connexons abolishes sucrose and Lucifer Yellow permeability, (c) the regulation of Cx43 by PKC is the direct result of phosphorylation of Ser-368 and does not involve intermediary regulatory factors, and (d) phosphorylation of Ser-368 produces a conformational change in purified Cx43 as demonstrated by changes in intrinsic Trp fluorescence and proteolytic digestion pattern. We conclude that phosphorylation of Ser-368 by PKC induces a conformational change of Cx43 that results in a decrease in connexon permeability. Indirect evidence suggests that the permeability of connexin 43 (Cx43) gap-junctional channels (connexons) to small organic molecules (Mr < 1,000) is decreased by protein kinase C (PKC)-mediated phosphorylation of Ser-368. However, it is currently unknown whether this effect is produced directly by phosphorylation of this residue or whether cytoplasmic regulatory factors are required for the decrease in Cx43 gap-junctional channel permeability. Here we studied the effects of PKC-mediated phosphorylation on purified recombinant wild-type Cx43 and a PKC-unresponsive mutant (S368A). Our studies show that (a) PKC phosphorylates Ser-368, (b) the phosphorylation by PKC of purified and reconstituted connexons abolishes sucrose and Lucifer Yellow permeability, (c) the regulation of Cx43 by PKC is the direct result of phosphorylation of Ser-368 and does not involve intermediary regulatory factors, and (d) phosphorylation of Ser-368 produces a conformational change in purified Cx43 as demonstrated by changes in intrinsic Trp fluorescence and proteolytic digestion pattern. We conclude that phosphorylation of Ser-368 by PKC induces a conformational change of Cx43 that results in a decrease in connexon permeability. Gap-junctional channels are aqueous channels responsible for cell-to-cell communication that allow for the exchange of solutes of Mr ≤ 1,000. Gap-junctional channels are formed by the docking of two connexons or gap-junctional hemichannels (GJHs) 1The abbreviations used are: GJH, gap-junctional hemichannel; Cx43, connexin 43; EGFP, enhanced green fluorescent protein; Cx43-EGFP, Cx43 with the EGFP fused to its C-terminal end; Cx43-S368A, Cx43 with Ser-368 substituted with Ala; OG, n-octyl-β-d-glucopyranoside; PMSF, phenylmethylsulfonyl fluoride; PKC, protein kinase C. , one from each neighboring cell; each connexon is formed by six connexin molecules that contain four transmembrane domains each (for a review, see Ref. 1Harris A.L. Q. Rev. Biophys. 2001; 34: 325-472Crossref PubMed Google Scholar). Cx43 is expressed in several cell types in organs such as brain, myocardium, and kidney as well as in vascular endothelial cells (2Berthoud V.M. Ledbetter M.L. Herzberg E.L. Sáez J.C. Eur. J. Cell Biol. 1992; 57: 40-50PubMed Google Scholar, 3Beyer E.C. Paul D.L. Goodenough D.A. J. Cell Biol. 1987; 105: 2621-2629Crossref PubMed Scopus (919) Google Scholar, 4Little T.L. Beyer E.C. Duling B.R. Am. J. Physiol. 1995; 37: H729-H739Google Scholar, 5Sainio K. Gilbert S.F. Lehtonen E. Nishi M. Kumar N.M. Gilula N.B. Saxen L. Development. 1992; 115: 827-837Crossref PubMed Google Scholar, 6Venance L. Premont J. Glowski J. Giaume C. J. Physiol. (Lond.). 1998; 510: 429-440Crossref Scopus (72) Google Scholar). Its functional significance in cell-to-cell communication has been clearly established (1Harris A.L. Q. Rev. Biophys. 2001; 34: 325-472Crossref PubMed Google Scholar). Cx43 GJHs have also been shown on the plasma membrane of a number of cell types (7Contreras J.E. Sánchez H. Eugenin E.A. Speidel D. Theis M. Willecke K. Bukauskas F. Bennett M.V.L. Sáez J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 495-500Crossref PubMed Scopus (479) Google Scholar, 8DeVries S.H. Schwartz E.A. J. Physiol. (Lond.). 1992; 445: 201-230Crossref Scopus (241) Google Scholar, 9John S.A. Kondo R. Wang S.Y. Goldhaber J.L. Weiss J.N. J. Biol. Chem. 1999; 274: 236-240Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 10Li H. Liu T.F. Lazrak A. Peracchia C. Goldberg G.S. Lampe P.D. Johnson R.G. J. Cell Biol. 1996; 134: 1019-1030Crossref PubMed Scopus (300) Google Scholar, 11Musil L.S. Goodenough D.A. J. Cell Biol. 1991; 115: 1357-1374Crossref PubMed Scopus (617) Google Scholar, 12Kondo R.P. Wang S.W. John S.A. Weiss J.N. Goldhaber J.I. J. Mol. Cell. Cardiol. 2000; 32: 1859-1872Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 13Vergara L. Bao X. Cooper M. Bello-Reuss E. Reuss L. J. Membr. Biol. 2003; 196: 173-184Crossref PubMed Scopus (53) Google Scholar), and there is evidence supporting a role of their activation in cell injury because of uncompensated water and solute fluxes that overwhelm the normal membrane transport mechanisms, thus altering cell composition. ATP depletion may result in a decrease in the phosphorylation state of GJHs, which activates these channels, leading to cell damage (7Contreras J.E. Sánchez H. Eugenin E.A. Speidel D. Theis M. Willecke K. Bukauskas F. Bennett M.V.L. Sáez J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 495-500Crossref PubMed Scopus (479) Google Scholar, 8DeVries S.H. Schwartz E.A. J. Physiol. (Lond.). 1992; 445: 201-230Crossref Scopus (241) Google Scholar, 9John S.A. Kondo R. Wang S.Y. Goldhaber J.L. Weiss J.N. J. Biol. Chem. 1999; 274: 236-240Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 12Kondo R.P. Wang S.W. John S.A. Weiss J.N. Goldhaber J.I. J. Mol. Cell. Cardiol. 2000; 32: 1859-1872Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 13Vergara L. Bao X. Cooper M. Bello-Reuss E. Reuss L. J. Membr. Biol. 2003; 196: 173-184Crossref PubMed Scopus (53) Google Scholar). It has been established that phosphorylation of specific Ser or Tyr residues in the C-terminal domain of Cx43 reduces gap-junctional intercellular communication (for reviews, see Refs. 14Lampe P.D. Lau A.F. Arch. Biochem. Biophys. 2000; 384: 205-215Crossref PubMed Scopus (471) Google Scholar and 15Lau A.F. Warn-Cramer B.J. Lin R. Peracchia C. Gap Junctions: Molecular Basis of Cell Communication in Health and Disease. Academic Press, San Diego, CA2000: 315-341Google Scholar) and that PKC phosphorylates Cx43 at Ser-368 and Ser-372 of the C-terminal domain (16Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Johnson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (470) Google Scholar, 17Sáez J.C. Nairn A.C. Czernk A.J. Fishman G.I. Spray D.C. Hertzberg E.L. J. Mol. Cell. Cardiol. 1997; 29: 2131-2145Abstract Full Text PDF PubMed Scopus (136) Google Scholar). Recent evidence suggests that decreased phosphorylation of Cx43 at Ser-368 of the C-terminal domain increases gap-junctional channel dye permeability in cells exposed to PKC inhibitors (16Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Johnson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (470) Google Scholar, 17Sáez J.C. Nairn A.C. Czernk A.J. Fishman G.I. Spray D.C. Hertzberg E.L. J. Mol. Cell. Cardiol. 1997; 29: 2131-2145Abstract Full Text PDF PubMed Scopus (136) Google Scholar). However, it is currently unknown whether phosphorylation of Ser-368 by PKC regulates Cx43 directly or whether this effect requires intermediary regulatory factor(s). In this context, it is believed that the decrease in Cx43 permeability produced by lowering intracellular pH, which also depends on the C-terminal domain (18Ek-Vitorin J.F. Calero G. Morley G.E. Coombs W. Taffet S.M. Delmar M. Biophys. J. 1996; 71: 1273-1284Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 19Liu S. Taffet S. Stoner L. Delmar M. Vallano M.L. Jalife J. Biophys. J. 1993; 64: 1422-1433Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 20Morley G.E. Taffet S.M. Delmar M. Biophys. J. 1996; 70: 1294-1302Abstract Full Text PDF PubMed Scopus (235) Google Scholar), requires a cytosolic component (21Nicholson B.J. Zhou L. Cao F. Zhu H. Chen Y. Werner R. Gap Junctions. IOS Press, Washington, D. C.1998: 3-8Google Scholar). Since studies on the regulation of Cx43 by PKC in cells are difficult to interpret because of the complexity of the PKC signaling pathways and the possible presence of cytosolic regulatory factors, we decided to assess the regulation of purified Cx43. Our results using purified recombinant Cx43 GJHs indicate that phosphorylation of Ser-368 produces a conformational change that reduces GJH permeability and that this effect does not require cytosolic factors. Recombinant Baculoviruses—Three constructs coding for Cx43 were created for these studies: (a) wild-type rat Cx43 (Cx43), (b) Cx43 fused to the enhanced green fluorescent protein (EGFP) at the C terminus (Cx43-EGFP), and (c) Cx43 in which Ser-368 was substituted with Ala (Cx43-S368A). Cx43 was amplified by PCR using the rat Cx43 DNA as template (a gift from Dr. Scott John, see Ref. 9John S.A. Kondo R. Wang S.Y. Goldhaber J.L. Weiss J.N. J. Biol. Chem. 1999; 274: 236-240Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) with XhoI and EcoRI restriction sites added at the 5′ and 3′ ends. The PCR products digested with XhoI and EcoRI were ligated to the plasmid pBlueBac 4.5/V5-His (Invitrogen) cut with the same enzymes. EGFP was amplified by PCR from pEGFP-C1 (Clontech) with EcoRI and HindIII flanking sites for in-frame cloning at the 3′ end of the Cx43 DNA. The PCR product digested with EcoRI and HindIII was ligated to Cx43 into pBlueBac 4.5/V5-His cut with the same enzymes to create the Cx43-EGFP fusion protein. The pBlueBac 4.5/V5-His vector was used as a source of the His6 tag added to the C-terminal end of all constructs for Ni2+ affinity purification. The DNAs coding for Cx43 and Cx43-EGFP, plus the His tag at the C terminus, were excised with BamHI and SalI and ligated into the baculovirus transfer vector pFastBac (Invitrogen). The S368A mutant was obtained by site-directed mutagenesis (QuikChange multisite site-directed mutagenesis kit, Stratagene, La Jolla, CA) using the following oligonucleotide: 5′-CGACCTTCCAGCAGAGCCGCCTCCCGCGCCAGCAGCAGGCCTCGG-3′. Recombinant baculoviruses were generated following the instructions of the Bac-to-Bac system (Invitrogen). The viruses were produced in Sf9 cells grown at 27 °C in Grace's medium supplemented with 10% fetal calf serum and 0.05 mg/ml gentamycin. Cell density was ∼106 cells/ml. A scheme of the Cx43s engineered is shown in Fig. 1A. DNA sequencing of the constructs was carried out at the Protein Chemistry Core Laboratory of The University of Texas Medical Branch. Western Blot Analysis—Western blots were performed as described previously (22Han E.S. Vanoye C.G. Altenberg G.A. Reuss L. Am. J. Physiol. 1996; 270: C1370-C1378Crossref PubMed Google Scholar) using a rabbit anti-rat Cx43 polyclonal antibody against the Cx43 C terminus (Zymed Laboratories Inc.) and a horseradish peroxidase-labeled goat anti-rabbit antibody. Purification of Wild-Type Cx43 and Cx43-S368A—Protein expression was carried out in High-Five insect cells in suspension, grown in 300-ml baffled flasks containing 100 ml of Excell 401 medium supplemented with 200 units/ml penicillin, 200 μg/ml streptomycin, and 2 mm glutamine. Cells (106 cells/ml) grown at room temperature were shaken at 200 rpm and infected at a multiplicity of infection of 10. Generally cells were harvested 72 h postinfection by centrifugation at 700 × g for 10 min at 4 °C. Cells were washed twice with phosphate-buffered saline solution, and the final pellet was frozen in liquid nitrogen. The frozen pellets were used for purification immediately or stored at -80 °C. For purification, cells were thawed in a buffer containing 1 mm bicarbonate and 1 mm phenylmethylsulfonyl fluoride (PMSF) and lysed with a Dounce homogenizer. The membranes were alkali-extracted as described previously (23Stauffer K.A. Kumar N.A. Gilula N.B. Unwin P.N. J. Cell Biol. 1991; 115: 141-150Crossref PubMed Scopus (95) Google Scholar, 24Zimmer D.B. Green C.R. Evans W.H. Gilula N.B. J. Biol. Chem. 1987; 262: 7751-7763Abstract Full Text PDF PubMed Google Scholar). Briefly, after addition of NaOH to a 20 mm final concentration, the lysate was sonicated with a probe sonicator, incubated on ice for 30 min, and then centrifuged at 35,000 × g for 30 min at 4 °C. In some experiments, purification was performed from native membranes without alkali extraction. The membranes were solubilized with 2.5% n-dodecyl-β-d-maltoside or 2.3% n-octyl-β-d-glucopyranoside (OG) in 2 m NaCl, 10 mm EDTA, 10 mm dithiothreitol, 10 mm PMSF, and 10 mm glycine/NaOH, pH 10, at a protein concentration <2 mg/ml. The suspension was sonicated and incubated for 2 h at 4 °C with gentle rotation. Unsolubilized material was separated by ultracentrifugation at 100,000 × g for 40 min at 4 °C. Solubilized Cx43s were diluted with 15 volumes of 10 mm β-mercaptoethanol, 1 mm PMSF, 100 mm HEPES, and 0.2% n-dodecyl-β-d-maltoside or 2.3% OG, pH 8.0. Diluted samples were loaded at a speed of 0.5 ml/min, at 4 °C, on a nickel-nitrilotriacetic acid column (Qiagen) pre-equilibrated with the dilution buffer. The column was washed with 10 volumes of buffer (10 mm KCl, 0.1 mm EDTA, and 10 mm HEPES/KOH, pH 7.4, containing 0.01% n-dodecyl-β-d-maltoside or 2.3% OG and 20 mm imidazole). Imidazole, 250 mm in washing buffer, was added for elution. For the isoosmolar sucrose gradient experiments (see below), 459 mm urea was present in the elution buffer. Analysis of the Oligomeric State of Purified Cx43—The oligomerization of solubilized Cx43 was determined by gel filtration on Sephacryl S300HR (16/60 prepacked column, Amersham Biosciences) using an ÄKTA FPLC system (Amersham Biosciences). For these experiments, Cx43-EGFP in 2.3% OG, 150 mm NaCl, 0.1 mm EDTA, and 10 mm HEPES/NaOH, pH 7.4, was run at a flow rate of 0.5 ml/min (0.5-ml total volume, ∼0.5 mg/ml Cx43-EGFP, with or without standard molecular weight markers added) on the column equilibrated with the same buffer. Molecular weights were calculated from the linear relationship between Kav and the log of the molecular weight. The standards were run under the same conditions as the Cx43-EGFP samples and had the following molecular masses: 669 kDa (thyroglobulin), 440 kDa (ferritin), 232 kDa (catalase), 158 kDa (aldolase), and 67 kDa (albumin). Blue dextran 2000 (∼2,000 kDa) was used to determine the void volume. Phosphorylation and Dephosphorylation of Cx43—Purified proteins were dephosphorylated as described by Kim et al. (26Kim D.Y. Kam Y. Koo S.K. Joe C.O. J. Biol. Chem. 1999; 274: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Approximately 30 μg of purified Cx43 or Cx43-S368A in 10 mm KCl, 0.1 mm EDTA, 2.3% OG, and 10 mm potassium phosphate, pH 7.4, were mixed with 0.1 volumes of agarose-conjugated calf intestinal phosphatase (50 units/ml, Sigma), and the mixture was incubated for 3 h at 25 °C with gentle agitation. Reactions were stopped by the removal of the enzyme-conjugated agarose beads by centrifugation. Dephosphorylated Cx43 and Cx43-S368A were phosphorylated by PKC as described by Sáez et al. (27Sáez J.C. Nairn A.C. Czernk A.J. Spray D.C. Hertzberg E.L. Greengard P. Bennett M.V. Eur. J. Biochem. 1990; 192: 263-273Crossref PubMed Scopus (158) Google Scholar) for Cx32 with minor modifications. Briefly 30 μg of dephosphorylated Cx43 were phosphorylated by rat brain PKC (1–2 μg, Calbiochem) in the presence of 200 μm ATP, 5 mm Ca2+,5 mm Mg2+,50 μg/ml phosphatidylserine (Avanti, Alabaster, AL), and 3 μg/ml 1,2-dioleoyl-sn-glycerol (Sigma). The reaction volume was generally 100 μl, and the reaction proceeded for 2 h at room temperature. Phosphorylation was confirmed by autoradiography using 10 nCi of [γ-32P]ATP (1,000 cpm/pmol, American Radiolabeled Chemicals, St. Louis, MO) in the reaction described above. For these experiments, either Cx43 or Cx43-S368A was precipitated in 10% trichloroacetic acid and then subjected to SDS-PAGE (16% Tris, glycine gels). Films were exposed at -80 °C before development. For quantitative assessments of the phosphorylation level, Cx43 at a concentration of 0.1 mg/ml was phosphorylated in the presence of 10 nCi of [γ-32P]ATP. The phosphorylated protein was then labeled with 100 μm fluorescein maleimide (Molecular Probes, Eugene, OR) for 15 min at 25 °C. The reaction was terminated by addition of a 5-fold molar excess of dithiothreitol, and then the free radioactive label and the free dithiothreitol-reacted fluorescein maleimide were removed from 50 μl of the reaction by filtration through a G-25 column (Amersham Biosciences). Measurements of radioactivity and fluorescence were performed in aliquots of the filtrate. The protein amount was estimated from the fluorescence and parallel Cx43 fluorescence/protein calibration curves using the BCA protein assay reagent from Bio-Rad with bovine serum albumin as standard. Reconstitution of Wild-Type Cx43 and Cx43-S368A—The reconstitution procedure for Cx43 and Cx43-S368A was a modification of published techniques (28Davey R.A. Hamson C.A. Healey J.J. Cunningham J.M. J. Virol. 1997; 71: 8096-8102Crossref PubMed Google Scholar, 29Harris A.L. Walter A. Paul D.L. Goodenough D.A. Zimmerberg J. Mol. Brain. Res. 1992; 15: 269-280Crossref PubMed Scopus (33) Google Scholar). Briefly proteins were reconstituted in a mixture of phosphatidylcholine and phosphatidylserine at a 2:1 molar ratio. The lipids in chloroform stocks were mixed and lyophilized overnight under argon. The dry film was rehydrated in 75 μl of OG-containing washing buffer/mg of lipid and warmed to 37 °C until it became transparent. OG-solubilized proteins were added to the lipid-detergent mixture and dialyzed through a Spectra/Pro 6,000–8,000 molecular weight cut-off membrane (Spectrum Laboratories, Rancho Dominguez, CA) for 24 h at room temperature against 500 ml of detergent-free washing buffer containing 10 ml of a 50% (w/v) suspension of Biobeads SM-2 (Bio-Rad). Large unilamellar vesicles for the transport and proteolysis experiments (∼100-nm diameter) were obtained by extrusion (Mini-Extruder, Avanti). For the sedimentation analysis, lissamine rhodamine B-labeled phosphatidylethanolamine was added (phosphatidylcholine:phosphatidylserine:phosphatidylethanolamine ratio of 2:1:0.05) to label the liposomes for easy identification. For these experiments, the buffer contained 459 mm urea. Sucrose Uptake—Sucrose permeability of the proteoliposomes containing purified Cx43 GJHs was assessed by the transport-specific density shift technique (26Kim D.Y. Kam Y. Koo S.K. Joe C.O. J. Biol. Chem. 1999; 274: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 30Harris A.L. Walter A. Zimmerberg J. J. Membr. Biol. 1989; 109: 243-250Crossref PubMed Scopus (19) Google Scholar, 31Harris A.L. Bevans C.G. Bruzzone R. Giaume C. Methods in Molecular Biology: Connexin Methods and Protocols. Humana Press, Totowa, NJ2001: 357-377Google Scholar) and by a rapid filtration assay. For the shift assay, the proteoliposomes containing lissamine rhodamine B-labeled phosphatidylethanolamine were layered onto a linear isoosmolar sucrose gradient (0–400 mm sucrose with a reverse urea gradient) in detergent-free buffer. The gradient was usually centrifuged on a swinging bucket Beckman SW55Ti rotor at 300,000 × g for 8 h at 4 °C, and the location of the proteoliposomes was determined in fractions collected from the top to the bottom of the gradient tubes. The fluorescence of samples excited at 560 ± 4 nm was measured at 590 ± 4 nm (Fluorolog-2, SPEX, Edison, NJ). In most of the experiments, the proteoliposomes were loaded with Lucifer Yellow by adding the fluorescent probe at a concentration of 2 mm during the extrusion procedure. The non-trapped Lucifer Yellow was removed by filtration through a PD-10 column (Amersham Biosciences). Lucifer Yellow fluorescence was measured in the sedimentation gradient fractions at an excitation of 430 ± 4 nm and an emission of 540 ± 4 nm. To measure sucrose uptake by rapid filtration, proteoliposomes were incubated at 25 °C in washing buffer containing 2 mm sucrose and 10 nCi/μl [14C]sucrose (American Radiolabeled Chemicals, specific activity of 7.5 mCi/mmol). Filtration was through 0.2-μm nitrocellulose filters (Whatman). Radioactivity of the filters was measured by liquid scintillation after washing with 45 ml of ice-cold washing buffer containing 10 μm Gd3+, a blocker of gap-junctional channels and GJHs (32Eskandari S. Zampighi G.A. Leung D.W. Wright E.M. Loo D.D. J. Membr. Biol. 2002; 185: 93-102Crossref PubMed Scopus (136) Google Scholar, 33Bao X. Altenberg G.A. Reuss L. Am. J. Physiol. 2004; 286: C647-C654Crossref PubMed Scopus (96) Google Scholar). Tryptophan Fluorescence Measurements—Tryptophan fluorescence emission spectra of OG-solubilized Cx43 and Cx43-S368A were obtained on a SPEX CMT1 with excitation at 295 ± 4 nm. Data were obtained at protein concentrations between 0.1 and 0.3 μm in 10 mm KCl, 0.1 mm EDTA, 2.3% OG, and 10 mm potassium phosphate, pH 7.4, in 1-ml quartz cuvettes. The spectra shown were subtracted from the background (buffer alone). Trypsin Digestion—Proteoliposomes were incubated with sequencing grade modified trypsin (V5111, Promega, Madison, WI) at a protein: trypsin ratio of 200:1 (w/w). Typically proteoliposomes containing 20 μg of Cx43 or Cx43-S368A were incubated with trypsin at 37 °C, and the reactions were stopped at varying times by adding SDS gel sample buffer and increasing the temperature to 95 °C. In the control experiments shown, 1 mm PMSF was added before trypsin. Other Techniques—Protein concentrations were determined using the BCA protein assay reagent from Bio-Rad using bovine serum albumin as standard. To detect possible contaminants, the highly sensitive protein gel stain SYPRO Ruby (Molecular Probes) was used according to the manufacturer's instructions. Statistics—Data shown are means ± S.E. Statistically significant differences were assessed by the Student's t test for unpaired data or one-way analysis of variance as appropriate. Purification of Cx43 Overexpressed in Insect Cells—Our general strategy was to take advantage of the fluorescence of Cx43-EGFP to determine the best conditions for expression, solubilization, and purification and then to assess whether those conditions were also the best ones for the non-fluorescent Cx43 and Cx43-S368A (see Fig. 1A for schematics of these proteins). We found that initial trials with Cx43-EGFP indeed facilitated the initial screening and that the biochemical and functional properties of Cx43-EGFP and Cx43 were essentially identical in terms of detergent solubility, oligomerization, reconstitution efficiency, and permeability regulation by PKC-mediated phosphorylation. In this section, we summarize the main results of the expression, purification, and reconstitution of the Cx43s. Fig. 1, B and C, shows that a good expression level of Cx43-EGFP in High-Five cells is reached 2–3 days after infection. This time course of Cx43 expression was similar to that previously published for the expression of Cx32 (23Stauffer K.A. Kumar N.A. Gilula N.B. Unwin P.N. J. Cell Biol. 1991; 115: 141-150Crossref PubMed Scopus (95) Google Scholar). There was a good correlation between the expression assessed by SDS-PAGE (Fig. 1B) and the cell fluorescence (Fig. 1C). The latter method has the advantages that it can be used in real time and is simpler. The results for the expression of Cx43 and Cx43-S368A were similar (data not shown). Fig. 1 also shows that the His-tagged Cx43 can be easily purified based on its affinity for Ni2+. The Coomassie Blue- and SYPRO Ruby-stained gels (Fig. 1, D and E, respectively) show that the preparation of Cx43 is highly purified, i.e. no contaminants were detected. The yield of Cx43 and Cx43-EGFP was 5–10 mg/liter of cells, whereas that of Cx43-S368A was ∼70% of that of Cx43 (data not shown). The purified preparations obtained from membranes that were not alkali-extracted (see “Experimental Procedures”) also appeared homogeneous, but the yield was somewhat lower. Structural, biochemical, and functional studies have shown that gap-junctional hemichannels are oligomers composed of six connexin molecules (for a review, see Ref. 1Harris A.L. Q. Rev. Biophys. 2001; 34: 325-472Crossref PubMed Google Scholar). To assess the oligomerization state of the purified Cx43 we performed gel filtration experiments on Cx43-EGFP solubilized in 2.3% OG. Fig. 1F shows that the major fluorescence peak (calculated molecular mass of 517 kDa) is compatible with a Cx43-EGFP hexamer with ∼18% of the complex apparent weight in bound detergent. The absorbance peaks labeled 1 and 2 correspond to the 669- and 440-kDa markers (which did not affect Cx43 elution) added to the Cx43-EGFP sample. The smaller fluorescence peak is compatible with monomeric Cx43-EGFP. Similar results were obtained with Cx43 in 0.3% decyl-maltopyranoside (not shown). The predominance of connexons in the detergent-solubilized preparation was confirmed by determination of the sedimentation coefficients on a sucrose gradient (data not shown), a technique routinely used for the assessment of connexin quaternary structure (see Ref. 25Falk M.M. Buehler L.K. Kumar N.M. Gilula N.B. J. Cell Biol. 1997; 16: 2703-2716Google Scholar). From the results in this section, we conclude that Cx43 in our preparation contains predominantly Cx43 assembled as connexons (70–80% of the total Cx43 from the gel filtration experiment data). Ser-368 of Cx43 Is Phosphorylated by PKC—To test Cx43 function, we compared the permeability of GJHs reconstituted in liposomes in the dephosphorylated and PKC-phosphorylated states. We dephosphorylated the purified proteins with alkaline phosphatase (see “Experimental Procedures”). Dephosphorylated protein was then phosphorylated by PKC. Thus, we studied dephosphorylated and PKC-phosphorylated Cx43s in the absence of endogenous phosphorylation by other kinases. Pilot time course experiments using radiolabeled ATP showed that, under the conditions of our experiments, phosphorylation of Cx43 by PKC was complete by 1 h. Fig. 2A shows a representative autoradiography of Cx43 and Cx43-S368A phosphorylated by PKC in the presence of radiolabeled ATP. It is clear that the phosphorylation by PKC was substantially reduced when Ser-368 was substituted with Ala. The results of quantitative Pi incorporation studies show a maximal incorporation of Pi of ∼2 and 1 mol/mol of Cx43 and Cx43-S368A, respectively (Fig. 2B). In control experiments performed in the absence of PKC Cx43 phosphorylation was <8% of the value in the presence of PKC. The calculated decrease in phosphorylation by the S368A mutation (∼60 and ∼75% from the stoichiometry and densitometry analyses, respectively) is consistent with previous observations that indicate that Ser-368 is the major phosphorylation target of PKC and that phosphorylation also occurs at Ser-372 (16Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Johnson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (470) Google Scholar, 17Sáez J.C. Nairn A.C. Czernk A.J. Fishman G.I. Spray D.C. Hertzberg E.L. J. Mol. Cell. Cardiol. 1997; 29: 2131-2145Abstract Full Text PDF PubMed Scopus (136) Google Scholar). We have recently shown that changes in the phosphorylation state of Ser-372 are not involved in the activation of Cx43 GJH carboxyfluorescein permeability by PKC blockers (33Bao X. Altenberg G.A. Reuss L. Am. J. Physiol. 2004; 286: C647-C654Crossref PubMed Scopus (96) Google Scholar). The observation that the decrease in Pi incorporation in Cx43-S368A is ∼1 mol/mol of protein suggests that under the conditions of these experiments all Cx43 Ser-368 residues are phosphorylated. Phosphorylation of Ser-368 by PKC Reduces the Permeability of Cx43 GJHs—Previous studies using mutagenesis and pharmacological agents that affect the PKC signaling pathway suggested that phosphorylation of Ser-368 reduces the permeability of Cx43 gap-junctional channels to small hydrophilic solutes (16Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Johnson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (470) Google Scholar). However, it is not known whether the effects of PKC-mediated phosphorylation are direct (i.e. due to phosphorylation of Cx43) or indirect (i.e. via other signaling pathways affected by the PKC activation or involving regulatory factors or phosphorylation by PKC of one or more cytosolic factors). To determine whether or not the regulation of Cx43 GJH permeability is direct, we carried out functional studies with purified Cx43 GJHs reconstituted into liposomes. Purified Cx43 was reconstituted in phosphatidylcholine and phosphatidylserine liposomes by dialysis/extrusion as described under “Experimental Procedures.” At protein:lipid ratios of 1:25, 1:60, 1:100, and 1:280 (w/w) essentially all the protein was incorporated into the liposomes. The reconstitution efficiency for Cx43-EGFP and Cx43-S368A was also >90%. In all experiments Cx43 amounts were measured in the proteoliposomes (i.e. after reconstitution, not inferred from the reconstitution efficiency). We assessed sucrose permeability by measuring the migration of the proteoliposomes on a linear isoosmolar sucrose/urea gradient, a metho" @default.
• W2146581722 created "2016-06-24" @default.
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• W2146581722 date "2004-05-01" @default.
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