(Received for publication, October 3, 1994; and in revised form, December 14, 1994)
From the
Two different types of gap junction proteins, -
and
-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.
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, -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,
-connexin(8) , while it appears to be possible
to solubilize the whole channel with, for instance, lens
connexins(9) . We have found that
- and
-connexons exhibit slightly different hydrodynamic
properties, and that it is therefore possible to investigate the
behavior of mixed populations of
- and
-connexins in gel filtration. The results suggest that
- and
-connexins can indeed form
heteromeric channels.
The hybridoma cell line producing a monoclonal antibody
against -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.
Fig. 1shows SDS-PAGE from samples of
- (a) and
(b)-connexons after solubilization and chromatography on
Superose 6. Aliquots of the same fractions, corresponding to R
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
-connexin were mixed with membranes from
cells expressing
-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
-connexons, peaking at an R
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
-connexons, which peak at
an R
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
and
, 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
-connexin solubilized as
described under ``Materials and Methods.'' The
band is indicated. b, SDS-PAGE of the same fractions as a of a sample of
-connexin. c,
SDS-PAGE of the same fractions as a and b of a sample
of
- and
-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
-connexin (upper bands) or
-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 - (a) and
-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 - and
-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
-protein now runs
in the same position as the
-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
-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
over
.
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 - and
-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
peak from R
= 0.53 to R
= 0.47 to co-purify with
-connexin.
In order to investigate if the
hydrodynamic properties of the presumed heteromeric connexons could be
changed if the ratio of expressed :
was changed, a number of experiments were performed using ratios
of
:
-virus between 1:1.5 and 1:10 to
infect the insect cells. Fig. 4a was obtained from a culture
infected with
-virus and
-virus at a
ratio of approximately 1:2, and Fig. 4b from a culture
infected with a ratio of 1:3
-:
-virus, a ratio which resulted in
approximately equal expression levels of the two proteins. In both
cases, the distribution of
is more or less unchanged,
while
appears to form a very broad band, suggesting
that it is partly associated with
-connexin and partly
assembled into homomeric connexons.
-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
-connexin do not change significantly upon
co-expression with
-connexin, but comparison of Fig. 5, a and b, reveals the shift in the
-peak. Fig. 5, c and d,
demonstrates a nearly equal distribution of
-connexin
at R
values between 0.47 and 0.55 (Fig. 5c) and an accumulation of extra
-connexin at higher R
values (Fig. 5d). These results suggest that the stoichiometry
of the heteromeric connexon is such that
must be the
major constituent, i.e. (
)
(
) or
(
)
(
)
, with
the excess
connexin assembled into homomeric
connexons.
Figure 4:
SDS-PAGE of samples from cells co-infected
with :
-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
-connexin is essentially unchanged while
-connexin is spread out, possibly into a population
co-purifying with
-connexin and a population running
as pure
-connexin.
Figure 5:
Plots of integrated optical density of the
silver-stained bands shown in the previous figures versus
R values.
-bands are
represented by filled bars,
-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.
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
-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,
. At the same
time, it was observed that
-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-
-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 - and
-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
- and
-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 - and
-connexins resulted in membrane preparations fairly
similar to those commonly obtained from rat liver, with
-connexin present in a large excess over
-connexin (see Fig. 3). Indeed, gel filtration
performed with rat liver gap junction preparations gives the same
distribution of connexins, i.e.
-connexin is
co-purified with
-connexin. (
)The excess of
- over
-connexin in the co-infected
preparation is mostly due to the fact that expression of
-connexin in insect cells reaches high levels more
quickly than expression of
. When we tried to incubate
infected cultures for longer periods of time after infection, we found
that
-connexin was at least partly degraded. In order
to obtain comparable expression levels of the two proteins, it was
necessary to infect cells with
:
-virus at a ratio of about 1:3.
The -connexon has a theoretical molecular mass of
192 kDa, and the
-connexon should amount to 156 kDa.
However, if connexons are solubilized in detergent and subjected to gel
filtration under nondenaturing conditions, the
-connexon runs at an apparent molecular mass of about
800 kDa, while the
-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
values. (
)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
-connexon. It was not possible to obtain heteromeric
connexons that exhibited a Stokes radius more similar to
-connexons. This suggests that the presence of
-connexin in a heteromeric connexon, no matter at
which
:
ratio, will determine its
overall hydrodynamic properties. Alternatively, only certain
stoichiometries may be permissible in heteromeric connexons, thus
forcing an excess of
over
, and
resulting in a complex with a Stokes radius so close to that of a
homomeric
-connexon that the two are not resolved on
the gel filtration column used here.
The experiments shown in Fig. 1prove that - and
-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
- and
-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.