Intercellular communication via gap junction channels1

Bioelectrochemistry and Bioenergetics 45 Ž1998. 55–65

Intercellular communication via gap junction channels

1

a,) c Dieter F. Hulser , Reiner Eckert b, Uwe Irmer a , Algimantas Krisciukaitis , ¨ ˇ a d a e Anja Mindermann , Jurgen Pleiss , Beate Rehkopf , Julia Sharovskaya , Otto Traub ¨

f

a

Biologisches Institut, Abt. Biophysik, UniÕersitat ¨ Stuttgart, 70550 Stuttgart, Germany b UniÕersity of Auckland, School of Biological Sciences, Auckland, New Zealand c Kaunas Medical Academy, Kaunas, Lithuania d Institut fur ¨ Technische Biochemie, UniÕersitat ¨ Stuttgart, 70569 Stuttgart, Germany e Belozersky Institute, Lomonossow UniÕersity, Moscow, Russian Federation f Institut fur ¨ Genetik, Abt. Molekulargenetik, UniÕersitat ¨ Bonn, 53117 Bonn, Germany Received 24 November 1997; accepted 23 December 1997

Abstract Gap junction channels maintain cell–cell communication and thus are essential for the functional co-ordination of tissues and organs. These channels are composed of connexins, a membrane protein family with more than a dozen members. Properties of gap junction channels are best studied with connexin deficient cells which are transfected with a selected connexin gene. We investigated connexin transfected HeLa cells with electronmicroscopical Žimmunogold labelling of freeze fractured replicas. and electrophysiological Žpatch clamp and dye transfer. techniques. Our results revealed functional gap junction channels outside of quasi-crystalline plaques and give further evidence for different regulations of channel gating for different connexin isoforms by voltage polarity of the transjunctional voltage and by intracellular proton concentration. Our model of gap junction channels indicates connexins that are integrated in the membrane lipids with different tilt angles and a ring of positive and negative charges within the pore wall that might act as a selectivity filter. q 1998 Elsevier Science S.A. Keywords: Gap junction channel; Connexin; Open probability; Immunogold labelling

1. Introduction Gap junction channels permit direct signal transfer between most vertebrate cells; they are essential for the functional co-ordination of tissues and organs. These channels bridge a gap of 2–4 nm distance between cells and thus enable short range interactions in multicellular organisms by allowing the passage of inorganic ions, small water–soluble molecules, and metabolites w1–3x. Gap junction channels are composed of connexins, a membrane protein family with more than a dozen members. A connexin ŽCx. is a membrane protein with four transmembrane a-helices, two extracellular loops, and three intracellular domains. The COOH-terminus and the intra-

) Corresponding author. Fax: q 49-711-685-5096; e-mail: [email protected] 1 Presented on F.E.B.S. Advanced Course, August, 1997, Bucharest.

0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 7 6 - 2

cellular loop account for the differences in connexin isoforms ŽFig. 1.. A connexin hexamer constitutes a gap junction hemichannel—the connexon. The connexon must dock to its counterpart in a neighbouring cell to initiate a gap junction channel. In contact regions of adjacent cells up to several hundreds of these channels are aggregated into gap junction plaques. A characterization of homotypic gap junction channels in cells with well defined Cx-types allows the functional identification of endogenous gap junction channels in different tissues and organs. This strategy has been employed by a number of laboratories using either a transient expression system such as Xenopus oocytes w4x or permanently transfected mammalian cell lines. Among these, three major recipient cell lines have so far been established, SkHep1 w5x, N2A w6x, and HeLa w7x. The most complete library of connexin isoforms was transfected into HeLa cells by the group of Willecke and co-workers w7–12x. We have used this extensive collection to compare the functional proper-

D.F. Hulser et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 55–65 ¨

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min and the replicas were put for 1 h on a droplet of primary antibody, washed again, treated with BSA, and put on a droplet of secondary antibody for 1 h. For double labelling the same procedure was followed with a mixture of diluted primary and secondary antibodies. Controls were treated with inappropriate antibodies or secondary antibodies only. After washing with PBS and distilled water replicas were positioned on a copper grid and investigated with a transmission electron microscope. 2.3. Antibodies

Fig. 1. Schematic topology of a connexin in a membrane.

ties of different connexin channels in the same cellular environment by utilising different biophysical techniques.

2. Experimental 2.1. Cell culture HeLa ŽECACC a96112022. is a permanently growing epithelioid cell line derived from a human cervix carcinoma. Wild type cells and transfectants were cultivated in Dulbecco’s modified Eagle’s medium Žplus 3.7 grl NaHCO 3 , 100 mgrl streptomycin sulfate, 150 mgrl penicillin G, supplemented with 10% calf serum. at pH 7.4 and 378C in a humidified incubator with an 8% CO 2rair mixture. Transfectants were kept with 1 m grml puromycin or geneticin, and Cx40r43 transfectants with 0.5 m grml puromycin and 0.3 m grml geneticin w8,9x. Medium was renewed at 2- to 3-day intervals, and cultures were passaged at confluence by a treatment with 0.25% trypsin in phosphate-buffered saline ŽPBS, pH 7.4. without Caqq and Mgqq. 2.2. Electron microscopy Freeze fracturing of plasma membranes cleaves gap junction channels and a replicated gap junction plaque is identified by distinct particles on the protoplasmic fracture face ŽP-face. and by pits Žextracted particles. on the extracellular fracture face ŽE-face.; both represent individual connexons. For immunogold labelling, cells were washed in PBS, collected, pelleted and placed between two gold specimen carriers and plunged into liquid propane. Carrier sandwiches were fractured and replicated by platinumrcarbon in a Balzers 301 instrument. Replicas were floated on PBS and treated at least for 60 min in 2.5% sodium dodecyl sulfate containing 10 mM Tris HCL plus 30 mM sucrose w13–15x. After washing in PBS unspecific reactions were blocked with 2% bovine serum albu-

Antibodies were prepared by immunising rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin. These specific peptide antibodies were directed against the last 22 amino acids of the COOH terminus of either Cx40 or Cx43 w7x. Antibodies directed against Cx45 were prepared with a fusion protein of glutathione S transferase and COOH terminus of Cx45 which was synthesized in E. coli w8x. Prior to use, these antibodies were diluted in PBS with 2% bovine serum albumin ŽBSA, Sigma A 4503.: anti-Cx40 or anti-Cx43 1:80 and anti-Cx45 1:10. The following antibodies were purchased from Biotrend ŽKoln, Germany.: Mouse-anti-Cx43 ŽNo. CT ¨ 1359., prior to use diluted 1:25 in PBS with 2% BSA; goat-anti-mouse IgG ŽH q L., 15 nm gold ŽNo. 115.022.; and goat-anti-rabbit IgG ŽH q L., 10 nm gold ŽNo. 110.011.. Prior to use, these antibodies were diluted 1:30 in PBS with 2% BSA. 2.4. Ionic coupling For patch clamp experiments cell pairs were prepared by trypsinizing almost confluent monolayer cells for 5–15 min at 378C. After transfer on glass cover slips, cells were allowed to attach for at least 30 min before performing the experiments. Cover slips with attached cells were removed from the dish, washed with PBS, and transferred into an experimental chamber containing PBS with 0.5 mM MgCl 2 and 2.6 mM CaCl 2 , pH 7.3. Intercellular currents were measured between isolated cell pairs using the double whole-cell recording technique w16,17x. Patch type pipettes were pulled from soft glass capillaries and filled with a solution containing 120 mM CsCl, 2.7 mM MgCl 2 , 2.6 mM CaCl 2 , 10 mM HEPES, 10 mM EGTA, pH 7.4 and pCa 7.56. Pipette resistance was in the range of 1–5 M V. All experiments were carried out at room temperature. Current recordings were made using two EPC-7 patch clamp amplifiers ŽHEKA, Lambrecht, FRG.. To obtain a voltage difference between the two cells, command voltage waveforms Žramps or pulses. were applied to one cell while the neighbouring cell was kept at a constant voltage near its resting potential. The apparent transjunctional voltage was calculated as the difference between these two potentials. Command voltage waveforms were generated

D.F. Hulser et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 55–65 ¨

via the DrA converter ŽData Translation DT-2821, Stemmer, Munchen, FRG or DAS-1602, Keithley, Germering, ¨ FRG. of a PC 486 computer using custom made programs from our laboratory. The resulting current recordings were low pass filtered at 250 or 500 Hz with six pole Bessel filter Žcustom built., recorded and stored on computer hard disk for further processing. Voltage dependence of the normalized conductance was fitted with a double Boltzmann function, resulting in estimates for minimum normalized steady-state junctional conductance Gss , equivalent gating charge z, and the voltage for half-maximal inactivation U0 . 2.5. Dye coupling The determination of dye spreading was carried out with Lucifer yellow ŽLY-dilithium salt, Sigma, L 0259, 4% Žwrv. in 1 M LiCl. backfilled into glass microelectrodes pulled from capillaries with inner filament. The fluorescent dye was injected iontophoretically with negative current using a List LrM 1 amplifier ŽList, Darmstadt, Germany.. Experiments were performed between 10 and 40 min after DMEM was replaced by phosphate- or carbonic acidbuffered physiological saline  130 mM NaCl, 5 mM KCl, 1.4 mM CaCl 2 , 1.0 mM MgCl 2 , 5.0 mM glucose, plus an appropriate concentration of KH 2 PO4 and Na 2 HPO4 Ž[PPS. or NaHCO 3rH 2 CO 3 Ž[CPS.4 . Coupling was determined with respect to cell density, intra- and extracellular pH, and concentration of carbonic acid by counting the number of fluorescent neighbours. Concentration of nondissociated carbonic acid in Ringer solution was determined by the Henderson–Hasselbalch equation: pH s p K q lg

HCOy 3

w H 2 CO 3 x

with the dissociation constant of carbonic acid p K s 6.1. The intracellular pH i was measured in a flow cytometer. Cell suspension was stained for 45 min at 378C in a PBS-solution containing 5 m M SNARF1rAM w18x. After washing, cells were transferred into pH buffered solutions that were used for the dye coupling experiments. The probes were excited at 530 " 15 nm, the emitted fluorescence was split close to the isobestic point of SNARF1 at 605 nm and its intensity was measured simultaneously at 575 " 13 nm and at 630 " 11 nm. The system was calibrated cellfree and with HeLa-clones which were permeabilized by 10 m M nigericin in solutions of different pH values in the range from 6.0 to 7.5. 2.6. Modelling of a gap junction pore Amino acid sequences of connexins were taken from the Internet SWISS-PROT database w19x and a gap junction inner pore was modelled on the basis of the third membrane spanning a-helix ŽM3.. This M3-model was

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computed with the Protgen program ŽO. Edholm, Royal Institute of Technology, Stockholm, Sweden. on a Silicon Graphics workstation. Six copies of the M3-helix were arranged with the program Mover ŽR.D. Kloth, MPI for Biology, Tubingen, Germany. in the corners of a hexagon ¨ and with the most hydrophilic sidechains pointing inside the pore. The outer eighteen helices, necessary for the stability of the pore, were formed as polyleucine helices and were arranged on a trigonal lattice with a distance of 1 nm. No water, lipids, or other parts of the protein were added for modelling. Energy minimization and molecular dynamics were performed with the GROMOS engine incorporated in Pro-Simulate w20x. Molecular dynamics simulations were performed without constraints at temperature levels of 5, 30, 100, 200, and 300 K. At 300 K, simulation was continued for 140 ps to test the stability of the models. Cx26 and Cx43 were generated on the basis of the Cx32 structure by changing the sidechains of the helices. An extension of this M3 model considers also cytoplasmic domains ŽM3CD model.. For this model we assumed a spherical shape of the C-terminus and the cytoplasmic loops and calculated their radii and volumes.

3. Results 3.1. Electronmicroscopy On freeze fractured membranes quasi-crystalline gap junction plaques are easily identified as is demonstrated for Cx45 transfected HeLa cells which are labelled with 10 nm immunogold ŽFig. 2.. However, on P-face areas dispersed particles were present which might not have been identified as gap junction channels without their labelling ŽFig. 3.. Gap junction plaques were not detected in HeLa wild type cells, but electrophysiological measurements with high resolution revealed that they are coupled through few channels which are characterized by a low single channel conductance w17x. Channels with similar electrical properties were also found in SkHep1 cells where they were identified as Cx45 gap junction channels w21x. We, therefore, explored HeLa wild type cells with antibody directed against Cx45 and found labelled dispersed particles, often associated with tight junctions, i.e. in areas of close contact w15x. As a control, these HeLa wild type cells were tested with antibody directed against Cx43 and no label was found even in tight junctional areas. To test whether two different connexins ŽCx40 and Cx43. are separated or co-localized in plaques, we investigated double-transfected HeLa cells. Both homomeric channel types form gap junction plaques in single transfected HeLa cells and these structures were also present in the double-transfected cells. Both connexins are co-localized in quasi-crystalline gap junction plaques ŽFig. 4., but also contact areas with dispersed particles which contained

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D.F. Hulser et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 55–65 ¨

Fig. 2. Two gap junction plaques between Cx45-transfected HeLa cells labelled with 10 nm immunogold particles. Bar: 100 nm.

Fig. 3. Contact areas between Cx45-transfected HeLa cells with dispersed gap junction channels labelled with 10 nm immunogold particles. Bar: 100 nm.

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Fig. 4. Double-transfected ŽCx40 and Cx43. HeLa cells. Cx40 Ž10 nm gold particles. and Cx43 Ž15 nm gold particles. are co-localized in a gap junction plaque. Bar: 100 nm.

only one channel type were found. The endogenous Cx45 channels in Cx43 transfectants could also be identified by double-labelling with antibodies directed against Cx43 and Cx45: here gap junction plaques were mainly labelled as Cx43—the transfected connexin—and dispersed particles were identified as Cx43 and Cx45. With antibody directed against ubiquitin, we could also show that gap junction plaques contain ubiquitinized proteins. 3.2. Electrophysiology The total junctional conductance Ž G j . of the connexintransfected HeLa cells ranged from 10–170 nS and thus was on average hundred times higher than in wild type cells. G j indicates the degree of connexin expression and varies from clone to clone. Physiological relevance, however, may be attributed to the single channel conductance g and the voltage dependency of intercellular conductance which is given by the voltage of half maximal inactivation U0 , i.e. the voltage at which 50% of the active channels are closed. For single channel analysis only weakly coupled cell pairs Ž G j - 2 nS. were investigated. An example for single channel currents is given in Fig. 5 for Cx45 gap junction channels. The results of our measurements in different Cx transfectants are summarized in Table 1 for wild type HeLa and seven Cx transfected clones. As can be seen, all transfectants form channels with more than one conductance state. An example for the distribution of

single channel conductances is presented in Fig. 6 for Cx37 channels. Positive and negative transjunctional voltage pulses with amplitudes greater than U0 close individual channels, which is observed as an exponential decay of the total junctional conductance from an instantaneous conductance G inst to a steady state conductance Gss . The response of Cx43 channels to transjunctional voltage steps ŽUj ) U0 . is presented in top of Fig. 7, where repetitive pulses of both polarities give the same decay, always starting with the same G inst which is proportional to the number of open channels at zero transjunctional voltage. This current relaxation was also observed in Cx37 transfectants, but does not occur in Cx32-, Cx40-, and Cx45-transfectants as is also shown in Fig. 7 for Cx45 where after polarity change of the transjunctional voltage, the junctional current always rises dur-

Fig. 5. Single channel gating of about 26 pS in Cx45 transfected HeLa cells.

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Table 1 Single channel conductance g and voltage of half maximal inactivation U0 in HeLa wild type cells and Cx-transfected HeLa clones Clone

g ŽpS.

U0 ŽmV.

wild type Cx26

26"6 35"17 102"49 144"9 59"15 94"24 100"20 50"10 100"11 110"36 240"17 315"50 121"7 153"5 40"2 60"5 27"5 50"13 100"14 180"20 40"2 59"2 88"10 124"5 150"8

49 92

Cx31

Cx32 Cx37

Cx40 Cx43 Cx45

Cx40r43

68

68 35

44 73 49 26 72

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ing the first pulse, indicating that channels reopen and close under these conditions. Since the maximum amplitude of the transjunctional current is always similar, the same number of active channels must be involved. This effect is independent of the total junctional conductance, it can be observed with only a few or with many active channels. In different connexin isoforms the number and sequence of amino acids in the first extracellular loop is highly conserved, whereas the second extracellular loop is significantly longer in Cx37 and Cx43 compared to Cx32, Cx40, and Cx45. Whether this difference could be attributed to the reopening was tested with the HeLa connexin transfectant Cx40)43E1,2 where both extracellular loops from Cx40 were replaced by Cx43 loops. Under

Fig. 6. Distribution of single gap junction channel conductances in Cx37 transfectants. Data Ž ns 435; ps probability. were fitted with three Gaussian equations, which gives conductance states of 110"36, 240"17, and 315"50 pS.

Fig. 7. Relaxation of junctional current in the driving cell demonstrated for Cx43-, Cx45-, and Cx40)43E1,2 transfected HeLa cells. Lower trace: 80 mVr3 s pulses for Cx43; 50 mVr5 s pulses for Cx45; 90 mVr5 s pulses for Cx40)43E1,2 transfectants.

these conditions, reopening was no longer observed ŽFig. 7.. 3.3. Dye transfer Another example for the different regulation of gap junction channels is the variation in Lucifer yellow spreading due to acidification. When Cx26- and Cx32-HeLa

Fig. 8. Junctional coupling of Cx26-transfectants. Number of Lucifer yellow coupled cells Ž N . decreases when the cultures were kept in the same medium for five days.

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Table 2 Extra- and intracellular pH and dye transfer in Cx26- and Cx32 HeLa transfectants

Fig. 9. Junctional coupling of Cx32-transfectants. Number of Lucifer yellow coupled cells Ž N . decreases when the cultures were kept in the same medium for five days.

transfectants grew as monolayers in DMEM, Lucifer yellow transfer is reduced with increasing cell density and decreasing pH of the medium ŽpH m .. For these measurements the cells were transferred in PPS, pH 7.5 but their status of coupling remained unchanged for a period of up to 4 h: at a pH m 6.7 on average about 7 neighbours were still coupled in Cx26 transfectants ŽFig. 8., whereas Cx32transfectants were almost entirely uncoupled ŽFig. 9.. When DMEM was replaced daily, the cell density was almost doubled and pH m did not fall below 6.9. Under these conditions only a minor decrease in coupling was observed in both Cx-transfectants as is demonstrated for Cx32 in Fig. 10. Similarly, the coupling was unaffected by PPS, pH 6.0. To test whether CO 2 has caused the observed block, we evaluated Lucifer yellow transfer at different concentrations of H 2 CO 3 . To discriminate between CO 2 and pH effects, we either measured dye spreading at constant pH with varying concentrations of non-dissociated carbonic acid, or at different pH 0 with constant wH 2 CO 3 x. As can be seen on Table 2, within ten minutes after replacing the

Fig. 10. Junctional coupling of Cx32-transfectants. Number of Lucifer yellow coupled cells Ž N . is almost unaffected when the culture medium is exchanged daily.

Solution

pH 0

PPS PPS CPS5r2 CPS12.5r5 CPS20r8 CPS2r2 CPS5r5 CPS10r10

7.5 6.0 6.5 6.5 6.5 6.1 6.1 6.1

Cx26

Cx32

pH i

Dye transfer

pH i

Dye transfer

7.8 7.2 7.0 6.9 6.6 7.0 6.8 6.4

q q " y y y y y

7.8 7.1 6.8 6.7 6.5 6.8 6.5 6.2

q q q q q q " y

Solutions: PPS: phosphate buffered physiological saline; CPS: carbonic acid buffered physiological saline pH-adjusted by varying the ratio of HCOy 3 rH 2 CO 3 .

medium with CPS, the dye transfer in Cx26 transfectants was blocked at pH 0 6.5 and 2 mM H 2 CO 3 . Even from pH 0 6.1 and 10 mM carbonic acid coupling could be re-established by changing the solution for PPS, pH 7.5. Cx32 transfectants uncoupled differently: only at pH i 6.1 and wH 2 CO 3 x s wNaHCO 3 x ) 5 mM was the dye transfer reversibly blocked. To test whether the intracellular proton concentration ŽpH i . was differently affected by these buffers, we determined pH i with the SNARF ratio method. As can be seen on Table 2, Cx26 transfectants close their gap junction channels at pH i - 7 independently of wH 2 CO 3 x. Cx32 transfectants do not uncouple before a pH i - 6.5 is reached and are also independent of wH 2 CO 3 x. 3.4. Modelling For the secondary structure of connexins a rough model is generally accepted ŽFig. 1. and we modelled a gap junction inner pore on the basis of the Cx32 amino acid sequence. With hydrophobicity and amphiphilicity analyses transmembraneous residues can be assigned and 19 amphiphilic amino acids of the M3-helix were selected to compute our M3 model. Six of these M3-helices line the inner pore ŽFig. 11. and the remaining 18 helices Žnot shown here. stabilize the pore and were modelled with leucine. Because of eight strong amphiphilic amino acids found in M3, one can assume that the inner pore is pasted by 6 = 8 hydrophilic amino acids. A stable condition is reached when the axes of opposite helices are 2 nm apart, so that the effective pore diameter is about 1 nm. Pore openings were only slightly different for Cx26, Cx32, and Cx43, since the M3 sequences are similar for most connexins. A ring of positive charge can be seen inside the pore which is due to arginine in Cx26- and Cx32-pores and due to lysine in Cx43-pores. Next to this positively charged ring, all connexins also formed a ring of negative charge by glutamic acid in the center of the pore.

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Fig. 11. M3-model of the Cx32 pore viewed from the cytoplasmic side Žleft. and cut open pore with the cytoplasmic side at top Žright.. Shading of the surface indicates charge distribution Ždark: negative; light: positive..

Assuming a spherical arrangement of the COOH terminus and the intracellular loop, one can approximate their volumes and show that these cytoplasmic domains further open the intracellular entry of the pore and cause a tilted arrangement of the helices ŽM3CD model; Fig. 12.. Different connexins can have different tilt angles. These cytoplasmic domains contain many charged amino acids which might also explain different pore permeabilities. Freeze

fracture pictures of Cx26 gap junction plaques do not reveal significant differences in particle packing when compared with Cx43 plaques. This indicates that the amino acid content of the COOH terminus might play a minor role in particle spacing, but it might well be that the intracellular loop influences the pore’s intracellular diameter. The extracellular loops are not as big and do not fold into a separate domain, so that the extracellular opening at

Fig. 12. Scheme of a Cx32 pore with COOH terminus and intracellular loop arranged as spheres ŽM3CD model.. v: positive charge; circle with vertical bars inside: negative charge; circle with horizontal bars inside: histidine; CL: cytoplasmic loop; CT: COOH terminus.

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the docking site is smaller, furthermore, the conserved cysteine residues indicate that no major structural variety may be expected on the extracellular side of the pore.

4. Conclusions A reliable identification of connexons in freeze fractured and replicated membranes of vertebrate cells requires tightly packed particles on a P-face and an array of pits on an E-face in the same plaque. A non-ordered arrangement with different particle to particle distances on a P-face may still be considered as a gap junction area. Whether this variation in pattern reflects different physiological states of cells andror cell-specific differences is controversially discussed ŽRefs. w22–30x; for review see Ref. w31x.. Indirect immunogold labelling with anti-connexin antibodies improves the identification of gap junction proteins in freeze fractured membranes and allows new insights in the life cycle of these membrane proteins. In HeLa wild type cells gap junction plaques were neither detected in thin sections w32x nor on freeze fracture replicas w32,33x, a result consistent with early electrophysiological measurements where no coupling could be detected w34x. However, with high resolution patch clamp measurements gap junction channels with a single channel conductance of about 26 pS were resolved w17x. Similar electrical properties were found for Cx45 gap junction channels in SkHep1 cells w21x. From these findings we assumed that Cx45 gap junction channels connect HeLa wild type cells. Indeed, after probing with antibody directed against Cx45 some non-typical gap junction areas were clearly labelled. These results indicate that functional gap junction channels are present as dispersed channels in contact areas. In doubletransfected HeLa cells a co-localization of Cx40 and Cx43 was occasionally detected in quasi-crystalline gap junction plaques whereas in contact areas with dispersed particles only one Cx-type was present. This result indicates that these connexins which are not compatible with each other to form functional gap junction channels may also be separated in the plasma membrane as long as they form functional gap junction channels. Interestingly, a calculation of access resistances revealed that the most favourable configuration of gap junction plaques is one in which channels are spaced as far from one another as possible w35x. This assumption does not consider other requirements for the formation of gap junctions, but it is clear that only a few percent of the electronmicroscopically identified gap junction channels are necessary to maintain the communicating paths. The total junctional conductances of Cx40- and Cx43-transfected HeLa cells were 140 and 70 nS at comparable open probabilities, their single channel conductance is 120 and 150 pS for Cx40 and 40, 60, and 90 pS for Cx43 w36x. From these figures one can conclude that on average about 1000 gap junction channels were open between two con-

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tacting cells. In Cx40 transfected HeLa cells very often several large quasi-crystalline gap junction plaques were found each containing up to 1000 and more channels, whereas in Cx43 transfected HeLa cells smaller plaques were regularly present w15x. Since the number and the size of gap junction plaques vary with the total amount and the life cycle of connexins, the degree of coupling is only indirectly reflected by plaques. It might well be, that dispersed gap junction channels in contact areas are sufficient to maintain the necessary communication between contacting cells and that gap junction plaques are structures of accumulated inactive channels which will be degraded not only by internalization but also by a ubiquitin dependent proteolysis. Our patch clamp measurements with Cx transfectants often revealed more than one conductance state for gap junction channels. Substates may be due to phosphorylation of the connexins w1x but for the non-phosphoprotein Cx26 we have also found substates so that other regulative processes might be active. The listed electrical properties illustrate the variability of gap junction channels from different connexin isoforms. Our single channel conductance data for murine Cx37 revealed three conductance levels of 110 pS, 240 pS, and 315 pS. The main state is comparable to the value described by Veenstra et al. w37x who found two conductance states with human Cx37 in N2A cells. In no case, we observed their low single channel conductance of 63 pS. Reed et al. w6x found four single channel conductance states for N2A transfectants, two of them are comparable to those found in our experiments Ž123 vs. 110 pS, and 219 vs. 240 pS.. In contrast to the human Cx37 channels, the murine Cx37 channels were totally closed at transjunctional voltages Uj ) 80 mV. The voltage for half-maximal inactivation of our murine Cx37–HeLa-transfectants is similar as for human Cx37– N2A-transfectants w6x. In both cases the values of the normalized steady state conductance were best fitted with a single Boltzmann function. Interestingly, when murine Cx37 was functionally expressed in Xenopus oocytes two Boltzmann functions were necessary to fit the data w38x. For oocytes this might be due to the voltage dependency of the initial junctional conductance in relaxation current recordings, which is not observed in transfected N2A- w6x and HeLa cells. The problem of unequivocal identification of conductance is also evident for Cx45. This is the endogenous connexin in coupling deficient HeLa wild type cells, where only one single channel conductance of g s 26 pS was found w17x. In Cx45 transfected HeLa cells, however, we resolved at least three additional conductance states and also a second U0 . Voltage dependent gating and single channel conductance are determined by the connexin type which forms the gap junction channel. Our patch clamp measurements with connexin transfected HeLa cells revealed also two groups of connexin isoforms which differ by their voltage dependent gating after changing polarity of the transjunctional

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voltage. The same exponential decay of the initial conductance G inst is observed for Cx37- and Cx43-channels but not for Cx32-, Cx40-, and Cx45-channels. The latter respond upon the first reversed pulse in a way which might be explained by a sudden closure of the channels, followed by a slow reopening, and a subsequent closing in a similar mode when the polarity is not changed. The results obtained with a Cx40)43E1,2 transfectant indicate that this effect is connected with a structural component which must be located in the extracellular loops. Since intracellular domains and transmembrane helices of this transfectant were part of Cx40 and the first extracellular loop is highly conserved, we hypothesize that the second extracellular loop is involved in the regulatory process for reopening. The pH sensitivity of Cx43 is influenced by the position rather than by the total number of histidine residues in its cytoplasmic loop w39x. As we have demonstrated for Cx26and Cx32- gap junction channels, it is not sufficient to decrease pH 0 by phosphate buffer or by CO 2 . Thus, two uncoupling mechanisms might have been involved: Ž1. a long lasting downregulation caused by a degradation of gap junction channels in ‘exhausted’ medium. Ž2. A fast and reversible blockage of junctional conductance caused by shifts in pH i . When cells are kept for several days in the same medium, not only the pH 0 will decrease but also several other medium ingredients will be depleted or enriched by cellular synthesis w40x. Under these conditions it takes hours before junctional coupling is re-established. This may be explained by an onset of connexin synthesis in fresh medium. The fast pH i induced blockage of junctional conductance is independent of wH 2 CO 3 x but may be caused by protonation of histidine residues in the cytoplasmic loop. Cx26 transfectants have only one histidine in this domain and uncouple at pH i 7.0, whereas Cx32 transfectants have six histidines and remain coupled till pH i 6.5. It should be noted, however, that lowering pH i will also trigger other regulatory events such as Caqq– calmodulin mediated mechanisms, which are known to uncouple gap junctions in Xenopus oocytes w41x. Our two models of a gap junction pore do not yet allow predictions on docking and gating processes and leave the question open, whether the rings of charged amino acids may act as a selectivity filter. The M3 model gives a minimal pore diameter of about 1 nm. Since loops, membrane lipids, and water are not included it represents the closest possible packing of helices in this arrangement. The M3CD model includes volumes of the COOH terminus and the cytoplasmic loop which cause main differences in channel structure and permeability properties of the pore. It explains different tilting of the 24 helices which form a connexon and it matches the electron crystallographic data of Perkins et al. w42x who found a cone ˚ at the shaped Cx32 pore with a minimum diameter of 16 A ˚ extracellular side and a maximum diameter of 26 A at the cytoplasmic entry. With similar methods, however, Unger

et al. w43x revealed a cylindrical pore with an overall ˚ for Cx43 channels deficient in diameter of about 24 A COOH-domains.

Acknowledgements These investigations were supported by grants from Deutsche Forschungsgemeinschaft, Boehringer Ingelheim Fonds, Landesgraduiertenforderung Baden-Wurttemberg, ¨ ¨ INTAS, and the Russian Foundation for Fundamental Research. Based on a presentation given at the ‘Thirteenth School on Biophysics of Membrane Transport’ in Ladek Zdroj, Poland, May 11–18, 1997.

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