©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Gap Junction Proteins -Connexin (Connexin-32) and -Connexin (Connexin-26) Can Form Heteromeric Hemichannels (*)

(Received for publication, October 3, 1994; and in revised form, December 14, 1994)

Kathrin A. Stauffer (§)

From the Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two different types of gap junction proteins, beta(1)- and beta(2)-connexin, were expressed in insect cells, either singly or together, using infection with recombinant baculovirus. Membrane fractions enriched in gap junction proteins were isolated, and connexons (hemichannels) were solubilized with detergent. These solubilized connexons were then run out on a gel filtration column which was capable of partially separating the two homomeric connexons. It was found that connexons from cells co-infected with both types of baculovirus ran together on this column, whereas connexons from cells infected separately and mixed before solubilization did not, suggesting that in the co-infected cells the two types of connexin are assembled into heteromeric hemichannels.


INTRODUCTION

Gap junctions are the regions of cell surfaces which are responsible for direct cell-to-cell communication by metabolic and electrical coupling. They consist of channels which span the plasma membranes of both participating cells as well as the intervening extracellular space. Each channel is composed of two hemichannels, or connexons, one from each cell, which join in the extracellular gap to form the complete cell-to-cell pathway. Each connexon in turn is composed of six polypeptides, or connexins, arranged in a ring around the central pore (for recent reviews, see (1) and (2) ).

Numerous cDNAs coding for connexins have been isolated and sequenced (for a review, see (3) ). It is clear that most if not all the animal tissues so far examined express more than one type of connexin. In view of this, and the pronounced homology between connexin species, the question has arisen of the composition of cell-to-cell channels. Could one channel be made up of an assembly of several different connexins (heteromeric channel; a schematic representation of possible assembly types is given in Fig. 6)? Or perhaps of two different hemichannels (heterotypic channel)? Or do connexins only ever form homo-oligomers?


Figure 6: Schematic drawing of possible assembly patterns of connexins into complete gap junction channels. Connexin designates a polypeptide species which forms a subunit of a gap junction channel. Connexons, or hemichannels, are hexamers of connexin and can be either Homomeric (i.e. composed of six identical connexin subunits) or Heteromeric (i.e. composed of more than one species of connexin). Connexon pairs, or whole cell-to-cell channels, can be Homomeric (composed of 12 identical connexin subunits) and therefore homotypic (composed of two identical connexons), Heterotypic (composed of two different homomeric connexons), or Heteromeric (composed of two different heteromeric connexons).



There is ample evidence for the existence of homomeric and homotypic gap junctions, since some tissues contain a predominant connexin species which makes up 90% or more of the total gap junction protein (see, for instance, (4) and (5) ). Data obtained from studies with recombinant connexins support the notion of heterotypic channels(6) . Such heterotypic gap junctions appear to show physiological properties which are quite different from those of the parent homotypic junctions. This enlarges the scope for possible electrical and regulatory properties of gap junctions, and one could imagine an even greater variety of different channels if the possibility of not only heterotypic but also heteromeric channels could be demonstrated. Evidence for the existence of such heteromeric gap junctions is presented in this work.

As reported earlier(7) , it has been found that the major connexin from liver, beta(1)-connexin, is solubilized in detergent as a connexon, or hemichannel, rather than as a whole channel. The same holds true for a slightly smaller but highly homologous species, beta(2)-connexin(8) , while it appears to be possible to solubilize the whole channel with, for instance, lens connexins(9) . We have found that beta(1)- and beta(2)-connexons exhibit slightly different hydrodynamic properties, and that it is therefore possible to investigate the behavior of mixed populations of beta(1)- and beta(2)-connexins in gel filtration. The results suggest that beta(1)- and beta(2)-connexins can indeed form heteromeric channels.


MATERIALS AND METHODS

Cell Culture and Membrane Preparation

Spodoptera cells (cell line Sf9) were grown in spinner flasks in TNM-FH medium supplemented with 10% fetal calf serum and containing 50 µg/ml gentamicin and 2.5 µg/ml amphotericin B(10) . Batches of up to 1 liter were grown. At a density of 2 times 10^6 cells/ml, they were infected with baculovirus containing the coding sequence for human beta(1)- or beta(2)- connexin under control of the polyhedrin promoter. Virus titers were approximately 10^9 plaque-forming units/ml, and cultures were infected with at least 100 ml, but not more than 200 ml, of virus, resulting in a multiplicity of infection of about 50. Cultures infected with beta(1)-virus were harvested typically after 64 h, while cultures infected with beta(2)-virus did not reach comparable levels of expression until about 90 h postinfection. Cultures co-infected with both beta(1)- and beta(2)-virus were harvested typically 60 h postinfection. Harvested cells were washed with isotonic buffer containing 2.5 µg/ml pepstatin A, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, and then frozen at -20 °C until further processing. Membranes were prepared from frozen cells as described previously using alkaline extraction(7) . Media, serum, antimicrobial agents, and protease inhibitors were obtained from Sigma. All other reagents were of the highest grade available.

The hybridoma cell line producing a monoclonal antibody against beta(1)-connexin (M12.13) was grown in RPMI medium supplemented with 10% fetal calf serum, at 37 °C, in spinner flasks up to a culture size of 600 ml. Cell supernatants were harvested by gentle centrifugation of the cells. Immediately after harvesting, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5 µg/ml pepstatin A, and 0.02% sodium azide were added. For Western blots, this supernatant was used at dilutions of 1:50 to 1:100.

Solubilization of Connexons

Samples of frozen membranes were thawed on ice, centrifuged to collect the membranes in a minimal volume, and dispersed in ice-cold solubilization buffer (100 mM glycine, pH 10, 2 M NaCl, 10 mM EDTA, 100 mM dithiothreitol, 4% LC-14 (^1)(Avanti Polar Lipids Inc.), 2.5 µg/ml pepstatin A, 5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) with the aid of a 23-gauge syringe needle. The clear solution was sonicated for about 20 s in a bath sonicator and left at 4 °C for at least 2 h or, more often, overnight.

Gel Filtration

An HR 16/60 column packed with Superose 6 prep grade (total volume 110 ml; Pharmacia Fine Chemicals) was equilibrated with 50 mM HEPES, pH 8.0, 500 mM NaCl, 5 mM EDTA, 10 mM dithiothreitol, 0.1% n-dodecyl-beta-D-maltopyranoside. About 200 µl of this buffer was added to solubilized membranes, and the sample was centrifuged in a Beckman TLA 100.3 rotor at 55,000 rpm (100,000 times g) for 60 min at 4 °C. The resulting supernatant was filtered through a syringe filter with a pore size of 0.2 µm and applied to the column. Sample volumes were kept to less than 2 ml. The column was developed at a flow rate of 1 ml/min. 1-ml fractions were collected. The R(F) value of a fraction was defined as the volume corresponding to that fraction divided by the total column volume.

Analysis

Aliquots (10 µl) of the fractions collected from the column were analyzed by SDS-PAGE according to Laemmli(11) . Total protein was visualized by silver stain. The gels were digitized using a video camera connected to a UVP Imagestore 500 digitizer, and appropriate bands were integrated using NIH Image software on a Macintosh computer. For Western blots, samples were prepared by precipitating aliquots (200 µl) with 15 volumes of ice-cold acetone followed by brief centrifugation at high speed on a benchtop microcentrifuge. The supernatants were discarded, and the pellets were dissolved in sample buffer. Each sample was then split in two halves for blotting against beta(1) and beta(2), respectively. Western blots (12) were developed using antibodies against beta(1)- or beta(2)-connexin which were known not to cross-react. Antibodies were detected using biotinylated secondary antibodies followed by avidin-coupled horseradish peroxidase (Vector Laboratories), with 4-chloronaphthol as the peroxidase substrate. The aggregation state of connexins after gel filtration was determined by examining specimens negatively stained with uranyl acetate in the electron microscope (Philips EM 420).


RESULTS

Fig. 1shows SDS-PAGE from samples of beta(1)- (a) and beta(2) (b)-connexons after solubilization and chromatography on Superose 6. Aliquots of the same fractions, corresponding to R(F) values of 0.41 to 0.55, were analyzed on SDS-PAGE and visualized by silver staining. In the experiment shown in Fig. 1, c and d, membranes from cells expressing beta(1)-connexin were mixed with membranes from cells expressing beta(2)-connexin, and the mixture was solubilized. After about 2 h on ice, the sample was run out on Superose 6. Again, the same fractions as in Fig. 1, a and b, were run on SDS-PAGE and analyzed for total protein by silver staining. The beta(1)-connexons, peaking at an R(F) value of about 0.47 (which would, in the absence of detergent, correspond to a molecular mass of about 800 kDa), appear to be much larger than the beta(2)-connexons, which peak at an R(F) value of about 0.53 (or 550 kDa for a detergent-free system). The difference corresponds to at least 5 ml on the 110-ml column and was found to be highly reproducible. In order to verify the identification of the bands, Western blots were made of the same fractions as in Fig. 1c and separately stained for beta(1) and beta(2), respectively (Fig. 1d). When gel filtration was performed with either protein on its own, the corresponding Western blots looked identical with the ones shown here.


Figure 1: a, SDS-PAGE of selected fractions (R values indicated above the corresponding lanes) of a Superose 6 HR16/60 column injected with a sample of beta(1)-connexin solubilized as described under ``Materials and Methods.'' The beta(1) band is indicated. b, SDS-PAGE of the same fractions as a of a sample of beta(2)-connexin. c, SDS-PAGE of the same fractions as a and b of a sample of beta(1)- and beta(2)-membranes mixed together before solubilization. The two proteins can be seen to run in the same positions as in a and b, respectively. d, the fractions shown in c, except the first one, were rerun, transferred onto nitrocellulose, and stained with antibodies against beta(1)-connexin (upper bands) or beta(2)-connexin (lower bands).



The distribution of the two species of connexons did not change even when the solubilized sample was left overnight in the cold before gel filtration (data not shown), suggesting that, in a population of connexons in solution, subunit exchange does not take place even after prolonged periods of exposure to high concentrations of detergent and reducing agent at pH 10. The fact that we are indeed looking at single connexons is illustrated in Fig. 2, which shows the particles typically found in the peak fractions of beta(1)- (a) and beta(2)-connexin (b).


Figure 2: An aliquot of the fraction with an R value of 0.47 of the sample shown in Fig. 1a (a) and an aliquot of the fraction with an R value of 0.53 of the sample shown in Fig. 1b (b) was deposited on a carbon-coated copper grid, stained with uranyl acetate, and viewed in the electron microscope. In both samples, typical doughnut-shaped particles can be seen which represent connexons, or hemichannels.



Fig. 3a shows the silver stain, and Fig. 3b, the Western blot of the same fractions as in Fig. 1, of a culture co-infected with roughly equal amounts of beta(1)- and beta(2)-encoding baculovirus and harvested 60 h postinfection. Comparison of this figure with Fig. 1shows that the two species no longer behave as independent particles. Instead, the beta(2)-protein now runs in the same position as the beta(1)-protein. This suggests that the two must be part of the same connexon which exhibits overall hydrodynamic properties very similar to those of a homomeric beta(1)-connexon. The stoichiometry of such a heteromeric connexon cannot of course be inferred from these data, although the relative quantities of the two species suggest an excess of beta(1) over beta(2).


Figure 3: SDS-PAGE (a) and Western blots (b) of membranes of cells co-infected with equal amounts of baculovirus containing the coding sequences for beta(1)- and beta(2)-connexin, solubilized as the preparations in Fig. 1and subjected to gel filtration on the same column. Again, the R values of the fractions shown are indicated above the corresponding lanes, and the two connexin bands are marked on the sides. Note especially the shift in the beta(2) peak from R = 0.53 to R = 0.47 to co-purify with beta(1)-connexin.



In order to investigate if the hydrodynamic properties of the presumed heteromeric connexons could be changed if the ratio of expressed beta(1):beta(2) was changed, a number of experiments were performed using ratios of beta(1):beta(2)-virus between 1:1.5 and 1:10 to infect the insect cells. Fig. 4a was obtained from a culture infected with beta-virus and beta-virus at a ratio of approximately 1:2, and Fig. 4b from a culture infected with a ratio of 1:3 beta-:beta-virus, a ratio which resulted in approximately equal expression levels of the two proteins. In both cases, the distribution of beta is more or less unchanged, while beta appears to form a very broad band, suggesting that it is partly associated with beta-connexin and partly assembled into homomeric connexons. beta-Connexin does not undergo a significant shift toward higher R values even when it is no longer present in excess. A semiquantitative display of the distribution of the two connexin species is given in Fig. 5, which shows the integrated optical densities of the silver-stained bands as a function of the R values. It illustrates that the hydrodynamic properties of beta-connexin do not change significantly upon co-expression with beta-connexin, but comparison of Fig. 5, a and b, reveals the shift in the beta-peak. Fig. 5, c and d, demonstrates a nearly equal distribution of beta-connexin at R values between 0.47 and 0.55 (Fig. 5c) and an accumulation of extra beta-connexin at higher R values (Fig. 5d). These results suggest that the stoichiometry of the heteromeric connexon is such that beta must be the major constituent, i.e. (beta)(beta) or (beta)(beta), with the excess betaconnexin assembled into homomeric connexons.


Figure 4: SDS-PAGE of samples from cells co-infected with beta(1):beta(2)-virus at a ratio of 1:2 (a) and 1:3 (b). The same fractions as in Fig. 1and Fig. 3are shown. Note that the position of beta(1)-connexin is essentially unchanged while beta(2)-connexin is spread out, possibly into a population co-purifying with beta(1)-connexin and a population running as pure beta(2)-connexin.




Figure 5: Plots of integrated optical density of the silver-stained bands shown in the previous figures versus R values. beta(1)-bands are represented by filled bars, beta(2)-bands by shaded bars. The optical densities are given in arbitrary units. a, taken from the gel in Fig. 1c. b, taken from the gel in Fig. 3a. c, taken from the gel in Fig. 4a. d, taken from the gel in Fig. 4b.




DISCUSSION

We have established previously (7) that an alkaline extraction procedure developed for the isolation of gap junctions from liver plasma membrane (13) can be applied, with minor alterations, to connexons expressed in insect cells. It removes most membrane proteins and leaves behind a fraction highly enriched in gap junction plaques. Solubilization experiments performed with beta(1)-connexin showed that it was not possible to solubilize the protein in the form of a whole cell-to-cell channel but only in the form of a hemichannel, or connexon. The same has been found to be true for the smaller homologue, beta(2). At the same time, it was observed that beta(2)-connexin, due to its greater overall hydrophobicity, required very long-chain detergents if it was to be solubilized at all. Therefore, the detergent monomyristoyl lysolecithin (LC-14) was routinely used in solubilization buffers. For gel filtration experiments, n-dodecyl-beta-D-maltopyranoside was chosen since connexons were found to be optimally stable in this detergent.

Many cell types are known to express several different species of connexins, yet the functional consequences of this remain largely unclear, partly because it is not known whether these connexins assemble into distinct, homomeric channels, or whether oligomers containing several different subunits can exist, as is the case with most ligand- and voltage-gated ion channels. If connexins are segregated, the total gap junctional conductivity found in a particular cell could be described as the sum of the individual conductivities, which are amenable to study in vitro. If, however, heterotypic and even heteromeric channels exist, the task of characterizing the functional properties of gap junctions present in a particular cell type may become much more challenging. Fig. 6shows a schematic drawing of possible mixed-subunit assemblies, resulting in heterotypic or heteromeric gap junction channels.

It has been shown that beta(1)- and beta(2)-connexin can occur in rodent liver gap junctions in the same plaque(14) . In addition, results were reported from immunprecipitation experiments (15) , which suggested that beta(1)- and beta(2)-connexins were not separable, but the aggregation state of connexins in these preparations seemed rather doubtful; under the conditions used to solubilize these gap junctions, quite large and unspecific aggregates of connexins and connexons could have been present. Based on previous biochemical characterization of gap junction proteins(7) , we are confident that the samples analyzed here are indeed homogeneous populations of single connexons, and thus the results presented here reflect the properties of such hemichannels.

Co-infection of insect cells with baculovirus encoding for both beta(1)- and beta(2)-connexins resulted in membrane preparations fairly similar to those commonly obtained from rat liver, with beta(1)-connexin present in a large excess over beta(2)-connexin (see Fig. 3). Indeed, gel filtration performed with rat liver gap junction preparations gives the same distribution of connexins, i.e. beta(2)-connexin is co-purified with beta(1)-connexin. (^2)The excess of beta(1)- over beta(2)-connexin in the co-infected preparation is mostly due to the fact that expression of beta(1)-connexin in insect cells reaches high levels more quickly than expression of beta(2). When we tried to incubate infected cultures for longer periods of time after infection, we found that beta(1)-connexin was at least partly degraded. In order to obtain comparable expression levels of the two proteins, it was necessary to infect cells with beta(1):beta(2)-virus at a ratio of about 1:3.

The beta(1)-connexon has a theoretical molecular mass of 192 kDa, and the beta(2)-connexon should amount to 156 kDa. However, if connexons are solubilized in detergent and subjected to gel filtration under nondenaturing conditions, the beta(1)-connexon runs at an apparent molecular mass of about 800 kDa, while the beta(2)-connexon runs as a particle of about 550 kDa. As we have shown previously, and as the electron micrographs in Fig. 3strongly suggest, these are nonetheless single connexons and not double ones, or aggregates, of connexons. This is also consistent with unpublished observations that whole channels exhibit substantially smaller R(F) values. (^3)The high apparent molecular mass of connexons is presumably due to the size of the enveloping detergent micelle; proteins which bind large amounts of detergent can exhibit substantial increases in Stokes radius(16) .

It appears from the data in Fig. 3and Fig. 4that the Stokes radius of a heteromeric connexon is very similar to that of a homomeric beta(1)-connexon. It was not possible to obtain heteromeric connexons that exhibited a Stokes radius more similar to beta(2)-connexons. This suggests that the presence of beta(1)-connexin in a heteromeric connexon, no matter at which beta(1):beta(2) ratio, will determine its overall hydrodynamic properties. Alternatively, only certain stoichiometries may be permissible in heteromeric connexons, thus forcing an excess of beta(1) over beta(2), and resulting in a complex with a Stokes radius so close to that of a homomeric beta(1)-connexon that the two are not resolved on the gel filtration column used here.

The experiments shown in Fig. 1prove that beta(1)- and beta(2)-connexons run independently from each other in gel filtration. The experiments shown in Fig. 3(and 4) show that connexons produced in co-infected insect cells lose this independent behavior. It seems difficult to think of another explanation for this if we do not postulate that the two polypeptides are localized in the same oligomeric assembly. Under the conditions used for these experiments, the oligomeric assemblies present are connexons, or hemichannels. Thus it appears that insect cells co-infected with the two connexins express heteromeric connexons, composed of both beta(1)- and beta(2)-connexin. There is no reason to assume that this phenomenon should be confined to recombinant connexons. It is therefore postulated that heteromeric gap junction channels can exist in vivo.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pathology, Cambridge University, Tennis Court Rd., Cambridge, UK. Tel.: 44-223-333-740; Fax: 44-223-333-732.

(^1)
The abbreviations used are: LC-14, monomyristoyl lysolecithin; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Cascio, N. M. Kumar, and N. B. Gilula, unpublished observations.

(^3)
N. C. Koenig, personal communication.


ACKNOWLEDGEMENTS

I acknowledge the generous gift of recombinant baculovirus, the cell line M12.13, and the antibody against the beta(2)-protein from Nalin Kumar, useful help with the culturing of M12.13 from Robert Safarik, and excellent secretarial assistance from Tim Green. Many helpful and stimulating discussions with Lukas Buehler, Bernie Gilula, Nalin Kumar, John Berriman, Tim Green, Nicola Koenig, and Olga Perisic were essential to this work, and I thank them all for their time and attention. Special thanks are due to Nigel Unwin for his unwavering support of this work.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.