September 2007
Volume 48, Issue 9
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Lens  |   September 2007
Purinergic Receptor–Mediated Regulation of Lens Connexin43
Author Affiliations
  • Monica M. Lurtz
    From the Department of Cell Biology and Neuroscience, University of California, Riverside, California.
  • Charles F. Louis
    From the Department of Cell Biology and Neuroscience, University of California, Riverside, California.
Investigative Ophthalmology & Visual Science September 2007, Vol.48, 4177-4186. doi:10.1167/iovs.06-0496
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      Monica M. Lurtz, Charles F. Louis; Purinergic Receptor–Mediated Regulation of Lens Connexin43. Invest. Ophthalmol. Vis. Sci. 2007;48(9):4177-4186. doi: 10.1167/iovs.06-0496.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine whether the purinergic receptor–mediated, delayed transient inhibition of lens cell-to-cell communication is due to the protein kinase C (PKC)–catalyzed phosphorylation of connexin (Cx)43.

methods. The functional activity of gap junctions was determined in the presence of various pharmacologic agents by injecting fluorescent dye into a single cell in a confluent monolayer of HeLa cells that had been stably transfected with either wild-type or mutant Cx43, and the number of cells taking up dye was determined.

results. Application of adenosine triphosphate (ATP) to Cx43-transfected HeLa cells resulted in a delayed, transient decrease in cell-to-cell transfer of fluorescent dye similar to the authors' previous report in sheep lens epithelial cell cultures. The ATP-mediated, delayed, transient decrease in dye transfer was prevented by the inhibition of PKC or phospholipase C, but not by calmodulin inhibition or by preloading the cells with BAPTA (bis-(o-aminophenoxy)-N,NN′-tetraacetic acid). This functional inhibition of Cx43 cell-to-cell dye transfer was sustained in the presence of the nonhydrolyzable ATP analogue AMP-PNP (adenyl-5′-yl imidophosphate), the ectonucleotidase inhibitor ARL 67156, or the protein phosphatase inhibitor okadaic acid. In experiments in HeLa cells transfected with Cx43Δ257, a Cx43 C terminus truncation mutant, or Cx43S368A, a Cx43 point mutant, cell-to-cell coupling was unaffected by the addition of ATP.

conclusions. The results indicate the essential role of serine 368 in the ATP-dependent inhibition of Cx43. This novel mechanism of regulating Cx43 most likely plays an important role in maintaining the microcirculation that is essential for the movement of water and solutes in the intact lens.

Gap junction intercellular channels allow for the direct movement of small molecules and metabolites of molecular mass up to ∼1000 Daltons between of the cytoplasm of adjacent cells. 1 In mammalian cells, gap junctions are composed of connexins, a family of proteins in which each of two adjacent cells contribute hexameric connexon hemichannels that, when closely opposed, form cell-to-cell channels. Over 20 human connexin genes have now been identified that exhibit significant tissue-specific expression patterns 2 and selectivity to small molecules and ions. Gap junctions are regulated by multiple mechanisms that include connexin phosphorylation, 3 4 intracellular pH, 5 6 7 intracellular [Ca2+], 8 9 10 and transjunctional voltage. 11 12 13 14  
This laboratory has demonstrated previously that purinergic receptor activation of primary cultures of sheep lens epithelial cells effects a delayed, transient reduction in cell-to-cell transfer of divalent cations and dye molecules. 8 Subsequently, we demonstrated that the regulation of lens cell-to-cell communication by purinergic receptor activation was mediated by the activation of PKC through a phospholipase (PL) C–mediated pathway. 15 We concluded that this action of purinergic receptor activation is the result of the phosphorylation of one or more of the connexins present in these lens cells. 
Connexin (Cx)43, the most widely distributed connexin in mammalian tissues, is the major connexin in lens epithelial cells. 16 Previous studies in our laboratory have shown that these sheep lens epithelial cells can be used to develop primary cell cultures that are well coupled by gap junctions. 17 However, as in the intact lens, these differentiating lens epithelial cell cultures express not only Cx43, but also the sheep orthologues of human Cx46 and Cx50. 18 19 Thus, this lens epithelial cell culture cannot be used to identify which of the three lens connexins is responsible for mediating the effect of purinergic agonists on lens epithelial cell-to-cell communication. Thus, to determine whether the effect of purinergic receptor activation on lens cell-to-cell communication is mediated in part by Cx43, studies were conducted with Cx43-transfected HeLa cells, as these cells have been shown to provide an excellent system to examine the properties of homotypic gap junctions. 20 21 Furthermore, like lens epithelial cells, HeLa cells have been shown to have purinergic receptors and PKC 22 and therefore provide an excellent model system to test the hypothesis that purinergic receptor activation effects a PKC-mediated inhibition of gap junctions composed solely of Cx43. 
In this study, we tested whether this purinergic receptor–mediated effect on lens cell-to-cell communication is due to the PKC-catalyzed phosphorylation of connexin 43. In addition we found an explanation for the transient nature of the purinergic receptor dependent inhibition of cell-to-cell communication previously reported in lens epithelial cell cultures. 8 15  
Materials and Methods
Untransfected HeLa cells and HeLa cells stably transfected with Cx43 were generous gifts from Klaus Willecke (University of Bonn, Germany). HeLa cells stably transfected with the Cx43 truncation mutant Cx43Δ257 were a gift from Erika TenBroek (University of Minnesota, Minneapolis, MN; currently at Medtronics Inc., Minneapolis, MN). HeLa cells stably transfected with the point mutant Cx43S368A were the kind gift of Paul Lampe (Fred Hutchinson Cancer Center and the University of Washington, Seattle, WA). Characterized fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). G418 (geneticin) and hygromycin B were obtained from Invitrogen (Carlsbad, CA). Fura-2 AM, BAPTA AM, and AlexaFluor 594 (AF594) were from Invitrogen-Molecular Probes (Eugene, OR). Monoclonal anti-mouse connexin43 primary antibody (MAB3068, IgG1), goat anti-mouse IgG, HRP-conjugated secondary antibodies, goat anti-rabbit polyclonal, HRP-conjugated secondary antibodies, and sheep anti-rabbit polyclonal, HRP-conjugated secondary antibodies were purchased from Chemicon International (Temecula, CA). Polyclonal anti-rabbit α1J antibody directed to the intracellular loop of Cx43 was the kind gift of Nalin Kumar (Department of Ophthalmology, University of Illinois, Chicago, IL). Cx43 serine 368-phosphospecific antibody (p368) and a mouse anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) were also kindly provided by Paul Lampe; bisindolylmaleimide I (Bim-1) was obtained from Calbiochem (San Diego, CA). Dulbecco's modified Eagle's medium (DMEM), medium 199, Hanks' balanced salt solution without divalent cations (HBSS) or with divalent cations (HBSS2+), ATP, dimethyl sulfoxide (DMSO), 1-octanol, PMA, staurosporine, carbenoxolone (CBX), ARL 67156 (ARL), U73122, U73343, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 
Cell Culture
HeLa cells were grown to confluence in 35-mm culture dishes on sterilized glass coverslips in DMEM (with 44 mM NaHCO3, pH 7.2) supplemented with 10% vol/vol FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Stable transfectants were grown under antibiotic selection as follows: Cx43, 0.4 mg/mL G418; Cx43Δ257, 0.2 mg/mL hygromycin B; and Cx43S368A, 0.1 mg/mL hygromycin B. All cells were grown in a humidified 37°C incubator with 5% CO2
Calcium Imaging
Cells grown on a glass coverslip to a confluent monolayer were loaded with the Ca2+ indicator Fura-2 AM (1 μM) in 2 mL HBSS2+ buffer (with 10 mM HEPES and 5 mM NaHCO3[pH 7.2]), then transferred to a microincubation chamber (model MSC-TD; Harvard Apparatus, Holliston, MA), as described previously. 15 Imaging of intracellular Ca2+ (Ca2+ i) was performed on an inverted microscope (model TE300; Nikon Inc., Melville, NY) that was equipped with filter blocks for Fura-2 emission and AF594 optics (Chroma Technology Corp, Rockingham, VT), a filter wheel (Metaltek Instruments, Raleigh, NC) housing excitation filters for Fura-2, a 75-W xenon short arc lamp, and a CCD digital camera (Hamamatsu Corp., Bridgewater, NJ) and was supported on a vibration isolation table (Technical Manufacturing, Peabody, MA). Ca2+ i was measured ratiometrically (λ340380) with Fura-2 throughout each experiment in the injected cell and the cells adjacent to the injected cell, and Ca2+ concentrations determined as described previously. 23 Data collection was accomplished with commercial software (MetaFluor; Universal Imaging Corp., ver. 3.5, Downingtown, PA). 
Microinjection and Assessment of Gap Junctional Communication
Micropipettes (borosilicate glass capillaries: 1 mm OD, 0.75 mm ID, 100 μm internal microfilament; Dagan Corp., Minneapolis, MN) backfilled with a 1 mM solution of AF594 were mounted on a half-cell with a chlorodized silver wire and were used when they had a resistance between 100 and 300 MΩ. Cell-to-cell transfer of AF594 dye was imaged 5 minutes after iontophoretic injection of dye with an electrometer (Duo 773, equipped with an A310 Accupulser; World Precision Instruments, Sarasota, FL) and a pulse protocol of 5 ms every 100 ms for 1 minute (3 seconds total injection time) at ambient temperature. Digitized images were recorded and subsequently used to determine the number of cells receiving dye, as described previously. 15  
Membrane Isolation
Cells were harvested from 100- or 150-mm culture dishes by adding 500 μL of ice-cold buffer 1 (in mM: 25 Tris base, 100 NaCl, 10 EDTA, 50 NaF, 0.5 Na3VO4 [pH 8.0], supplemented with protease inhibitors: 2 mM PMSF, and 1 mg/L each: aprotinin, leupeptin, and pepstatin) to each culture dish, removing the cells with a cell scraper (Sarstedt, Newton, NC), and transferring the suspended cells to a thick-walled 1.5-mL microfuge tube (Beckman/Coulter, Fullerton, CA). Culture dishes were washed twice with 200 μL of ice-cold buffer 1, and the final volume in each sample was brought to 1 mL. The cells were sedimented (5 minutes at 1260g at 4°C) in a centrifuge (TLA100.3; Beckman Instruments, Fullerton, CA). The supernatant was removed and the sedimented cells were resuspended in 500 μL ice-cold buffer 1 and incubated on ice for 30 minutes. The cells were homogenized with a 1-mL glass-on-glass Potter-Elvehjem tissue homogenizer (Bellco Glass Inc., Vineland, NJ). Homogenates were centrifuged at 100,000g at 4°C for 30 minutes in the centrifuge. The supernatant was removed and discarded, and the sedimented membranes were resuspended in 200 μL ice-cold buffer 2 (10 mM HEPES [pH 7.2]) by drawing the membranes through a 50-μL syringe (GASTIGHT 1700; Hamilton, Reno, NV) with cemented needle (Sigma-Aldrich, St. Louis, MO) until the membrane suspension was homogenous (∼30 times). The protein concentration of each membrane preparation was determined with the micro-BCA assay using bovine serum albumin standards. 24 The membranes were either used immediately or flash frozen in liquid nitrogen and stored at −80°C. Alternatively, the cells were lysed in sample buffer supplemented with inhibitors as just described. 
Western Immunoblot Analysis
After electrophoretic analysis of proteins on 10% PAGE gels with a 4% stacking gel in 0.1% SDS, 25 gels were equilibrated with transfer buffer for 15 minutes before electrophoretic transfer to nitrocellulose membranes (Schleicher & Schuell Inc., Keene, NH), as described previously, 15 by a transfer cell (Trans Blot; Bio-Rad, Richmond, CA), per the manufacturer's instructions. Nonspecific antibody binding sites on the nitrocellulose membranes were blocked in PBS-Tween (137 mM NaCl, 2.7 mM KCl, 1.8 mM Na2HPO4, and 0.1% vol/vol Tween-20 [pH 7.2]), containing 1% wt/vol dried milk for 30 minutes at ambient temperature, or in 3% wt/vol dried milk overnight at 4°C, with gentle agitation. After the nonspecific antibody binding sites were blocked, the nitrocellulose membranes were washed three times for 15 minutes each in fresh PBS-Tween without dried milk, before incubation with the primary antibody indicated in the figure legends. After removal of the primary antibody solution, the nitrocellulose membranes were washed three times for 15 minutes before addition of the appropriate HRP-conjugated secondary antibody. The immunoreactive components were detected on autoradiograph film (Biomax MS or MR; Eastman-Kodak, Rochester, NY) after incubation with the appropriate detection reagent (GE Healthcare, Piscataway, NJ, or Pierce Chemicals, Rockford, IL). Experiments were repeated a minimum of three times. Densitometry of the immunoreactive bands was performed on a gel documentation system (BioChemi EC(3) Documentation System; UVP, Inc., Upland, CA). 
Data Handling and Statistical Analysis of the Data
Dye injection data for identical treatments were pooled, averaged, and expressed as the mean ± SE. Statistical differences were tested between two samples by t-test. Differences from the control experiments were considered significant when P < 0.01. 
Results
Characterization of Protein in Connexin-Transfected HeLa Cells
To confirm the expression of transfected protein, cell membranes isolated from untransfected HeLa cells or HeLa cells stably transfected with full-length Cx43, Cx43 truncated at amino acid residue 257 (Cx43Δ257), or Cx43 containing a serine-to-alanine point mutation at residue 368 (Cx43S368A) were electrophoretically fractionated and analyzed by Western blot analysis. Using α1J, an antibody directed to the intracellular loop of Cx43, protein migrating at the predicted molecular weights for transfected Cx43 and Cx43Δ257 protein was confirmed (Fig. 1B) . Expression the of Cx43S368A protein was confirmed using the α-Cx43 monoclonal antibody MAB3068 (Fig. 1C)
Mediation of Cell-to-Cell Transfer of Dye in Stably Transfected HeLa Cells by Homotypic Gap Junctions Composed of Cx43, Cx43Δ257, or Cx43S368A
Formation of functional gap junctions in the transfected cells was demonstrated by injecting AF594 dye into a single cell in a confluent monolayer of Cx43-transfected HeLa cells, Cx43Δ257-transfected HeLa cells, or Cx43S368A-transfected HeLa cells in the absence or presence of the gap junction inhibitor CBX (35 μM, 20–30 minutes; Fig. 2 ). In Cx43-transfected HeLa cells, dye spread from the injected cell to an average of 21.8 ± 0.5 cells (n = 4) in the absence of CBX and to 1.0 ± 0.4 cells (n = 4) in the presence of 35 μM CBX. In Cx43Δ257-transfected HeLa cells, dye spread from the injected cell to an average of 17.2 ± 0.5 cells (n = 5) in the absence of CBX and to 0.6 ± 0.2 cells (n = 5) in the presence of 35 μM CBX, and in Cx43S368A transfected HeLa cells, dye spread from the injected cell to an average of 19.8 ± 0.6 cells (n = 5) in the absence of CBX and to 1.0 ± 0.5 cells (n = 8) in the presence of 35 μM CBX. Thus, cell-to-cell dye transfer was gap junction mediated in all three types of connexin-transfected HeLa cell lines, as it was inhibited by both CBX (Fig. 2G)and the classic gap junction inhibitor 1-octanol (data not shown). 17 23 In contrast, as shown in many other laboratories (for example, Hennemann et al., 26 and Martin et al., 27 ), there was no cell-to-cell transfer of dye between untransfected wild-type HeLa cells (data not shown). 
Effect of Purinergic Receptor Activation on Cell-to-Cell Dye Transfer in Cx43-Transfected HeLa Cells
We have shown previously that activation of purinergic receptors by addition of adenosine triphosphate (ATP) to lens epithelial cell cultures results in a delayed, transient reduction in cell-to-cell dye transfer. 8 15 Cx43 is the predominant connexin in these cells, which in the intact lens are bathed with the aqueous humor into which ATP can be released from ciliary epithelial cells. 28 29 In this study, the HeLa cell line, a well-characterized epithelial cell line that contains the purinergic receptor signaling system, 22 was stably transfected with Cx43. These cells provide an ideal approach to the examination of the effect of purinergic receptor activation on Cx43-containing gap junction-mediated cell-to-cell dye transfer. 30 It was demonstrated that, as previously reported in lens epithelial cells, 8 15 5 minutes after addition of ATP to these cells, cell-to-cell dye transfer between Cx43-transfected HeLa cells was significantly reduced (to 4.7 ± 0.5 cells [n = 7] from 17.6 ± 0.6 cells [n = 17]; P < 0.01; Fig. 3 ). Cell-to-cell dye transfer returned to control levels by 20 minutes after the addition of ATP (dye transferred to 17.3 ± 0.5 cells, n = 8; P > 0.01). 
Role of Purinergic Receptor Activation in the Ca2+-Independent Activation of PKC
Although HeLa cells, like the previously studied lens cells, are epithelial cells, it was important to demonstrate that the activation of purinergic receptors in HeLa cells was coupled to a signal transduction pathway similar to the one we have described previously in lens epithelial cells. 8 15 By using inhibitors of both PKC (staurosporine and Bim-1) and PLC (U73122; Table 1 ), we determined that this purinergic receptor-mediated, delayed, transient reduction in cell-to-cell dye transfer in Cx43-transfected HeLa cells was indeed effected through a signal transduction pathway similar to that in the lens epithelial cells. 15 Thus, inhibition of either protein kinase C (PKC) or PLC prevented this ATP-dependent decrease in cell-to-cell dye transfer; the inactive PLC analogue U73343 was without effect on dye transfer. Furthermore, it was demonstrated by preloading the Cx43-transfected HeLa cells with BAPTA ((bis-(o-aminophenoxy)-N,NN′-tetraacetic acid) to prevent the transient increase in [Ca2+]i resulting from the release of Ca2+ from stores in the endoplasmic reticulum (ER) that this signal transduction pathway leading to the activation of PKC was not dependent on elevation of [Ca2+]i
The Mechanism that Mediates the Purinergic Receptor–Dependent, Delayed, Transient Inhibition of Gap Junction-Mediated Cell-to-Cell Dye Transfer
Given the delayed, transient nature of this inhibition of cell-to-cell dye transfer, there are at least two mechanisms by which this agonist-mediated signaling pathway may be regulated at the level of the receptor: namely, either by agonist hydrolysis, or by purinergic receptor desensitization. To differentiate between these two possible mechanisms, experiments were conducted in which either AMP-PNP (adenyl-5′-yl imidodiphosphate), a nonhydrolyzable analogue of ATP that is known to activate purinergic receptors, 31 was used as the agonist, or hydrolysis of ATP was prevented by inhibition of ectonucleotidases 32 by ARL, which has been shown to be effective in other systems. 33 Purinergic receptor activation using the nonhydrolyzable ATP analogue AMP-PNP (50 μM) 31 34 resulted in a significant reduction in cell-to-cell dye transfer 5 minutes after the addition of this agonist (Fig. 4A) . This reduction in Cx43-mediated cell-to-cell dye transfer persisted for at least 30 minutes after the addition of AMP-PNP (Fig. 4A) . Thus, the addition of a nonhydrolyzable analogue of ATP resulted in a sustained reduction in Cx43-mediated cell-to-cell dye transfer, evidence that agonist hydrolysis and not purinergic receptor desensitization regulates this signaling pathway at the receptor level. 
It was important to demonstrate that this ATP analogue does not produce some irreversible change but is effective solely because of its inability to be hydrolyzed by ectonucleotidases (i.e., that, similar to the removal of ATP from the extracellular medium by hydrolysis, the effect of AMP-PNP on cell-to-cell dye transfer is reversible). Therefore, after inhibition of cell-to-cell dye transfer by AMP-PNP, this ATP analogue was removed from the bathing medium by washing the cells with buffer containing no AMP-PNP and then incubating them in AMP-PNP-free buffer for 20 minutes, to simulate the timeframe required for cell-to-cell dye transfer to return to basal levels after the addition of ATP. After AMP-PNP washout, Cx43-mediated cell-to-cell dye transfer did indeed return to pre-AMP-PNP levels (Fig. 4B) . Subsequent addition of 25 μM ATP to these cells resulted in the characteristic significant reduction in cell-to-cell dye transfer 5 minutes after agonist addition and a return to pre-ATP addition levels of dye transfer within 20 minutes (Fig. 4B) . That this action of AMP-PNP on purinergic receptors is similar to ATP was confirmed by inhibiting PKC activation before AMP-PNP addition to Cx-43-transfected HeLa cells. The inhibition of PKC with Bim-1 (100 nM, 20–30 minutes) prevented the significant decrease in cell-to-cell dye transfer 5 to 30 minutes after purinergic receptor activation by AMP-PNP (Fig. 4C)
The ectonucleotidase inhibitor ARL 67156 (30 μM) 33 was used to confirm that the ATP-dependent delayed, transient inhibition of Cx43-mediated cell-to-cell dye transfer requires unhydrolyzed ATP. If a sustained reduction in Cx43-mediated cell-to-cell dye transfer was effected by a nonhydrolyzable analogue of ATP, then a similar sustained reduction should be observed after inhibition of the enzyme responsible for the hydrolysis of extracellular ATP. As depicted in Figure 4D , the addition of the ectonucleotidase inhibitor ARL (30 μM, 20–30 minutes) alone had no affect on Cx43-mediated cell-to-cell dye coupling (dye transferred to 19.4 ± 0.4 cells, n = 5, versus 20.2 ± 0.5 cells, n = 6 in the presence of ARL). However, in the presence of this inhibitor, cell-to-cell dye transfer was significantly reduced 5 minutes after ATP (25 μM) addition (dye transferred to 3.3 ± 0.7 cells, n = 3; P < 0.01) and remained significantly reduced in the continued presence of ARL for up to 30 minutes after addition of ATP (dye transferred to 2.4 ± 0.8 cells, n = 5). 
Regulation of Cx43-Mediated Cell-to-Cell Dye Transfer by Protein Dephosphorylation
The results presented in this study demonstrate that agonist hydrolysis rather than receptor desensitization was responsible for the transient nature of the reduction in cell-to-cell dye transfer after addition of ATP to Cx43-transfected HeLa cells. The data presented in Table 1and Figure 4confirm that, as in lens epithelial cells, PKC activation is necessary for this reduction in cell-to-cell dye transfer. 15 Cx43 is a known PKC substrate (see Lampe and Lau 35 and Wam-Cramer and Lau 36 for recent reviews), and many protein phosphatases are constitutively active. 37 38 It was therefore pertinent to determine whether the reversibility of this ATP-dependent reduction in cell-to-cell coupling is due in part to the dephosphorylation of a PKC substrate. To address this question, cells were incubated with the protein phosphatase inhibitor okadaic acid (OA; 250 nM, 20–40 minutes) before the addition of 25 μM ATP (Fig. 5A) . AF594 transferred to 19.2 ± 0.5 cells (n = 5) in the absence of any additions, and dye transfer was not affected by the presence of OA (dye transferred to 19.8 ± 0.7 cells, n = 6). As expected, in either the absence or presence of OA, cell-to-cell dye transfer was significantly reduced 5 minutes after addition of 25 μM ATP (dye transferred to 3.3 ± 0.7 cells, n = 4; P < 0.01). Consistent with the hypothesis that a return to pre-ATP addition levels of cell-to-cell dye transfer requires the dephosphorylation of protein, Cx43-mediated cell-to-cell dye transfer remained significantly reduced 30 minutes after ATP addition when OA was present throughout the period of incubation (dye transferred to 2.4 ± 0.8 cells, n = 5; P < 0.01). 
Role of Cx43 C Terminus Amino Acid(s) in the Purinergic Receptor–Dependent Inhibition of Cx43-Mediated Cell-to-Cell Dye Transfer
The data presented in the present study demonstrate that purinergic receptor activation results in the activation of PKC, which effects the phosphorylation of a protein, and that this phosphorylation event is an essential step in the pathway leading to the inhibition of Cx43-mediated cell-to-cell dye transfer. It has been well documented that Cx43 is a substrate for PKC and that phosphorylation of Cx43 by PKC is associated with an inhibition of cell-to-cell communication. 35 39 40 41 Cx43 contains several PKC consensus sites in its C terminus region, 42 and it was therefore important to determine whether the purinergic receptor-dependent PKC-mediated phosphorylation of Cx43 is necessary to effect the inhibition of cell-to-cell dye transfer described in this study. This question was investigated by using the C terminus truncation mutant Cx43Δ257, stably transfected into HeLa cells (see Fig. 1Afor topology and Fig. 1Bfor Western blot). If the phosphorylation of a residue in the C terminus of Cx43 was involved in this purinergic receptor–mediated transient reduction in cell-to-cell dye transfer, then removal of the portion of the C terminus of Cx43 containing the likely phosphorylated residue(s) would result in gap junctions that were now insensitive to this purinergic receptor-activated pathway. As depicted in Figure 5B , Cx43Δ257-mediated cell-to-cell dye transfer remained at pre-ATP addition levels (15.3 ± 0.5 cells, n = 24) at all times measured after ATP (25 μM) addition (i.e., truncation of Cx43 results in gap junctions that are insensitive to purinergic receptor activation). Of particular importance are the levels of cell-to-cell dye transfer 5 to 10 minutes after the addition of ATP (15.6 ± 0.8 cells, n = 9) and more than 20 minutes after the addition of ATP (15.4 ± 0.4 cells, n = 5), as these two time points demonstrate that the C-terminal portion of Cx43 is necessary for the ATP-dependent reduction of Cx43-mediated cell-to-cell dye transfer. 
Role of Serine 368 in the C Terminus of Cx43 in the Reduction in Cell-to-Cell Dye Transfer after Purinergic Receptor Activation
Of the PKC consensus serine residues located in the C terminus of Cx43, the serine at amino acid residue 368 has been shown to be one of the major PKC substrates in this protein. 43 Therefore, a Cx43 point mutant in which serine 368 is mutated to an alanine (Cx43S368A) was used as a stable transfectant in HeLa cells. As demonstrated by Western blot analysis in Figure 1 , this Cx43 mutant protein is expressed in transfected HeLa cells. Furthermore, this Cx43 mutant forms functional gap junctions that are fully inhibited by CBX (Figs. 2K 2L 2M 2N 2O)and 1-octanol (data not shown). Therefore, if gap junctions composed of Cx43S368A are resistant to inhibition after ATP-mediated purinergic receptor activation, then this Cx43 serine residue is most likely an essential substrate for this PKC-mediated pathway in wild-type Cx43-transfected HeLa cells. As demonstrated in Figure 5C , in the absence of purinergic receptor activation, dye was transferred to 22.4 ± 1.0 cells (n = 11). Subsequent to the addition of ATP (25 μM), cell-to-cell dye transfer remained unchanged at both 5 minutes (21.2 ± 1.6 cells, n = 5) and 20 minutes (25.0 ± 1.8 cells, n = 5) after purinergic receptor activation. 
To establish a direct correlation between the regulation of Cx43 by serine 368 and purinergic receptor activation, we incubated Cx43-transfected HeLa cells in either the presence or absence of AMP-PNP, harvested them, and immunoblotted them with an antibody (p368) that is specific for Cx43 phosphorylated at serine 368. 34 In Figure 6 , which shows the results of a replicate of triplicate experiments, it is demonstrated that there was no change in the absolute amount of phospho-368 after purinergic receptor activation with AMP-PNP (lane 1) compared with unstimulated cells (lane 2). Furthermore, immunolocalization of phospho-368 in unstimulated and stimulated cells demonstrated no consistent changes in the amount of phospho-368 or cellular location of phospho-368 (data not shown). Thus, although these data demonstrate that serine at residue 368 in Cx43 is absolutely essential for the purinergic receptor-dependent reduction in Cx43–mediated cell-to-cell dye transfer, this requirement does not appear to necessitate a change in the phosphorylation state of this residue. 
Discussion
This study was undertaken to elucidate the mechanism by which purinergic receptor activation by ATP effects a reduction in Cx43-mediated cell-to-cell communication, as assessed by measuring cell-to-cell transfer of the dye AF594. In this study, ATP-mediated purinergic receptor activation resulted in the regulation of Cx43 gap junctions via a PKC-dependent signal transduction pathway in HeLa cells, an immortalized epithelial cell line. That we have demonstrated a similar pathway in a lens epithelial primary cell culture system 8 15 indicates the likely general nature of this mechanism of Cx43 regulation in different cell types (Fig. 7) . Furthermore, we identified serine 368 in the C terminus of Cx43, as an essential component in the mechanism leading to the decrease in Cx43-mediated cell-to-cell communication after purinergic receptor activation. Agonist hydrolysis and the subsequent dephosphorylation of yet to be identified PKC substrate(s) determine the duration of the decrease in Cx43-mediated cell-to-cell communication. 
To confirm that ATP rather than a hydrolysis product of this nucleotide is indeed the agonist for this physiologic Cx43 regulatory pathway in HeLa cells, experiments were conducted using either the hydrolysis-resistant ATP AMP-PNP 31 34 or the ectonucleotidase inhibitor ARL. 33 That the inhibition of Cx43-mediated cell-to-cell communication was sustained when either the hydrolysis-resistant analogue was substituted for ATP or the ectonucleotidase inhibitor was included before addition of ATP is consistent with results with lens cell primary cultures indicating that ATP itself is the agonist responsible for the transient closure of gap junctions in lens epithelial cells. 8 Furthermore, in the absence of agonist hydrolysis, the observed reduction in Cx43-mediated cell-to-cell communication was now sustained, consistent with agonist removal by hydrolysis, rather than purinergic receptor desensitization or internalization, as proposed by others. 44  
The mechanism by which ATP effects this delayed, transient inhibition of Cx43-mediated cell-to-cell communication at the level of the gap junction is unknown. Because the period between the decrease in cell-to-cell communication after the initial purinergic receptor activation by ATP and the return of cell-to-cell dye transfer to pre-ATP levels was approximately 20 minutes and Cx43 gap junction turnover rates are in the range of several hours, 45 it appears unlikely that ATP effected the internalization of Cx43-containing gap junctions from the plasma membrane and their subsequent replacement with newly synthesized Cx43 gap junctions within this relatively short period. A second possible mechanism, which fits the observed time course more accurately, was that the phosphorylation status of Cx43 serine residue(s) determined the permeability of Cx43 gap junctions. There are constitutively active protein phosphatases in most cell types that can dephosphorylate Cx43. 46 47 Protein phosphatase (PP) 1 and PP2A, expressed in both the ocular lens cells 48 and the HeLa cells, 49 50 51 have been implicated in Cx43 dephosphorylation and are inhibited by OA. 47 Thus, the sustained reduction of Cx43 cell-to-cell dye transfer after purinergic receptor activation in the presence of OA is consistent with either PP1 or -2A being responsible for this dephosphorylation of a Cx43 phospho substrate and the reopening of Cx43 gap junctions after ATP addition in the absence of OA. 
Because the ATP-dependent reduction in cell-to-cell communication was sustained in the presence of OA, these experiments also delineate a downstream mechanism for the reversibility of this Cx43 gap junction regulatory pathway. Indeed, that the Cx43 residue 368 serine-to-alanine point mutation was now insensitive to the purinergic receptor-mediated activation of PKC and inhibition of cell-to-cell communication provides strong support for the proposal that a serine at residue 368 is absolutely essential for this effect of purinergic agonists on the permeability of Cx43 gap junctions. However, in the present study (Fig. 6)Ser 368 was not necessary as a PKC substrate per se, but rather appeared to play some type of structural role in effecting the permeability of Cx43 gap junctions. Thus, PKC must be phosphorylating other substrates (possibly within Cx43) that require a partially phosphorylated serine 368 to mediate this action of purinergic agonists on Cx43 permeability. Together, these data demonstrate that protein dephosphorylation, and not gap junction turnover, is most likely responsible for this agonist-dependent reduction in cell-to-cell communication. 
That this pathway is common in lens epithelial cells, 8 15 astrocytes, 44 and HeLa cells, as demonstrated in this study, suggests that gap junction regulation by this purinergic receptor pathway may be a conserved physiologic mechanism for regulating gap junctions. Indeed, that such regulation is not unique either to this receptor or to Cx43-containing gap junctions is indicated by the recent observations that histamine effects a similar PKC-mediated reduction of gap junctional communication between human tonsil high endothelial cells and that metabotropic glutamate receptor activation of inhibitory neurons in the mammalian thalamus and neocortex effect a reduction of electrical communication between these cell types that is mediated by Cx36. 52  
There is considerable evidence that ATP is present in the aqueous humor fluid secreted from the ciliary epithelium and that this aqueous humor bathes the avascular tissue of the anterior segment of the eye that includes the lens, cornea, and trabecular meshwork. 53 Furthermore, stress stimuli have been shown to increase the amount of ATP released into this aqueous humor. 28 54 That changes in ATP concentration in the aqueous humor could be sensed by the lens is indicated by our previous identification of purinergic receptors in epithelial cell cultures derived from the lens epithelial region, 55 and by Collison and Duncan, 56 who have shown in the intact lens that these receptors are more abundant in the epithelial cells in the lens equatorial region than in the central anterior region. 
The data herein provide evidence of an agonist-mediated pathway by which Cx43-containing gap junctions would be regulated in the epithelial cell layer of the intact lens where they play an important role in maintaining the unique lens microcirculatory system that has been proposed by Mathias et al. 57 as maintaining fiber cell homeostasis and therefore lens transparency. 58 In this, a standing flow of ionic current is directed inward at the poles and outward at the equator. 57 The lens equatorial cells have been proposed to play an essential role in mediating the cell-to-cell flow of electrical current carried primarily by Na+ toward the lens surface via gap junction channels that are concentrated at the equator. 58 Thus, the intracellular current is highly concentrated at the lens equator where it leaves the lens such that the net current flow is outward, whereas at the poles there is very little intracellular current, and so the current flow is inward along the interstices of the extracellular spaces. This current creates a net flux of solute that generates a fluid flow inward at the poles and outward at the equatorial epithelia cells. 
The lens microcirculation is driven by differences in cellular electromotive potentials to move water and solutes 58 ; thus, a change in the osmolarity of the aqueous humor could disrupt this flow, changing the regulatory volume of the lens fiber cells, leading to either cellular swelling or a reduction in cell volume. This change in cell volume may reflect a change in intracellular solute concentrations. For example, any decrease in fiber cell volume would require an increased influx of water and a decreased epithelial cell osmolyte permeability to restore normal cell volume and osmolarity. Conditions of stress that lead to a reduction in fiber cell volume would also be likely to affect ciliary epithelial cells, leading to a secretion of ATP into the aqueous humor. Binding of ATP to purinergic receptors on the equatorial lens epithelial cells would result in a PKC-mediated decrease in gap junction permeability of these Cx43-containing epithelia cells, helping restore osmolyte levels in the lens and in turn fiber cell volume. Certainly other channels in cells in other regions of the lens must also play a role in maintaining lens osmolarity. However, with the ability of ciliary epithelial cells to increase ATP levels in the aqueous humor in response to stress, 28 54 the preferential localization of purinergic receptors in the Cx43-containing equatorial epithelial cells of the intact lens, 56 and the ability of ATP to effect a sustained reduction of Cx43-mediated gap junction communication (Fig. 3) , the eye has all the components required to effect this temporal regulation of the circulating lens current, indicating that this purinergic system probably plays a significant role in maintaining lens homeostasis. 
Besides providing a protective mechanism in the lens, the inhibition of Cx43 after purinergic receptor activation may also play a significant role in other tissues. For example, it has been demonstrated in the heart that purinergic receptor activation 59 and altered Cx43-mediated gap junction communication 60 61 62 are associated with cardiac preconditioning, although the precise mechanism(s) by which this occurs has not been elucidated. Même et al. 44 postulated that purinergic receptor regulation of Cx43 plays a role in the normal physiologic regulation of astrocytes and a protective role under inflammatory conditions, albeit through a different subset of purinergic receptors and connexin protein. 
In conclusion, we have defined an ATP/purinergic receptor–mediated signal transduction pathway that is capable of reducing the Cx43-mediated cell-to-cell communication in Cx43-transfected HeLa cells and is conserved in lens epithelial cells. This novel mechanism of regulating Cx43 may play an important role in regulating the microcirculation that is essential for the movement of water and solutes in the intact lens. 
 
Figure 1.
 
HeLa cells stably transfected with connexin43, Cx43Δ257, or Cx43S368A expressed functional gap junctions. (A) Schematic representation of the full-length Cx43, indicating where the carboxyl terminus is truncated in Cx43Δ257 and the site of the Cx43S368A mutation. The electrophoretically fractionated membrane proteins isolated from HeLa cell cultures were immunostained with an antibody directed to the intracellular loop of Cx43 (B) or to the C terminus of Cx43 (C). (B) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from untransfected HeLa cells. Lane 3: membranes isolated from Cx43Δ257-transfected HeLa cells. (C) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from Cx43S368A-transfected HeLa cells.
Figure 1.
 
HeLa cells stably transfected with connexin43, Cx43Δ257, or Cx43S368A expressed functional gap junctions. (A) Schematic representation of the full-length Cx43, indicating where the carboxyl terminus is truncated in Cx43Δ257 and the site of the Cx43S368A mutation. The electrophoretically fractionated membrane proteins isolated from HeLa cell cultures were immunostained with an antibody directed to the intracellular loop of Cx43 (B) or to the C terminus of Cx43 (C). (B) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from untransfected HeLa cells. Lane 3: membranes isolated from Cx43Δ257-transfected HeLa cells. (C) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from Cx43S368A-transfected HeLa cells.
Figure 2.
 
CBX inhibited cell-to-cell dye transfer between stably transfected HeLa cells expressing Cx43, Cx43Δ257, or Cx43S368A. HeLa cells stably transfected with Cx43, Cx43Δ257, or Cx43S368A were visualized with Fura-2 AM at λex380 (A, C, F, H, K, M) and cell-to-cell dye transfer was imaged by using AF594 (B, D, G, I, L, N). (A–E) In Cx43-transfected HeLa cells, dye transferred from the injected cell to 19 cells in the absence of inhibitor (B) and to one cell in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (D). (E) Summary data for Cx43-mediated cell-to-cell dye transfer in the absence (21.8 ± 0.5 cells, n = 4; P ≪ 0.001) and the presence of CBX (1.0 ± 0.4 cells, n = 4; P ≪ 0.001). (F–I) In Cx43Δ257, transfected HeLa cells dye transferred from the injected cell to 18 cells in the absence of inhibitor (G) and to one cell in the presence of CBX (35 μM; 20 minutes) (I); (J) Summary data for Cx43Δ257-mediated cell-to-cell dye transfer in the absence (17.2 ± 0.5 cells, n = 5) and presence of CBX (0.6 ± 0.2 cells, n = 5; P ≪ 0.001). (K–N) In Cx43S368A-transfected HeLa cells dye transferred from the injected cell to 19 cells in the absence of inhibitor (L) and to 0 cells in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (N). (O) Summary data for Cx43S368A-mediated cell-to-cell dye transfer in the absence (19.8 ± 0.6 cells, n = 5) and presence (1.0 ± 0.5 cells, n = 8; P ≪ 0.001) of CBX.
Figure 2.
 
CBX inhibited cell-to-cell dye transfer between stably transfected HeLa cells expressing Cx43, Cx43Δ257, or Cx43S368A. HeLa cells stably transfected with Cx43, Cx43Δ257, or Cx43S368A were visualized with Fura-2 AM at λex380 (A, C, F, H, K, M) and cell-to-cell dye transfer was imaged by using AF594 (B, D, G, I, L, N). (A–E) In Cx43-transfected HeLa cells, dye transferred from the injected cell to 19 cells in the absence of inhibitor (B) and to one cell in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (D). (E) Summary data for Cx43-mediated cell-to-cell dye transfer in the absence (21.8 ± 0.5 cells, n = 4; P ≪ 0.001) and the presence of CBX (1.0 ± 0.4 cells, n = 4; P ≪ 0.001). (F–I) In Cx43Δ257, transfected HeLa cells dye transferred from the injected cell to 18 cells in the absence of inhibitor (G) and to one cell in the presence of CBX (35 μM; 20 minutes) (I); (J) Summary data for Cx43Δ257-mediated cell-to-cell dye transfer in the absence (17.2 ± 0.5 cells, n = 5) and presence of CBX (0.6 ± 0.2 cells, n = 5; P ≪ 0.001). (K–N) In Cx43S368A-transfected HeLa cells dye transferred from the injected cell to 19 cells in the absence of inhibitor (L) and to 0 cells in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (N). (O) Summary data for Cx43S368A-mediated cell-to-cell dye transfer in the absence (19.8 ± 0.6 cells, n = 5) and presence (1.0 ± 0.5 cells, n = 8; P ≪ 0.001) of CBX.
Figure 3.
 
Purinergic receptor activation by ATP effected a delayed, transient reduction in cell-to-cell dye transfer between Cx43 stably transfected HeLa cells. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 before and at various times after the addition of 25 μM ATP to the bathing medium. In the absence of ATP, AF594 dye transferred to 17.6 ± 0.5 cells (n = 14). At 1 minute after addition of ATP, dye transferred to 19.0 ± 0.4 cells (n = 6), at 5 minutes dye transfer was reduced (P < 0.01, Student's t-test) to 4.7 ± 0.5 cells (n = 7), and at 20 minutes it returned to the control level of 17.3 ± 0.5 cells (n = 8).
Figure 3.
 
Purinergic receptor activation by ATP effected a delayed, transient reduction in cell-to-cell dye transfer between Cx43 stably transfected HeLa cells. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 before and at various times after the addition of 25 μM ATP to the bathing medium. In the absence of ATP, AF594 dye transferred to 17.6 ± 0.5 cells (n = 14). At 1 minute after addition of ATP, dye transferred to 19.0 ± 0.4 cells (n = 6), at 5 minutes dye transfer was reduced (P < 0.01, Student's t-test) to 4.7 ± 0.5 cells (n = 7), and at 20 minutes it returned to the control level of 17.3 ± 0.5 cells (n = 8).
Table 1.
 
Purinergic Receptor-Mediated Regulation of Cx43 Biochemical Permeability
Table 1.
 
Purinergic Receptor-Mediated Regulation of Cx43 Biochemical Permeability
Treatment No ATP ATP (25 μM, 5 min)
Number of Cells Receiving Dye n Number of Cells Receiving Dye n
Control 16.7 ± 0.5 12 5.2 ± 0.6* 5
U73122 19.4 ± 0.8 5 19.8 ± 1.2 5
U73343 18.3 ± 0.5 3 3.7 ± 0.9 3
Stau 19.4 ± 0.5 7 19.2 ± 0.7 5
Bim-1 18.8 ± 0.4 5 18.6 ± 0.8 5
BAPTA 18.4 ± 0.5 5 4.8 ± 0.6* 5
Figure 4.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a nonhydrolyzable analogue of ATP or by ATP in the presence of an ecto-ATPase inhibitor. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 both before and at various times after the addition of 50 μM {conpict1}AMP-PNP to the bathing medium or after the addition of 25 μM ATP to the bathing medium in the presence of the ecto-ATPase inhibitor ARL-67156 (30 μM). (A) In the absence of AMP-PNP, AF594 dye transferred to 21.4 ± 0.7 cells (n = 7). At 5 minutes after addition of AMP-PNP, dye transfer was significantly reduced (P < 0.01, Student's t-test) to 2.4 ± 0.7 cells (n = 5) and remained significantly reduced for the duration of the experiment (20 minutes: 1.7 ± 0.7 cells, n = 3; 30 minutes: 1.6 ± 0.2 cells, n = 5). (B) Twenty minutes after the washout of AMP-PNP from the Cx43-transfected HeLa cell cultures in (A), cell-to cell dye transfer returned to basal levels (20.5 ± 1.3 cells, n = 4). Five minutes after the addition of ATP (25 μM) to the bathing medium, cell-to-cell dye transfer was reduced (P < 0.01, Student's t-test) to 0.8 ± 0.5 cells (n = 4), which returned to basal levels by 20 minutes after ATP addition (21.0 ± 1.3 cells, n = 4). (C) Inhibition of PKC by Bim-1 (100 nM) did not affect basal cell-to-cell dye transfer (22.2 ± 0.5 cells, n = 5), but did prevent the AMP-PNP-mediated decrease in cell-to-cell dye transfer for up to 30 minutes (22.2 ± 0.7 cells, n = 5). (D) In the absence of ARL, AF594 dye transferred to 19.4 ± 0.4 cells (n = 5). In the presence of 30 μM ARL (20–30 minutes), dye transfer was unchanged (20.2 ± 0.5 cells, n = 6). Five minutes after the addition of ATP (25 μM) in the presence of ARL (30 μM), dye transfer was significantly reduced (P < 0.01, Student's t-test) to 3.3 ± 0.7 cells (n = 3) and remained significantly reduced for the duration of the experiment (30 minutes: 2.4 ± 0.8 cells, n = 5).
Figure 4.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a nonhydrolyzable analogue of ATP or by ATP in the presence of an ecto-ATPase inhibitor. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 both before and at various times after the addition of 50 μM {conpict1}AMP-PNP to the bathing medium or after the addition of 25 μM ATP to the bathing medium in the presence of the ecto-ATPase inhibitor ARL-67156 (30 μM). (A) In the absence of AMP-PNP, AF594 dye transferred to 21.4 ± 0.7 cells (n = 7). At 5 minutes after addition of AMP-PNP, dye transfer was significantly reduced (P < 0.01, Student's t-test) to 2.4 ± 0.7 cells (n = 5) and remained significantly reduced for the duration of the experiment (20 minutes: 1.7 ± 0.7 cells, n = 3; 30 minutes: 1.6 ± 0.2 cells, n = 5). (B) Twenty minutes after the washout of AMP-PNP from the Cx43-transfected HeLa cell cultures in (A), cell-to cell dye transfer returned to basal levels (20.5 ± 1.3 cells, n = 4). Five minutes after the addition of ATP (25 μM) to the bathing medium, cell-to-cell dye transfer was reduced (P < 0.01, Student's t-test) to 0.8 ± 0.5 cells (n = 4), which returned to basal levels by 20 minutes after ATP addition (21.0 ± 1.3 cells, n = 4). (C) Inhibition of PKC by Bim-1 (100 nM) did not affect basal cell-to-cell dye transfer (22.2 ± 0.5 cells, n = 5), but did prevent the AMP-PNP-mediated decrease in cell-to-cell dye transfer for up to 30 minutes (22.2 ± 0.7 cells, n = 5). (D) In the absence of ARL, AF594 dye transferred to 19.4 ± 0.4 cells (n = 5). In the presence of 30 μM ARL (20–30 minutes), dye transfer was unchanged (20.2 ± 0.5 cells, n = 6). Five minutes after the addition of ATP (25 μM) in the presence of ARL (30 μM), dye transfer was significantly reduced (P < 0.01, Student's t-test) to 3.3 ± 0.7 cells (n = 3) and remained significantly reduced for the duration of the experiment (30 minutes: 2.4 ± 0.8 cells, n = 5).
Figure 5.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a protein phosphatase inhibitor, but was prevented by the deletion of a large portion of the C terminus of Cx43 or by a Cx43 C terminus mutation. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with wild-type Cx43, the C terminus deletion mutant Cx43Δ257, or the point mutant Cx43S368A. (A) In Cx43-transfected HeLa cells, AF594 dye transferred to 19.2 ± 0.5 cells (n = 5). Incubation with the protein phosphatase inhibitor OA (250 nM) did not affect cell-to-cell dye transfer (19.8 ± 0.7 cells, n = 6). Addition of ATP to the bathing medium resulted in a sustained, statistically significant decrease (P < 0.01, Student's t-test) in cell-to-cell dye transfer (5 minutes: 1.8 ± 0.8 cells, n = 4; 30 minutes: 1.4 ± 0.5 cells, n = 5). (B) In Cx43Δ257-transfected HeLa cells, AF594 dye transferred to 15.3 ± 0.5 (n = 24) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 17.0 ± 0.6 cells, n = 5; 5–10 minutes: 15.6 ± 0.8 cells, n = 9; 11–20 minutes: 14.6 ± 0.6 cells, n = 7; >20 minutes: 15.4 ± 0.4 cells, n = 5). (C) In Cx43S368A-transfected HeLa cells, AF594 dye transferred to 22.4 ± 1.0 cells (n = 11) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 23.0 ± 1.4 cells, n = 5; 5 minutes: 21.2 ± 1.6 cells, n = 5; 20 minutes: 25.0 ± 1.8 cells, n = 5).
Figure 5.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a protein phosphatase inhibitor, but was prevented by the deletion of a large portion of the C terminus of Cx43 or by a Cx43 C terminus mutation. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with wild-type Cx43, the C terminus deletion mutant Cx43Δ257, or the point mutant Cx43S368A. (A) In Cx43-transfected HeLa cells, AF594 dye transferred to 19.2 ± 0.5 cells (n = 5). Incubation with the protein phosphatase inhibitor OA (250 nM) did not affect cell-to-cell dye transfer (19.8 ± 0.7 cells, n = 6). Addition of ATP to the bathing medium resulted in a sustained, statistically significant decrease (P < 0.01, Student's t-test) in cell-to-cell dye transfer (5 minutes: 1.8 ± 0.8 cells, n = 4; 30 minutes: 1.4 ± 0.5 cells, n = 5). (B) In Cx43Δ257-transfected HeLa cells, AF594 dye transferred to 15.3 ± 0.5 (n = 24) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 17.0 ± 0.6 cells, n = 5; 5–10 minutes: 15.6 ± 0.8 cells, n = 9; 11–20 minutes: 14.6 ± 0.6 cells, n = 7; >20 minutes: 15.4 ± 0.4 cells, n = 5). (C) In Cx43S368A-transfected HeLa cells, AF594 dye transferred to 22.4 ± 1.0 cells (n = 11) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 23.0 ± 1.4 cells, n = 5; 5 minutes: 21.2 ± 1.6 cells, n = 5; 20 minutes: 25.0 ± 1.8 cells, n = 5).
Figure 6.
 
Purinergic receptor activation by AMP-PNP did not effect a change in immunolabeling of the serine 368 residue of Cx43 in Cx43 stably transfected HeLa cells. Western blot analysis of whole cell lysates from Cx43-transfected HeLa cells in the absence and presence of purinergic receptor activation with the ATP analogue AMP-PNP (50 μM, 20 minutes) was conducted. Phosphoserine 368 was detected by a p368-specific antibody. Lane 1: protein from AMP-PNP-treated cells; lane 2: protein from untreated cells.
Figure 6.
 
Purinergic receptor activation by AMP-PNP did not effect a change in immunolabeling of the serine 368 residue of Cx43 in Cx43 stably transfected HeLa cells. Western blot analysis of whole cell lysates from Cx43-transfected HeLa cells in the absence and presence of purinergic receptor activation with the ATP analogue AMP-PNP (50 μM, 20 minutes) was conducted. Phosphoserine 368 was detected by a p368-specific antibody. Lane 1: protein from AMP-PNP-treated cells; lane 2: protein from untreated cells.
Figure 7.
 
Model of the mechanism by which Cx43 gap junctions is regulated by purinergic receptor activation. Top: Purinergic receptor activation by ATP activates PKC through G-protein-coupled PLC and diacyl glycerol (DAG). Cx43 gap junctions become phosphorylated at serine 368, and biochemical coupling is significantly reduced. Bottom: Basal biochemical communication is restored after purinergic receptor activation as a result of PP's removing the phosphate group from Cx43 serine 368. At the purinergic receptor level, this second-messenger signaling system is turned off by the hydrolysis of ATP by endogenous ectonucleotidases (E-NTPDase).
Figure 7.
 
Model of the mechanism by which Cx43 gap junctions is regulated by purinergic receptor activation. Top: Purinergic receptor activation by ATP activates PKC through G-protein-coupled PLC and diacyl glycerol (DAG). Cx43 gap junctions become phosphorylated at serine 368, and biochemical coupling is significantly reduced. Bottom: Basal biochemical communication is restored after purinergic receptor activation as a result of PP's removing the phosphate group from Cx43 serine 368. At the purinergic receptor level, this second-messenger signaling system is turned off by the hydrolysis of ATP by endogenous ectonucleotidases (E-NTPDase).
The authors thank Paul Lampe and Joell Solan for assistance with the Cx43 p368 Western immunoblots and discussions regarding the data derived using this antibody and Sheela Thomas for assistance with the data. 
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Figure 1.
 
HeLa cells stably transfected with connexin43, Cx43Δ257, or Cx43S368A expressed functional gap junctions. (A) Schematic representation of the full-length Cx43, indicating where the carboxyl terminus is truncated in Cx43Δ257 and the site of the Cx43S368A mutation. The electrophoretically fractionated membrane proteins isolated from HeLa cell cultures were immunostained with an antibody directed to the intracellular loop of Cx43 (B) or to the C terminus of Cx43 (C). (B) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from untransfected HeLa cells. Lane 3: membranes isolated from Cx43Δ257-transfected HeLa cells. (C) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from Cx43S368A-transfected HeLa cells.
Figure 1.
 
HeLa cells stably transfected with connexin43, Cx43Δ257, or Cx43S368A expressed functional gap junctions. (A) Schematic representation of the full-length Cx43, indicating where the carboxyl terminus is truncated in Cx43Δ257 and the site of the Cx43S368A mutation. The electrophoretically fractionated membrane proteins isolated from HeLa cell cultures were immunostained with an antibody directed to the intracellular loop of Cx43 (B) or to the C terminus of Cx43 (C). (B) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from untransfected HeLa cells. Lane 3: membranes isolated from Cx43Δ257-transfected HeLa cells. (C) Lane 1: membranes isolated from Cx43-transfected HeLa cells. Lane 2: membranes isolated from Cx43S368A-transfected HeLa cells.
Figure 2.
 
CBX inhibited cell-to-cell dye transfer between stably transfected HeLa cells expressing Cx43, Cx43Δ257, or Cx43S368A. HeLa cells stably transfected with Cx43, Cx43Δ257, or Cx43S368A were visualized with Fura-2 AM at λex380 (A, C, F, H, K, M) and cell-to-cell dye transfer was imaged by using AF594 (B, D, G, I, L, N). (A–E) In Cx43-transfected HeLa cells, dye transferred from the injected cell to 19 cells in the absence of inhibitor (B) and to one cell in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (D). (E) Summary data for Cx43-mediated cell-to-cell dye transfer in the absence (21.8 ± 0.5 cells, n = 4; P ≪ 0.001) and the presence of CBX (1.0 ± 0.4 cells, n = 4; P ≪ 0.001). (F–I) In Cx43Δ257, transfected HeLa cells dye transferred from the injected cell to 18 cells in the absence of inhibitor (G) and to one cell in the presence of CBX (35 μM; 20 minutes) (I); (J) Summary data for Cx43Δ257-mediated cell-to-cell dye transfer in the absence (17.2 ± 0.5 cells, n = 5) and presence of CBX (0.6 ± 0.2 cells, n = 5; P ≪ 0.001). (K–N) In Cx43S368A-transfected HeLa cells dye transferred from the injected cell to 19 cells in the absence of inhibitor (L) and to 0 cells in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (N). (O) Summary data for Cx43S368A-mediated cell-to-cell dye transfer in the absence (19.8 ± 0.6 cells, n = 5) and presence (1.0 ± 0.5 cells, n = 8; P ≪ 0.001) of CBX.
Figure 2.
 
CBX inhibited cell-to-cell dye transfer between stably transfected HeLa cells expressing Cx43, Cx43Δ257, or Cx43S368A. HeLa cells stably transfected with Cx43, Cx43Δ257, or Cx43S368A were visualized with Fura-2 AM at λex380 (A, C, F, H, K, M) and cell-to-cell dye transfer was imaged by using AF594 (B, D, G, I, L, N). (A–E) In Cx43-transfected HeLa cells, dye transferred from the injected cell to 19 cells in the absence of inhibitor (B) and to one cell in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (D). (E) Summary data for Cx43-mediated cell-to-cell dye transfer in the absence (21.8 ± 0.5 cells, n = 4; P ≪ 0.001) and the presence of CBX (1.0 ± 0.4 cells, n = 4; P ≪ 0.001). (F–I) In Cx43Δ257, transfected HeLa cells dye transferred from the injected cell to 18 cells in the absence of inhibitor (G) and to one cell in the presence of CBX (35 μM; 20 minutes) (I); (J) Summary data for Cx43Δ257-mediated cell-to-cell dye transfer in the absence (17.2 ± 0.5 cells, n = 5) and presence of CBX (0.6 ± 0.2 cells, n = 5; P ≪ 0.001). (K–N) In Cx43S368A-transfected HeLa cells dye transferred from the injected cell to 19 cells in the absence of inhibitor (L) and to 0 cells in the presence of the gap junction inhibitor CBX (35 μM; 20 minutes) (N). (O) Summary data for Cx43S368A-mediated cell-to-cell dye transfer in the absence (19.8 ± 0.6 cells, n = 5) and presence (1.0 ± 0.5 cells, n = 8; P ≪ 0.001) of CBX.
Figure 3.
 
Purinergic receptor activation by ATP effected a delayed, transient reduction in cell-to-cell dye transfer between Cx43 stably transfected HeLa cells. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 before and at various times after the addition of 25 μM ATP to the bathing medium. In the absence of ATP, AF594 dye transferred to 17.6 ± 0.5 cells (n = 14). At 1 minute after addition of ATP, dye transferred to 19.0 ± 0.4 cells (n = 6), at 5 minutes dye transfer was reduced (P < 0.01, Student's t-test) to 4.7 ± 0.5 cells (n = 7), and at 20 minutes it returned to the control level of 17.3 ± 0.5 cells (n = 8).
Figure 3.
 
Purinergic receptor activation by ATP effected a delayed, transient reduction in cell-to-cell dye transfer between Cx43 stably transfected HeLa cells. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 before and at various times after the addition of 25 μM ATP to the bathing medium. In the absence of ATP, AF594 dye transferred to 17.6 ± 0.5 cells (n = 14). At 1 minute after addition of ATP, dye transferred to 19.0 ± 0.4 cells (n = 6), at 5 minutes dye transfer was reduced (P < 0.01, Student's t-test) to 4.7 ± 0.5 cells (n = 7), and at 20 minutes it returned to the control level of 17.3 ± 0.5 cells (n = 8).
Figure 4.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a nonhydrolyzable analogue of ATP or by ATP in the presence of an ecto-ATPase inhibitor. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 both before and at various times after the addition of 50 μM {conpict1}AMP-PNP to the bathing medium or after the addition of 25 μM ATP to the bathing medium in the presence of the ecto-ATPase inhibitor ARL-67156 (30 μM). (A) In the absence of AMP-PNP, AF594 dye transferred to 21.4 ± 0.7 cells (n = 7). At 5 minutes after addition of AMP-PNP, dye transfer was significantly reduced (P < 0.01, Student's t-test) to 2.4 ± 0.7 cells (n = 5) and remained significantly reduced for the duration of the experiment (20 minutes: 1.7 ± 0.7 cells, n = 3; 30 minutes: 1.6 ± 0.2 cells, n = 5). (B) Twenty minutes after the washout of AMP-PNP from the Cx43-transfected HeLa cell cultures in (A), cell-to cell dye transfer returned to basal levels (20.5 ± 1.3 cells, n = 4). Five minutes after the addition of ATP (25 μM) to the bathing medium, cell-to-cell dye transfer was reduced (P < 0.01, Student's t-test) to 0.8 ± 0.5 cells (n = 4), which returned to basal levels by 20 minutes after ATP addition (21.0 ± 1.3 cells, n = 4). (C) Inhibition of PKC by Bim-1 (100 nM) did not affect basal cell-to-cell dye transfer (22.2 ± 0.5 cells, n = 5), but did prevent the AMP-PNP-mediated decrease in cell-to-cell dye transfer for up to 30 minutes (22.2 ± 0.7 cells, n = 5). (D) In the absence of ARL, AF594 dye transferred to 19.4 ± 0.4 cells (n = 5). In the presence of 30 μM ARL (20–30 minutes), dye transfer was unchanged (20.2 ± 0.5 cells, n = 6). Five minutes after the addition of ATP (25 μM) in the presence of ARL (30 μM), dye transfer was significantly reduced (P < 0.01, Student's t-test) to 3.3 ± 0.7 cells (n = 3) and remained significantly reduced for the duration of the experiment (30 minutes: 2.4 ± 0.8 cells, n = 5).
Figure 4.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a nonhydrolyzable analogue of ATP or by ATP in the presence of an ecto-ATPase inhibitor. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with Cx43 both before and at various times after the addition of 50 μM {conpict1}AMP-PNP to the bathing medium or after the addition of 25 μM ATP to the bathing medium in the presence of the ecto-ATPase inhibitor ARL-67156 (30 μM). (A) In the absence of AMP-PNP, AF594 dye transferred to 21.4 ± 0.7 cells (n = 7). At 5 minutes after addition of AMP-PNP, dye transfer was significantly reduced (P < 0.01, Student's t-test) to 2.4 ± 0.7 cells (n = 5) and remained significantly reduced for the duration of the experiment (20 minutes: 1.7 ± 0.7 cells, n = 3; 30 minutes: 1.6 ± 0.2 cells, n = 5). (B) Twenty minutes after the washout of AMP-PNP from the Cx43-transfected HeLa cell cultures in (A), cell-to cell dye transfer returned to basal levels (20.5 ± 1.3 cells, n = 4). Five minutes after the addition of ATP (25 μM) to the bathing medium, cell-to-cell dye transfer was reduced (P < 0.01, Student's t-test) to 0.8 ± 0.5 cells (n = 4), which returned to basal levels by 20 minutes after ATP addition (21.0 ± 1.3 cells, n = 4). (C) Inhibition of PKC by Bim-1 (100 nM) did not affect basal cell-to-cell dye transfer (22.2 ± 0.5 cells, n = 5), but did prevent the AMP-PNP-mediated decrease in cell-to-cell dye transfer for up to 30 minutes (22.2 ± 0.7 cells, n = 5). (D) In the absence of ARL, AF594 dye transferred to 19.4 ± 0.4 cells (n = 5). In the presence of 30 μM ARL (20–30 minutes), dye transfer was unchanged (20.2 ± 0.5 cells, n = 6). Five minutes after the addition of ATP (25 μM) in the presence of ARL (30 μM), dye transfer was significantly reduced (P < 0.01, Student's t-test) to 3.3 ± 0.7 cells (n = 3) and remained significantly reduced for the duration of the experiment (30 minutes: 2.4 ± 0.8 cells, n = 5).
Figure 5.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a protein phosphatase inhibitor, but was prevented by the deletion of a large portion of the C terminus of Cx43 or by a Cx43 C terminus mutation. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with wild-type Cx43, the C terminus deletion mutant Cx43Δ257, or the point mutant Cx43S368A. (A) In Cx43-transfected HeLa cells, AF594 dye transferred to 19.2 ± 0.5 cells (n = 5). Incubation with the protein phosphatase inhibitor OA (250 nM) did not affect cell-to-cell dye transfer (19.8 ± 0.7 cells, n = 6). Addition of ATP to the bathing medium resulted in a sustained, statistically significant decrease (P < 0.01, Student's t-test) in cell-to-cell dye transfer (5 minutes: 1.8 ± 0.8 cells, n = 4; 30 minutes: 1.4 ± 0.5 cells, n = 5). (B) In Cx43Δ257-transfected HeLa cells, AF594 dye transferred to 15.3 ± 0.5 (n = 24) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 17.0 ± 0.6 cells, n = 5; 5–10 minutes: 15.6 ± 0.8 cells, n = 9; 11–20 minutes: 14.6 ± 0.6 cells, n = 7; >20 minutes: 15.4 ± 0.4 cells, n = 5). (C) In Cx43S368A-transfected HeLa cells, AF594 dye transferred to 22.4 ± 1.0 cells (n = 11) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 23.0 ± 1.4 cells, n = 5; 5 minutes: 21.2 ± 1.6 cells, n = 5; 20 minutes: 25.0 ± 1.8 cells, n = 5).
Figure 5.
 
Purinergic receptor–mediated reduction in cell-to-cell dye transfer was sustained in the presence of a protein phosphatase inhibitor, but was prevented by the deletion of a large portion of the C terminus of Cx43 or by a Cx43 C terminus mutation. Cell-to-cell dye transfer was measured in HeLa cells stably transfected with wild-type Cx43, the C terminus deletion mutant Cx43Δ257, or the point mutant Cx43S368A. (A) In Cx43-transfected HeLa cells, AF594 dye transferred to 19.2 ± 0.5 cells (n = 5). Incubation with the protein phosphatase inhibitor OA (250 nM) did not affect cell-to-cell dye transfer (19.8 ± 0.7 cells, n = 6). Addition of ATP to the bathing medium resulted in a sustained, statistically significant decrease (P < 0.01, Student's t-test) in cell-to-cell dye transfer (5 minutes: 1.8 ± 0.8 cells, n = 4; 30 minutes: 1.4 ± 0.5 cells, n = 5). (B) In Cx43Δ257-transfected HeLa cells, AF594 dye transferred to 15.3 ± 0.5 (n = 24) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 17.0 ± 0.6 cells, n = 5; 5–10 minutes: 15.6 ± 0.8 cells, n = 9; 11–20 minutes: 14.6 ± 0.6 cells, n = 7; >20 minutes: 15.4 ± 0.4 cells, n = 5). (C) In Cx43S368A-transfected HeLa cells, AF594 dye transferred to 22.4 ± 1.0 cells (n = 11) in the absence of ATP. Dye transfer remained unchanged after addition of ATP (1 minute: 23.0 ± 1.4 cells, n = 5; 5 minutes: 21.2 ± 1.6 cells, n = 5; 20 minutes: 25.0 ± 1.8 cells, n = 5).
Figure 6.
 
Purinergic receptor activation by AMP-PNP did not effect a change in immunolabeling of the serine 368 residue of Cx43 in Cx43 stably transfected HeLa cells. Western blot analysis of whole cell lysates from Cx43-transfected HeLa cells in the absence and presence of purinergic receptor activation with the ATP analogue AMP-PNP (50 μM, 20 minutes) was conducted. Phosphoserine 368 was detected by a p368-specific antibody. Lane 1: protein from AMP-PNP-treated cells; lane 2: protein from untreated cells.
Figure 6.
 
Purinergic receptor activation by AMP-PNP did not effect a change in immunolabeling of the serine 368 residue of Cx43 in Cx43 stably transfected HeLa cells. Western blot analysis of whole cell lysates from Cx43-transfected HeLa cells in the absence and presence of purinergic receptor activation with the ATP analogue AMP-PNP (50 μM, 20 minutes) was conducted. Phosphoserine 368 was detected by a p368-specific antibody. Lane 1: protein from AMP-PNP-treated cells; lane 2: protein from untreated cells.
Figure 7.
 
Model of the mechanism by which Cx43 gap junctions is regulated by purinergic receptor activation. Top: Purinergic receptor activation by ATP activates PKC through G-protein-coupled PLC and diacyl glycerol (DAG). Cx43 gap junctions become phosphorylated at serine 368, and biochemical coupling is significantly reduced. Bottom: Basal biochemical communication is restored after purinergic receptor activation as a result of PP's removing the phosphate group from Cx43 serine 368. At the purinergic receptor level, this second-messenger signaling system is turned off by the hydrolysis of ATP by endogenous ectonucleotidases (E-NTPDase).
Figure 7.
 
Model of the mechanism by which Cx43 gap junctions is regulated by purinergic receptor activation. Top: Purinergic receptor activation by ATP activates PKC through G-protein-coupled PLC and diacyl glycerol (DAG). Cx43 gap junctions become phosphorylated at serine 368, and biochemical coupling is significantly reduced. Bottom: Basal biochemical communication is restored after purinergic receptor activation as a result of PP's removing the phosphate group from Cx43 serine 368. At the purinergic receptor level, this second-messenger signaling system is turned off by the hydrolysis of ATP by endogenous ectonucleotidases (E-NTPDase).
Table 1.
 
Purinergic Receptor-Mediated Regulation of Cx43 Biochemical Permeability
Table 1.
 
Purinergic Receptor-Mediated Regulation of Cx43 Biochemical Permeability
Treatment No ATP ATP (25 μM, 5 min)
Number of Cells Receiving Dye n Number of Cells Receiving Dye n
Control 16.7 ± 0.5 12 5.2 ± 0.6* 5
U73122 19.4 ± 0.8 5 19.8 ± 1.2 5
U73343 18.3 ± 0.5 3 3.7 ± 0.9 3
Stau 19.4 ± 0.5 7 19.2 ± 0.7 5
Bim-1 18.8 ± 0.4 5 18.6 ± 0.8 5
BAPTA 18.4 ± 0.5 5 4.8 ± 0.6* 5
×
×

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