Selective Permeability of Different Connexin Channels to the Second Messenger Cyclic AMP
2005; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m511235200
ISSN1083-351X
AutoresPeter Bedner, Heiner Niessen, Benjamin Odermatt, Markus Kretz, Klaus Willecke, Hartmann Harz,
Tópico(s)Ion channel regulation and function
ResumoGap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern. Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern. Gap junction channels formed by docking of two hemichannels in the plasma membranes of contacting cells mediate the exchange of small molecules (<1 kDa) and ions between cells. Hemichannels are formed by six protein subunits called connexins (Cx). 3The abbreviations used are: Cx, connexin; CNG, cyclic nucleotide-gated; IP3, inositol 1,4,5-trisphosphate; PBS, phosphate-buffered saline; DiI, 1-1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; NPE, P1-(2-nitrophenyl)ethyl ester; HBS, Hepes-buffered saline; [Ca2+]i, cytoplasmatic calcium concentration; [cAMP]i, cytoplasmatic cAMP concentration; pS, picosiemens. Gap junction-mediated intercellular communication is thought to play a crucial role in maintenance of homeostasis, morphogenesis, cell differentiation and growth control in multicellular organisms (1Willecke K. Eiberger J. Degen J. Eckardt D. Romualdi A. Guldenagel M. Deutsch U. Sohl G. Biol. Chem. 2002; 383: 725-737Crossref PubMed Scopus (1007) Google Scholar). To date, 20 mouse and 21 human cx genes have been tentatively identified (2Söhl G. Willecke K. Cell Commun. Adhes. 2003; 10: 173-180Crossref PubMed Scopus (375) Google Scholar). It has been shown that each cx gene is expressed in a distinct spatial and temporal pattern and that most cell types express more than one connexin protein. The diverse expression patterns of the different connexins suggest functional differences, including selective permeability for biologically relevant molecules such as second messengers. Selective permeability of different Cx channels to ions, fluorescent dyes, metabolites, and the second messenger inositol 1,4,5-trisphosphate (IP3) has been described (3Elfgang C. Eckert R. Lichtenberg-Frate H. Butterweck A. Traub O. Klein R.A. Hülser D.F. Willecke K. J. Cell Biol. 1995; 129: 805-817Crossref PubMed Scopus (734) Google Scholar, 4Veenstra R.D. J. Bioenerg. Biomembr. 1996; 28: 327-337Crossref PubMed Scopus (203) Google Scholar, 5Cao F. Eckert R. Elfgang C. Nitsche J.M. Snyder S.A. Hulser D.F. Willecke K. Nicholson B.J. J. Cell Sci. 1998; 111: 31-43Crossref PubMed Google Scholar, 6Goldberg G.S. Lampe P.D. Nicholson B.J. Nat. Cell Biol. 1999; 1: 457-459Crossref PubMed Scopus (269) Google Scholar, 7Goldberg G.S. Moreno A.P. Lampe P.D. J. Biol. Chem. 2002; 277: 36725-36730Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Niessen H. Harz H. Bedner P. Kramer K. Willecke K. J. Cell Sci. 2000; 113: 1365-1372PubMed Google Scholar, 9Weber P.A. Chang H.C. Spaeth K.E. Nitsche J.M. Nicholson B.J. Biophys. J. 2004; 87: 958-973Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). For instance, Niessen et al. (8Niessen H. Harz H. Bedner P. Kramer K. Willecke K. J. Cell Sci. 2000; 113: 1365-1372PubMed Google Scholar) showed by microinjection of IP3 into monolayers of different HeLa transfectants that Cx32 channels were able to propagate IP3-induced Ca2+ waves 2.5 times better than Cx43 channels and 3-4 times better than Cx26 channels. Cyclic AMP (cAMP) is a ubiquitous intracellular second messenger that affects cell physiology by directly interacting with effector molecules that include cAMP-dependent protein kinases, cyclic nucleotide-gated ion channels (CNG channels), and hyperpolarization activated channels. In turn, these effectors regulate diverse biological processes such as cardiac inotropy and chronotropy, glycogenolysis and lipolysis, vascular tone, neurotransmitter and hormone release as well as cell growth and differentiation (for review, see Refs. 10Dremier S. Kopperud R. Doskeland S.O. Dumont J.E. Maenhaut C. FEBS Lett. 2003; 354: 103-107Crossref Scopus (62) Google Scholar, 11Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (261) Google Scholar, 12Kaupp U.B. Seifert R. Physiol. Rev. 2002; 82: 769-824Crossref PubMed Scopus (943) Google Scholar). The permeability of gap junction channels for cAMP was already demonstrated more than 30 years ago (13Tsien R.W. Weingart R. J. Physiol. (Lond.). 1974; 242: 95-96Google Scholar) and confirmed in several subsequent studies (14Lawrence T.S. Beers W.H. Gilula N.B. Nature. 1978; 272: 501-506Crossref PubMed Scopus (379) Google Scholar, 15Murray S.A. Fletcher W.H. J. 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For this purpose, we established six HeLa cell lines, each of them stably expressing one type of Cx protein and mutant CNG ion channels (T537S rCNGα3), which were used as highly sensitive sensors for cAMP concentrations (19Bedner P. Niessen H. Odermatt B. Willecke K. Harz H. Exp. Cell Res. 2003; 291: 25-35Crossref PubMed Scopus (32) Google Scholar). With this method we show here that Cx43 channels are about 3 times more permeable to cAMP than Cx26 channels, 5-6 times more permeable than Cx32 or Cx45 channels, 8-9 times more permeable than Cx47 channels, and more than 30 times more permeable than Cx36 channels. Cell Culture and Transfection—For transfection, DNA coding for the T537S-mutated form of the rat olfactory CNG channel α3-subunit (T537S rCNGα3) (20Altenhofen W. Ludwig J. Eismann E. Kraus W. Bonigk W. Kaupp U.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9868-9872Crossref PubMed Scopus (134) Google Scholar) was inserted into the transfection vector pcDNA3.1/Zeo(+). HeLa wild type cells and the connexin channel expressing cell lines HeLa-Cx26C, -Cx32H, -Cx36K5, -Cx43K7, -Cx45A, and -Cx47K21 (3Elfgang C. Eckert R. Lichtenberg-Frate H. Butterweck A. Traub O. Klein R.A. Hülser D.F. Willecke K. J. Cell Biol. 1995; 129: 805-817Crossref PubMed Scopus (734) Google Scholar, 21Butterweck A. Gergs U. Elfgang C. Willecke K. Traub O. J. Membr. Biol. 1994; 141: 247-256Crossref PubMed Scopus (78) Google Scholar, 22Teubner B. Degen J. Sohl G. Guldenagel M. Bukauskas F.F. Trexler E.B. Verselis V.K. De Zeeuw C.I. Lee C.G. Kozak C.A. Petrasch-Parwez E. Dermietzel R. Willecke K. J. Membr. Biol. 2000; 176: 249-262Crossref PubMed Scopus (197) Google Scholar, 23Teubner B. Odermatt B. Güldenagel M. Sohl G. Degen J. Bukauskas F.F. Kronengold J. Verselis V.K. Jung Y.T. Kozak C.A. Schilling K. Willecke K. J. Neurosci. 2001; 21: 1117-1126Crossref PubMed Google Scholar, 24Traub O. Eckert R. Lichtenberg-Frate H. Elfgang C. Bastide B. Scheidtmann K.H. Hülser D.F. Willecke K. Eur. J. Cell Biol. 1994; 64: 101-112PubMed Google Scholar) were stably transfected by lipofection (Tfx™-50 reagent, Promega, Madison, WI) following the protocol provided by the manufacturer. Forty-eight hours after transfection, 100 μg/ml zeocin were added to the medium. Clones were picked after 3-4 weeks and grown under selective conditions. The cells were cultured as previously described (19Bedner P. Niessen H. Odermatt B. Willecke K. Harz H. Exp. Cell Res. 2003; 291: 25-35Crossref PubMed Scopus (32) Google Scholar). For experiments, cells were plated at low density on glass coverslips placed in a 35-mm plastic Petri dish. Cells were grown for 24-36 h and used for experiments at a confluence level of 10-20%. For cocultures, HeLa-Cx45/CNG transfectants were plated on a 35-mm plastic dish and grown to confluency. The cells were then incubated with isotonic glucose solution containing 5 μg/ml DiI (Molecular Probes, Eugene, OR) for 15 min at 37 °C. After two washes with phosphate-buffered saline (PBS), the DiI-labeled HeLa-Cx/CNG double transfectants and unstained HeLa-Cx45-transfected cells were trypsinized, centrifuged, and resuspended. DiI-labeled cells were mixed with unlabeled cells at a ratio of 1:1, plated onto glass coverslips, and grown for 48 h. Immunofluorescence Analyses—Cells grown for 24 h on coverslips were fixed in 100% ethanol (-20 °C) for 5 min, blocked with 4% bovine serum albumin (BSA, PAA Laboratories GmbH, Linz, Austria), PBS and incubated with antibodies diluted in 0.4% BSA in PBS overnight at 4 °C. Afterward, cells were washed with 0.4% bovine serum albumin in PBS and incubated with Alexa-conjugated antibodies directed to primary antibodies for 1 h at room temperature. Cells were stained with the following dilutions of primary antibodies: rabbit polyclonal anti-Cx43 (1:2000) (24Traub O. Eckert R. Lichtenberg-Frate H. Elfgang C. Bastide B. Scheidtmann K.H. Hülser D.F. Willecke K. Eur. J. Cell Biol. 1994; 64: 101-112PubMed Google Scholar), polyclonal rabbit anti-Cx26 (1:500; Zytomed, Berlin, Germany), polyclonal rabbit anti-Cx32 (1:250; Zytomed), monoclonal mouse anti-Cx45 (1:100; Chemicon), polyclonal rabbit anti-Cx36 (1:100; Zytomed), and polyclonal guinea pig anti-Cx47 (1:500) (25Odermatt B. Wellershaus K. Wallraff A. Seifert G. Degen J. Euwens C. Fuss B. Bussow H. Schilling K. Steinhauser C. Willecke K. J. Neurosci. 2003; 23: 4549-4559Crossref PubMed Google Scholar). Primary antibodies were detected with Alexa488-conjugated goat anti-rabbit immunoglobulin (1:2000; MoBiTech, Goettingen, Germany), Alexa488 goat anti-mouse (1:2000; MoBiTech), and Alexa594 goat anti-guinea pig (1:3000; MoBiTech), respectively. Nuclear staining was performed by 15 min of incubation with 0.2 μg/ml Hoechst 33258 fluorescent dye in PBS (Sigma). Cells were mounted with fluorescent mounting medium (Dako, Glostrup, Denmark). Antibody-stained cells were analyzed using the photomicroscope Axiophot (Zeiss, Jena, Germany). Fluorometric Measurements and Flash Photolysis—If not stated otherwise, cells were loaded with 8 μm Fluo-4FF (Molecular Probes), 200 μm cyclic AMP, P1-(2-nitrophenyl)ethyl ester (NPE-caged cAMP, Calbiochem), and 0.025% PLURONIC F127 (Molecular Probes) in Hepes-buffered saline (HBS) containing 140 mm NaCl, 5 mm KCl, 3 mm CaCl2, 1mm MgCl2, 10 mm Hepes, and 10 mm glucose, pH 7.4 (NaOH) for 45 min at 37 °C. After loading, cells were rinsed with HBS without MgCl2 (HBS-), the coverslip was transferred to the experimental chamber, filled with 500 μl of HBS- containing 200 μm caged cAMP, and mounted onto an inverse epifluorescence microscope (Axiovert 100, Zeiss Oberkochen, Germany). Fluorescence measurements were performed using a commercial imaging system (TILL Photonics, Gräfelfing, Germany) consisting of a monochromator (Polychrom I) and a peltier-cooled 12 bit charged coupled device (CCD) camera connected to a personal computer equipped with the calcium imaging software Fucal (Version 5.12C) (26Messler P. Harz H. Uhl R. J. Neurosci. Methods. 1996; 69: 137-147Crossref PubMed Scopus (30) Google Scholar). The monochromatic light was coupled via an epifluorescence condenser (dual port) to the microscope. All CCD images were background-corrected by subtraction of a dark picture. For photolysis experiments, a high pressure mercury lamp (Osram HBO 100W/2) equipped with a shutter (LS6, uniblitz, Rochester, NY) and a short pass filter (SP410) was used. The UV light from the mercury lamp was connected via the second port of the epifluorescence condenser to the microscope and focused to a rectangular area (30 × 30 μm) in the object plane. UV light pulses of 200-ms duration were used for the photolysis of all caged cAMP molecules in one cell. For the normalization procedure, UV light pulses of 2.5 and 4.5 ms duration were used. The average increase of the fluorescence intensity evoked by these light pulses was found to be about 40 and 60%, respectively, of maximum. To obtain reproducible results, the UV light intensity was measured by a UV-sensitive photodiode and held constant by regular readjustment. The light of the two lamps was combined in the dual port condenser by a dichroic mirror (Fura) and reflected into the objective by a second dichroic mirror (fluorescein isothiocyanate). For [Ca2+]i measurements, Fluo-4FF fluorescence was monitored at 470 nm excitation wavelength. In most experiments, 20 frames were recorded at a frequency of 1 Hz, with the first frame preceding the UV flash. The average pixel fluorescence within regions of interest of about 10 × 10 μmwas used for further analyses. To increase the sensitivity of the experiments performed on cocultures, Fluo-4 (Ca2+ binding constant, 345 nm; Molecular Probes) was used instead of the Fluo-4FF (Ca2+ binding constant, 9.7 μm). Additionally, a more sensitive CCD camera (Sensicam QE, PCO Kelheim, Germany) was used during these experiments. The sensitivity was further increased by combining an appropriate operation mode (analog gain, on; low light mode, on) with the 2 × 2 on chip binning option. Electrical Recordings—Pipettes were pulled from borosilicate glass capillaries (1.5 mm outer diameter × 0.86 mm inner diameter, Harvard Apparatus LTD, Edenbridge, UK) with a horizontal pipette puller (Sutter Instruments Inc., Eugene, OR) and filled with 140 mm KCl, 2 mm MgCl2, 1 mm CaCl2, 11 mm EGTA, 10 mm Hepes, pH 7.2 (KOH). The resistance of patch pipettes was measured to be in the range of 2-4 megaohms. Pipettes were lowered onto the cells by a motor-driven micromanipulator (SM1, Luigs & Neumann, Ratingen, Germany). All experiments were performed at room temperature (∼22 °C). Double whole-cell patch clamp measurements were performed on cell pairs bathed in HBS. Two patch pipettes were connected to two synchronized, single-electrode voltage clamp amplifiers (SEC-05LX, NPI Electronic, Tamm, Germany). These amplifiers switch between voltage measurement and current injection, thereby avoiding artifacts by the series resistance (27Muller A. Lauven M. Berkels R. Dhein S. Polder H.R. Klaus W. Am. J. Physiol. 1999; 276: C980-C987Crossref PubMed Google Scholar). Experiments were performed with a switching frequency of 35 kHz. The test pulses were generated, and currents were recorded on a computer equipped with a 12 bit A/D and D/A converter board by using the program Cell Work (NPI Electronic). After achievement of the whole-cell patch clamp configuration in both cells of a pair, the cells were clamped to a common holding potential (V1 = V2 = -60 mV). Transjunctional voltages Vj were applied by changing the membrane potential in one cell and keeping the other constant (Vj = V2 - V1). The resulting junctional current Ij was observed as a change in current in the unstepped cell. Junctional conductance gj was determined from Ij/Vj. The number of channels per cell pair was calculated by dividing the total conductance between the two investigated cells by the unitary conductance of the corresponding Cx channel (Cx26 = 135 pS, Cx32 = 55 pS, Cx36 = 15 pS, Cx43 = 115 pS, Cx45 =32 pS, or Cx47 =55 pS) (22Teubner B. Degen J. Sohl G. Guldenagel M. Bukauskas F.F. Trexler E.B. Verselis V.K. De Zeeuw C.I. Lee C.G. Kozak C.A. Petrasch-Parwez E. Dermietzel R. Willecke K. J. Membr. Biol. 2000; 176: 249-262Crossref PubMed Scopus (197) Google Scholar, 23Teubner B. Odermatt B. Güldenagel M. Sohl G. Degen J. Bukauskas F.F. Kronengold J. Verselis V.K. Jung Y.T. Kozak C.A. Schilling K. Willecke K. J. Neurosci. 2001; 21: 1117-1126Crossref PubMed Google Scholar, 28Bukauskas F.F. Elfgang C. Willecke K. Weingart R. Pflügers Arch. Eur. J. Physiol. 1995; 429: 870-872Crossref PubMed Scopus (97) Google Scholar, 29Bukauskas F.F. Angele A.B. Verselis V.K. Bennett M.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7113-7118Crossref PubMed Scopus (113) Google Scholar, 30Bukauskas F.F. Bukauskiene A. Verselis V.K. J. Gen. Physiol. 2002; 119: 171-185Crossref PubMed Scopus (69) Google Scholar). Cyclic AMP-induced currents through CNG channels were measured on single cells in the whole-cell configuration. Caged cAMP was diluted to a final concentration of 200 μm in the pipette solution. After achieving whole-cell configuration, the cell interior was equilibrated for at least 5 min with the pipette solution containing the caged compound. Membrane voltage was held at -60 mV. The extracellular solution contained 120 mm NaCl, 3 mm KCl, 10 mm EGTA, 10 mm glucose, 10 mm Hepes, pH 7.4 (NaOH). The caged component was photocleaved by UV light pulses of 4- or 100-ms duration, and the resulting inward currents were recorded. Immunofluorescence analyses with specific antibodies to the corresponding Cx proteins were used to determine the expression of the connexins in different HeLa transfectants. Fig. 1 shows that the transfected cells expressed Cx26, Cx32, Cx36, Cx43, Cx45, or Cx47 proteins according to the expected punctate immunostaining of gap junction plaques on contacting membranes. To confirm the specificity of the immuno signals, each connexin-transfected HeLa cell clone was also treated with the other Cx antibodies used in this analysis. None of these controls yielded a signal (data not shown). To confirm the presence of functional gap junction channels between HeLa-Cx cell pairs, we injected the fluorescent dye Lucifer Yellow into one cell of a cell pair. As expected, connexin-transfected HeLa clones showed dye transfer from the injected cell into the neighboring cell. No intercellular diffusion of Lucifer Yellow was detected in HeLa cells not transfected with Cx coding DNA (results not shown). The expression of CNG channels was shown by whole-cell patch clamp measurements and by application of 8-bromo-cGMP, as previously described (19Bedner P. Niessen H. Odermatt B. Willecke K. Harz H. Exp. Cell Res. 2003; 291: 25-35Crossref PubMed Scopus (32) Google Scholar). The relationship between the number of photolysed NPE-caged cAMP molecules and the number of applied UV light photons was measured in HeLa-Cx32/CNG double-transfected cells in a previous study (31Munck S. Bedner P. Bottaro T. Harz H. Eur. J. Neurosci. 2004; 19: 191-197Crossref Scopus (17) Google Scholar). In these experiments, CNG channel-mediated inward currents were measured during photorelease of cAMP by 9 successive UV light pulses of equal length (2 ms). The amplitudes of the resulting current steps corresponded to the published dose response curve of the channel (20Altenhofen W. Ludwig J. Eismann E. Kraus W. Bonigk W. Kaupp U.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9868-9872Crossref PubMed Scopus (134) Google Scholar). This result demonstrates that there is a linear relationship between the number of applied photons and the number of photoreleased cAMP molecules. Moreover it shows directly the relationship between flash duration and CNG channel current. To determine the selective permeability of different gap junctions to cAMP, we used a method that consisted of the following steps. First, cAMP was photoreleased from its NPE-caged analogue in one cell of the investigated cell pair by focusing UV light exclusively onto that cell. Next, the amount of cAMP that had diffused from cell 1 (where cAMP was photoreleased) through the Cx channels into the neighboring cell (cell 2) was quantified. Finally, the number of gap junction channels that connected the investigated cell pair was measured (Fig. 2; see also Ref. 19Bedner P. Niessen H. Odermatt B. Willecke K. Harz H. Exp. Cell Res. 2003; 291: 25-35Crossref PubMed Scopus (32) Google Scholar). First Step; Photorelease of cAMP from Its Caged Analogue—A prerequisite to measuring cAMP transfer between cells coupled by gap junction channels is the generation of a reproducible cAMP concentration difference between these cells. Uncaging technology is well suited for the generation of a reproducible concentration gradient, because the final concentration of the released compound can be easily controlled by the number of applied UV light photons. Before investigation, HeLa cells were loaded with 200 μm NPE-caged cAMP as described under "Experimental Procedures." The light of a mercury lamp was focused onto a small spot (30 × 30 μm), limiting photolysis of caged cAMP to a single cell. Complete photolysis of caged cAMP in the irradiated cell was achieved by UV light pulses of 200-ms duration. To avoid unwanted photorelease of cAMP in the adjacent cell, the UV light spot was oriented such that there was no overlap with the contacting cell or the area immediately surrounding it. Second Step; Quantification of the Amount of cAMP That Diffused to Cell 2—To measure the amount of cAMP that diffused through the gap junction channels into cell 2 after photorelease in cell 1, HeLa cells stably expressing a high sensitivity variant of the CNG channel (T537S rCNGα3 (20Altenhofen W. Ludwig J. Eismann E. Kraus W. Bonigk W. Kaupp U.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9868-9872Crossref PubMed Scopus (134) Google Scholar)) in addition to the corresponding connexin were used. Gating of this Ca2+ conducting channel by cAMP was monitored by Ca2+ imaging. The cells were, therefore, incubated with NPE-caged cAMP as well as 8 μm low affinity Ca2+ indicator dye Fluo-4FF/AM before the start of the experiment. Fig. 3 shows the changes in the cytoplasmatic Ca2+ concentration ([Ca2+]i) after flash photolysis of caged cAMP in one cell of a coupled HeLa-Cx32/CNG double-transfected cell pair. The UV light induced a strong increase of the [Ca2+]i in the irradiated cell that propagated into the contacting cell. Typically, this intercellular propagation started after a latency period of 1-10 s at the site of contact. The delay represents the time needed to increase the cAMP concentration in cell 2 to a level where significant CNG channel gating occurs. In some experiments, however, the irradiated and adjacent cells showed simultaneous Ca2+ transients. These cell pairs were excluded, since uncaging events in cell 2 due to cytoplasmatic bridges, overlapping processes, diffuse cell boundaries, or light reflections could not be excluded. Such simultaneous transients were also detected in 2 of 100 control experiments performed on HeLa-CNG cell pairs not transfected with Cx channels. In these two experiments not only the starting point but also the magnitude and the shape of the Ca2+ transients were similar in the irradiated and neighboring cell. Therefore, the most probable explanation for this result was that the cell pairs were coupled by cytoplasmic bridges. The occurrence of cytoplasmic bridges in HeLa cells has been extensively reported (32Bukauskas F.F. Kempf C. Weingart R. Exp. Physiol. 1992; 77: 903-911Crossref PubMed Scopus (21) Google Scholar, 33Valiunas V. Manthey D. Vogel R. Willecke K. Weingart R. J. Physiol. (Lond.). 1999; 519: 631-644Crossref Scopus (67) Google Scholar). However, in 98% of control experiments performed on HeLa-CNG cell pairs, a UV light pulse onto cell 1 resulted in an increase in [Ca2+]i in the irradiated cell, but this Ca2+ signal remained restricted to the stimulated cell (see Fig. 6; see also Ref. 19Bedner P. Niessen H. Odermatt B. Willecke K. Harz H. Exp. Cell Res. 2003; 291: 25-35Crossref PubMed Scopus (32) Google Scholar). We conclude that the cell-to-cell propagation of the Ca2+ signals was dependent on the expression of gap junction channels and that the expression level of endogenous Cx channels in our HeLa cell lines was not significant.FIGURE 6Measurements of cAMP diffusion through gap junction channels composed of different connexins. HeLa wild type cells and HeLa cells expressing the indicated connexins expressed in addition CNG channels as a cAMP sensor system. Cells were loaded with caged cAMP and Fluo-4FF/AM. Caged cAMP was photolysed by a 200-ms UV light pulse that was focused onto one cell (white squares) of the corresponding cell pair. The time course of Ca2+ concentration increase in the cell pairs is shown by three images recorded at the indicated time points. The color bar indicates the intracellular calcium concentration that increases from dark blue to red. White bar, 30 μm. The amount of cAMP that diffused from the first to the second cell was quantified as described in Fig. 2. Double whole-cell patch clamp measurements performed on the same cell pairs were used to quantify the number of Cx channels. The membrane potential of the two cells was clamped for a short time (200 ms) to two different values (V1 = -60, V2 = -20), and the current evoked by this voltage difference was measured (I1). The measured currents correspond to 270 Cx26 channels, 510 Cx32 channels, 3126 Cx36 channels, 93 Cx43 channels, 450 Cx45 channels, and 651 Cx47 channels. In wild type cells, no transjunctional current was detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next we investigated whether the Ca2+ transients in cell 2 were actually caused by cAMP-dependent gating of CNG channels and not by intercellular diffusion of Ca2+ ions. This question was addressed using cocultures of HeLa-Cx45 transfectants with HeLa-Cx45/CNG double transfectants. The latter were prestained with 5 μm DiI to differentiate between the two cell lines. Before the start of the experiment, DiI fluorescence was used to select HeLa-Cx45/CNG double transfectants that were in contact with Cx45 single transfectants. Selected cells were irradiated for 200 ms with UV light, and the change in intracellular Ca2+ concentration was monitored. After a recovery period of 5-10 min, the contacting Cx45 expressing cells were irradiated with the UV light. A typical example of the 12 measurements performed is shown in Fig. 4. The initial cAMP release in the Cx45/CNG-expressing cell induced a considerable Ca2+ transient in this cell, but no significant Ca2+ increase was detected in the neighboring cell that did not express CNG channels (Fig. 4C). In the second part of the experiment (Fig. 4D) it is shown that both cells were coupled by gap junction channels. The irradiated Cx45 transfectant exhibited no Ca2+ transient, whereas the neighboring Cx45/CNG channel-expressing cell showed a significant increase in the intracellular Ca2+ concentration. This result strongly indicates that the Ca2+ transient in cell 2 of Cx45-coupled cell pairs was based on the intercellular diffusion of cAMP molecules and not of Ca2+ ions. It is typical for experiments where the high affinity Ca2+ indicator Fluo-4 is used that no complete recovery of the resting Ca2+ signal can be reached after the massive Ca2+ influx through the CNG channels. However, it should be noted that stray from Cx45/CNG-transfected light the cell exhibiting the large Ca2+ transient may be misinterpreted as a small Ca2+ transient in the neighboring cell. Moreover, these experiments cannot be used to draw a conclusion on the Ca2+ permeability of Cx channels, since a significant part of the cytoplasmic Ca2+ is bound to Fluo-4 (927.09 Da), which may not permeate well through Cx channels. Control experiments on single cells expressing CNG channels revealed a considerable variation in both the amplitude of [Ca2+]i transients and the whole cell CNG channel current after cAMP photorelease (Fig. 5). Most likely this was caused by differences in the expression level of the CNG channels and varying cellular volume. Moreover, we found only a weak correlation between the whole cell current and the maximum amplitude of the respective [Ca2+]i transient. This indicates that the expression level of the CNG channel was at most weakly correlated with the cellular volume. To compensat
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