(Received for publication, February 21, 1995; and in revised form, May 1, 1995)
From the
Enriched subcellular fractions of double membrane gap junctions
(plaques) from rat livers were treated under reducing conditions with
high salt and non-ionic detergent concentrations at high pH to obtain a
preparation of structural 80-90 Å complexes of oligomers
(connexons). The isolated oligomers were chromatographically purified,
and subsequently characterized immunologically, morphologically by
electron microscopy, hydrodynamically by gel filtration and
ultracentrifugation, spectroscopically by circular dichroism, and
chemically via cross-linking studies. The physical characteristics of
these isolated gap junction complexes were compared to those of native
membrane-bound gap junctions in rat liver. These analyses indicate that
the isolated complex (connexon) principally contains a hexameric
arrangement of gap junction protein to form a single membrane
hemi-channel.
Paired protein channels located in specialized areas of closely
apposed plasma membranes, termed gap junctions, act to connect the
interiors of adjacent cells in a tissue. These channels allow for
direct intercellular communication between cells by acting as a conduit
for the passage of ions and small molecules, and they are important for
the regulation of development and cell growth (for reviews, see Refs.
1, 2). It is now recognized that a family of gap junction proteins
exists, members of which are expressed differentially in virtually all
cell types(3, 4) . This study has focused on the
gap junction channel of rat livers which is comprised primarily of a
single 32-kDa protein component(5, 6, 7) ,
with less than 5% of the gap junction protein being a related 26-kDa
protein species(8) . The mRNAs for both proteins have been
cloned and sequenced(9, 10, 11) . These are
referred to as Electrophoretically eluted (from
SDS-polyacrylamide gels) rat liver Using conditions similar to those described
by Stauffer et al.(19) , we have isolated and purified
the rat liver
Markers used in the Stokes radius determination were
horse heart myoglobin (Sigma), bovine heart malate dehydrogenase
(Sigma), bovine heart lactate dehydrogenase (Sigma), horse spleen
ferritin (Gallard Schlesinger), Isolated gap
junctions were solubilized by resuspension of the pellet (15 min at
12,000 Larger scale
separations (>500 µg) were conducted on a Pharmacia FPLC system
equipped with a P-500 pump and a UV-1 monitor with a 280-nm filter
using a Superose 6 HR 10/30 column (Pharmacia). Samples were prepared
as above except the sample was solubilized in 100 mM glycine,
pH 10.0, 2 M NaCl, 100 mM DTT, 10 mM EDTA,
and 2.0% LDAO to give a final concentration of Eluant of HPLC and FPLC were monitored
for gap junction content by electron microscopy, SDS-PAGE, and/or
immunoblotting.
The isolated
gap junction oligomers were purified by FPLC prior to CD examination.
The samples were dialyzed against 10 mM Tris, pH 9.2, 125
mM NaCl, 5 mM DTT, and 0.05% LDAO and concentrated
using an Amicon microconcentrator and characterized as above. Aliquots
of the dialysate were used as blanks. CD spectra were recorded on an
Aviv 61DS spectropolarimeter. The spectropolarimeter was calibrated for
wavelength using benzene vapor. The optical rotation was calibrated
with D-10-camphorsulfonic acid and checked with sperm whale
myoglobin. Optical measurements of ellipticity were routinely made
at room temperature using a demountable quartz cell (Hellma Kuvetten,
Mullheim/Baden, Germany) of pathlength 0.5 mm for the junctions and 0.1
mm for the isolated oligomers. Data was collected at 0.2-nm intervals
in the wavelength range between 300 and 185 nm for the former, and
between 300 and 195.4 nm for the latter (the presence of high salt,
DTT, and LDAO in the solubilization buffer precluded collection at
lower wavelengths). At least 15 reproducible scans were collected for
each sample, averaged, and then smoothed using a Savitzky-Golay filter (27) . The CD spectra were analyzed in the wavelength range
from 190 to 240 nm using a non-linear least-squares curve-fitting
procedure, with the values of the fractions of secondary structure
constrained to be non-negative and normalized, as described
previously(28) . The reference data set (29) was
augmented with a basement membrane collagen data set (22) to
correct for any collagen content. A helix length of 20 residues, the
minimum length of helix necessary to span the lipid bilayer, was used
in all analyses. The quality of the fit of the reference spectra to the
experimental data was evaluated by calculating a fit parameter, the
normalized root mean standard deviation (NRMSD) (28) . The
spectrum of a polypeptide in the near UV region is a sum of the
individual absorbances of three peptide transitions in this region: a
single n to
Figure 1:
Electron micrographs of gap junction
preparations. Negatively stained images of gap junction-enriched
plaques isolated from rat liver tissue (A), and
detergent-solubilized preparations isolated after gel filtration (B). These isolated protein-detergent complexes had physical
characteristics consistent with hexameric assemblies. The bar represents 200 nm.
Figure 2:
Immunoblots of fractions from gel
filtration of detergent-solubilized gap junctions. 1-1.5 mg of
protein were applied to a Superose 6 HR 10/30 column and eluted at 0.5
ml/min. Fractions were run on a 15% SDS-polyacrylamide gel and
transferred to nitrocellulose. Immunoblots were treated with antibodies
to
where V
Figure 3:
Plot
of ln R
The linearity of the gradients was confirmed by measurement
of the refractive indices of the fractions. The density, The average viscosity experienced by the gap junction complex
in each gradient was interpolated from linear plots of and is reported in Table 1. The sedimentation coefficient
corrected to water at 20 °C was then determined from both the
H
These values are reported in Table 1. Using this
information, along with the calculated R and the frictional ratio from:
These values are reported in Table 1. To determine the
molecular weight of the protein alone, we can use the relationship that
where f
For soluble proteins, the The averaged CD spectrum of the
isolated oligomers was similar, but not superimposable, to that of the
intact gap junction plaques (Fig. 4) and appeared to be
relatively red-shifted (e.g. the large positive peak at low
wavelength, which is indicative of helix-content, appeared red-shifted
2.0 nm relative to that of the membrane-bound sample). Analysis of the
membrane plaques indicated that there was 52%
Figure 4:
CD spectra of sonicated gap junction
patches (A) and solubilized gap junction complexes (B). The magnitude
of the mean residue ellipticity of the spectra were determined using
the normalization factor calculated in our
analyses.
The extent of agreement
between the calculated net structure and the measured CD spectrum is
reflected in the NRMSD parameter(42) . The NRMSD of the
analyzed spectrum of solubilized gap junction complexes was fairly high
(0.133). The absorption bands of the spectra of the reference basis
set, modeled as Gaussians, were shifted independently to correct for
solvent effects, resulting in a substantially improved fit to the
experimental data(32) . Shifting of the two
Figure 5:
Chemical cross-linking of Gap junction
oligomers. A, immunoblot of cross-linked
In this study, gap junction oligomers (connexons) were
isolated from enriched rat liver gap junctions by treatment with
non-ionic detergent, high pH, and high salt under reducing conditions.
These oligomers were then physically characterized as being principally
a The 6-fold
symmetry observed in x-ray diffraction studies (21) and image
analysis(17, 19) , suggests that the hemi-channel is
hexameric. Incubation of the isolated complexes with DMS yielded
increasing higher order cross-linked In the
hydrodynamic studies, the calculated size of the isolated oligomer was
also consistent with it being a hexamer similar in shape to that of the
hemi-channel. Previous x-ray diffraction studies of gap junctions
determined that each hexameric channel extends There are some differences in the
secondary structure of the solubilized and ``native'' gap
junction proteins. CD spectroscopic studies indicate that the
solubilized gap junction protein is slightly enriched in In
summary, these results indicate that the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(or Cx32) and
(or
Cx26) gap junction proteins.
protein has been
reported to form voltage-dependent channels in reconstituted lipid
bilayers (12) , and the topology of the molecule has been
partially mapped (13, 14, 15, 16) .
Upon isolation and purification, hepatic gap junction proteins form
extended two-dimensional crystalline arrays. Low resolution
three-dimensional structural information has been obtained from
electron micrographs of tilted specimens (17, 18, 19) and low angle x-ray diffraction
studies of stacked arrays(20, 21) . These studies have
suggested that each channel is composed of two hexameric assemblies
which span the opposing membranes and meet at the center of the gap.
Additionally, the secondary structure of the rat liver gap junction
channel in sonicated membrane patches has been determined by circular
dichroism (CD) studies(22) . Unfortunately, the presence of the
membrane bilayer in which the oligomers are embedded has precluded
structural examination of the oligomer at a molecular level. Conditions
have been described (19) under which the
connexin protein may be solubilized by detergent while retaining
a native-like structure.
gap junction protein in a
``native'' oligomeric state. These isolated complexes were,
in turn, characterized to determine the physical properties of the
oligomeric unit.
Materials
Female retired breeder Sprague-Dawley
rats, ranging from 200 to 300 g were obtained from Holtzman
Laboratories (Madison, WI). N-N-Dimethyldodecylamine-N-oxide (LDAO) ()was purchased from Fluka (Switzerland). Dodecyl maltoside
and dithiothreitol (DTT) were purchased from Calbiochem (San Diego,
CA). Tris, HEPES, and triethanolamine (TEA) were purchased from Sigma.
EDTA was purchased from Fisher.
Specimen Preparation
Subcellular fractions of
enriched rat liver gap junctions were prepared by alkali-extraction
(NaOH) of rat liver plasma membrane fractions according to
Hertzberg(7) , as modified by Zimmer et
al.(13) . The isolated gap junction fractions were stored
in 2 mM bicarbonate buffer, pH 7.5, at -70 °C.Gel Filtration
Gel filtration was carried out on a
Waters 510 HPLC system equipped with an LKB Uvicord SII model 2238
detector and a 277-nm filter. Separations were performed at 0.5 ml/min
on a 7.5 300 mm TSK G3000SW column with a 7.5
75 mm TSK
SWP pre-column (Toyo Soda, Japan) by isocratic elution with 1 M NaCl, 20 mM Tris, pH 7.2, 5 mM DTT, and
0.05% LDAO.
-galactosidase
(Worthington Biochemicals), and blue dextran (Sigma).
g at 4 °C) in 0.4% LDAO, 2 M NaCl,
10 mM Tris, pH 9.4, and 10 mM DTT at a final
concentration of 1 mg/ml. The suspension was vortexed for at least 2 h
at 4 °C. The sample was then diluted with an equal volume of 100
mM Tris at pH 7.2. Particulates were pelleted as before, and
200 µl of the supernatant was injected onto the column. In this
paper, the non-pelleting material in the presence of detergent will be
referred to as gap junction complexes or oligomers.
0.5 mg/ml, and then
concentrated 2-3-fold using a Centricon 10 microconcentrator
(Amicon, Danver, MA). A 1.0-ml aliquot was injected onto the column,
using a running buffer consisting of 50 mM HEPES, pH 8.0, 1.0 M NaCl, 10 mM DTT, 10 mM EDTA, and 0.5% LDAO
at a flow rate of 0.5 ml/min.
Ultracentrifugation
Linear 2-ml 5-20% or
10-25% sucrose gradients (Schwarz/Mann UltraPure grade) in
HO or D
O in 20 mM Tris, pH 9.2, 1 M NaCl, 5 mM DTT, and 0.05% LDAO were generated by
freezing and thawing a gradient containing five steps of sucrose.
Marker proteins or solubilized gap junction samples (separated by HPLC
or FPLC) were applied to the top of the gradients. All marker proteins
were purchased from Sigma, and their respective partial specific
volumes and corrected sedimentation coefficients were: horse heart
myoglobin, 0.741 ml/g and 2.04 s, chicken egg ovalbumin, 0.744
ml/g and 3.53 s, bovine transferrin, 0.725 ml/g and 4.92 s, rabbit muscle aldolase, 0.735 ml/g and 7.35 s, and
bovine liver catalase, 0.730 ml/g and 11.30 s(23) .
Gradients were centrifuged at 55,000 for 4 h at 4 °C (no brake) in
a TLS55 swinging bucket rotor in a TL100 ultracentrifuge (Beckman
Instruments). Gradients were fractionated by piercing with a 25-gauge
needle. The refractive indices of the fractions were determined with an
Atago R5000 hand refractometer. The sedimentation of the markers was
determined by SDS-PAGE and the presence of gap junction complexes by
electron microscopy.
SDS-PAGE
Preparations were analyzed by SDS-PAGE
according to Laemmli(24) , but without boiling. Bands were
visualized by Coomassie (Brilliant) Blue or silver
staining(13) .Immunoblotting
Affinity-purified antibodies
prepared against synthetic peptides corresponding to proposed
cytoplasmic loops of the human liver protein (14) and the rat
liver protein (25) were used for immunoblotting. These antibodies were
prepared, purified, and characterized as described
previously(14) . Western transfers were conducted using a
modification of the procedure of Towbin et al.(26) ,
as described by Milks et al.(14) . Antibody binding
was detected by
I-protein A and visualized by
autoradiography. Where noted, band intensities were digitized with an
LKB Ultroscan XL laser densitometer.
Electron Microscopy
Acetone-washed 400 mesh copper
grids (Ernest Fullam Co.) were coated with 5% collodion in amyl acetate
(Ted Pella, Inc.) and stabilized with a carbon film using an Edwards
E306A Coating System. The collodion was then removed with acetone, and
the grids glow-discharged using an EMscope model 350 unit. Samples were
applied to the grids for 1 min and then blot dried using filter paper
applied to the grid edge. Each grid was washed three times by inversion
onto a drop of 10 mM Tris-buffered solution, pH 9.2, for 1
min, with blotting between washes, and stained by inverting on a drop
of 1% uranyl acetate solution for 1-2 min and blot-dried. Samples
were examined on a Philips CM-12 electron microscope at 100 kV.Circular Dichroism Spectroscopy
Native
double-membrane junctions were pelleted as before, resuspended in 10
mM Tris, pH 9.2, and sonicated to clarity with a Branson
Sonifier microprobe using repeated 8-12-s bursts. During this
procedure, the sample vial was placed in an ice water-jacketed chamber
to prevent heating. Typically, five bursts were sufficient to render
the solution transparent. The membrane suspensions were centrifuged at
12,800 g for 15 min and the supernatant containing the
smaller membranes fragments used for further analysis.
, and two
to
transitions, one parallel and one perpendicular to the optical
beam. Since the energy, and thus the location of the absorption peak,
of each transition dipole is affected by the dipole moment of its local
environment, the CD bands of the various peptide transitions of
membrane proteins may be differentially red- or blue-shifted relative
to their location in the spectrum of an aqueous protein(30) .
Accordingly, additional analyses were conducted in which the reference
basis sets were deconvoluted to yield three gaussian absorption bands,
and these bands shifted independently to optimize the fit of the
reference data base to the experimental spectrum as reflected by
minimization of the NRMSD(22, 31, 32) . The
calculated secondary structures determined by this type of analysis may
reflect the actual secondary structure present more accurately and are
included in the calculations as a limit for the variability in our
calculations of net protein conformation.
Cross-linking
Gap junction membranes were
solubilized in 2% Dodecyl maltoside, 20 mM TEA, pH 9.2, 20
mM EDTA, and 10 mM DTT, at a concentration of 0.5
mg/ml. To cross-link, a 100 mM DMS (Pierce) stock solution in
200 mM TEA, pH 9.2, was diluted 10-fold in the buffer and
allowed to react at 4 °C. The reaction was quenched at various time
points with excess glycine. Reaction products were analyzed by
immunoblotting.
Preparation of the Isolated Gap Junction
Oligomers
Conditions for dissociating the isolated gap junction
membranes into oligomeric units were adapted from Stauffer et
al.(19) , using LDAO as the detergent. Oligomers were then
isolated by gel filtration on either HPLC or FPLC systems. Buffer
conditions were slightly different for HPLC and FPLC since restrictions
due to the chemical nature of the silica matrix of the HPLC column
required the sample pH to be reduced before chromatography on this
system. The elution volume, V, of gap
junction complexes isolated under these conditions was determined by
examining electron micrographs of negatively stained aliquots of the
eluant. Aliquots containing gap junction oligomers were identified by
the appearance of 70-80 Å circular structures containing a
central 15-20-Å electron-dense center (i.e. 70-80 Å diameter ``doughnuts'') (Fig. 1B); structures which are similar to those
observed in electron micrographs of isolated gap junction plaques (Fig. 1A). The presence of
protein in
these samples was confirmed by immunoblotting. Immunoblots indicated
that
protein was present also in the samples before
injection (but, relative to
protein, at much lower
concentrations). This protein was also detected in the FPLC eluant but
not in the HPLC eluant. The
protein was observed to
co-elute with
protein (Fig. 2). The absence of
detectable
protein in the HPLC eluant was probably
due to less material being loaded on the HPLC column resulting in
dilution below the detection limit during chromatography.
liver connexins or
liver
connexins. The position of the monomers of
and
gap junction protein is indicated on the left-hand
side.
Stokes Radius (R
Size information of the gap junction complex
isolated in detergent was obtained by gel filtration on HPLC in three
independent experiments. The partition coefficient, K)
Determination
, of a species is defined
as(33) :
is the void volume of the
column and V
is the total volume of the
column. In these studies, the V
was 5.77
± 0.15 ml as measured with blue dextran, and the V
was 18.19 ± 0.65 ml by salt
elution. Le Maire et al.(34) have shown that for HPLC
gels in general, and TSK3000 SW columns in particular, the porous media
of the gel may best be described as surface fractals, and there exists
a linear relationship between ln R
and ln
(1 - K
). By measuring the V
(by A
) of
various marker proteins of known R
, the
gap junction complex was determined to have an R
of 41.0 ± 0.6 Å (Fig. 3).
versus ln (1 - K
) as determined by gel filtration on a
TSK3000 SW column. The marker proteins used with their corresponding R
values (in A) were myoglobin,
18.9, malate dehydrogenase, 35.1, lactate dehydrogenase, 42.0,
ferritin, 63.0, and
-galactosidase,
68.6(23, 48) . Except where drawn, the associated error bars were smaller than the symbol dimensions. The arrow corresponds to the observed elution of the
gap junction oligomers characterized in later studies as being a
hexameric hemi-channel.
Determination of s
The sedimentation coefficient, s, and
partial specific volume, and
Molecular Weight
, of the isolated complex was determined
by using markers of known
and s
in
ultracentrifugation experiments on parallel linear gradients in
H
O and D
O(35, 36) . The
composition of the fractions was determined by SDS-PAGE of the markers
and by electron microscopy, and confirmed by immunoblotting, as
described above, for the solubilized gap junctions. It was assumed that
detergent binding is equivalent in H
O and D
O;
this assumption results in less than 10% error in the calculated
molecular weight of other membrane proteins(35) . The
similarity of the s
calculated from
H
0 and D
0 gradients (8.76 and 8.74 s,
respectively) indicate that this is a valid assumption. The
sedimentation coefficients for the markers and the gap junctions were
determined by measuring the half-distance of migration, r
, and from the relationship:
, at any
point in the gradient was determined by interpolation of the measured
density at the top and bottom of the gradients. The average viscosity,
, experienced by the marker proteins, was determined from the
relationship:
for the markers versus gradient fraction. The partial
specific volume of the complex,
, can then be calculated:
O and D
O gradients since:
from gel filtration, we can then determine the molecular weight
of the complex:
and f
are the fractional weight composition of protein and
detergent, respectively;
for LDAO is 1.112 ml/g and
was calculated from the amino acid
composition to be 0.748 ml/g(37) .
CD Spectroscopy
The secondary structures of the
isolated gap junction oligomers and the gap junction membranes may be
directly compared by CD spectroscopy, since the lipid and detergent
molecules are not optically active in the low UV range. Artifacts
caused by differential scattering (38) and absorption
flattening (39) have been shown to be negligible in sonicated
gap junction plaques(22) . Contamination of the samples by
collagen was corrected by subtraction of the calculated spectral
contribution of collagen to the samples (22) . No collagen was
detected in the samples of isolated solubilized oligomers, but the
junction membrane samples contained 2.4-3.7% collagen and were
corrected by subtracting the spectral contribution of this contaminant. -helix content calculated from the CD
spectrum using a non-linear least-squares fitting of the reference data
set (29) has been shown to strongly correlate with the helical
content as determined by x-ray crystallography. This type of analysis
has also been successful in describing the helix content of two
membrane-associated polypeptides, bacteriorhodopsin (40) and
crambin (41) . The discussion of the experimentally determined
secondary structure will focus primarily on the
-helix content
since it is not possible to determine other secondary structures with
high reliability by this technique.
-helix content in
this protein, consistent with the value determined in previous CD
studies(22) . The isolated detergent-solubilized oligomers
contained 66%
-helix (Table 2).
to
*
transitions and the single n to
* transitions of the
reference basis spectra by -5, 0, and -3
mm
, respectively, lowered the NRMSD to 0.074 in the
case of detergent solubilized gap junctions, but did not appreciably
affect the calculated secondary structure (67%
-helix). This type
of analysis did not improve the fit for the membrane bound samples
(NRMSD = 0.025).
Chemical Cross-linking
The oligomeric state of an
aggregate may be examined using chemical cross-linking agents to
identify the statistical distribution of neighboring molecules. DMS, a
membrane permeant, homobifunctional 11-Å linker which reacts with
primary amines, has been shown to effectively cross-link large globular
oligomers in solution (43) and in membranes(44) . The
gap junction plaques, resuspended in TEA buffer at pH 9.2, were
cross-linked by DMS. Even at reaction times as short as 1 min, these
samples were so extensively cross-linked that they failed to enter an
8% SDS-polyacrylamide gel (data not shown). This is probably due to the
close proximity of the individual hexamers within a gap junction
plaque. However, upon incubation of isolated gap junction oligomers
with this cross-linker, protein in higher molecular
weight aggregates was detected by immunoblotting. Regularly spaced
bands representing the gap junction monomer and oligomers (dimer,
trimer, . . . ) up to and including the hexamer were observed, as well
as higher order multimers corresponding to cross-linked hexamers (Fig. 5A). The assignment of oligomer stoichiometry was
confirmed by the linearity of the semilogarithmic plot of aggregation
state versus electrophoretic mobility of the species (Fig. 5B). Assuming that each species is comprised of
integral multiples of the
polypeptide, the
correlation coefficient was closest to unity when the major bands were
assigned as being oligomers of 1, 2, 3, 4, 6, 12, and 18 units.
Pentameric assemblies were observed in overloaded gels before
cross-linking (lane 3), but could not be resolved in lanes
containing cross-linked samples due to the high density of observed
hexamers (lane 4). The higher order aggregates observed at
longer reaction time had molecular weights consistent with those of
cross-linked hexamers (12- and 18-mers).
gap
junction oligomers. Samples were applied to a gradient 4-15%
SDS-polyacrylamide gel, transferred to nitrocellulose, and treated with
antibodies to
rat liver connexin. Samples were
treated as follows: lane 1, untreated sample (enriched
membranes); lane 2, detergent-solubilized sample; lane
3, detergent solubilized samples, preincubated with excess glycine
prior to cross-linking with 10 mM DMS; lane 4,
detergent-solubilized sample cross-linked with 10 mM DMS. The
reaction was quenched with excess glycine after 20 h. The arrowheads on the right side from bottom to top, indicate
position of monomers, dimers, trimers, tetramers, pentamers, and
hexamers. The lines on the left side indicate the position of
molecular mass markers from top to bottom: 205, 116.5, 80, and 32.5
kDa. The positions of the
monomer and hexamer are
denoted by the letters on the right side by M and H, respectively. B, semilogarithmic plot of
aggregation state versus mobility. Lane 4 of A was densitized; a linear relationship was observed for (r = 0.9995) with the peak densities corresponding to
monomers, dimers, trimers, tetramers, hexamers, dodecamers, and
18-mers.
hexamer with properties equivalent to a single
membrane hemi-channel. Immunoblots of eluant from HPLC indicated that
the
gap junction protein is present in a number of
fractions (data not shown, but the
elution profile is
similar to that observed in FPLC, shown in Fig. 2). However, the
concentration of this protein is greatest in the eluant fraction which
subsequent studies characterized as having physical parameters
consistent with a hexamer. Some gap junction protein also elutes with
the void volume as partially solubilized membrane patches or large
micelles (observed in micrographs as double membranes and amorphous
blobs, and present at greater concentration when the DTT was not
fresh), or as paired hexamers at slightly later volumes (also seen as
``doughnuts'' in micrographs, data not shown). Immunoblots
indicate that
protein is also present at higher V
, presumably as lower order aggregates (e.g. monomers, dimers, and trimers). Samples prepared for
injection contained primarily
protein, but a small
amount of
protein was also present. In FPLC eluant,
the
protein was observed to co-elute with the
species, suggesting a close association between
and
connexins, since the calculated
molecular mass of a
hexamer is different from a
hexamer (192 versus 156 kDa).
oligomers with
time. The molecularity of the reaction products increased step-wise to
six, and higher order aggregates were multiples of this hexameric unit (i.e. 12-, 18-, and 24-mers) (Fig. 5). It is possible
upon cross-linking of native channels, the pentameric units have
shifted molecular weights, such that the dominant band in lane 4 of Fig. 5A corresponds to pentameric units. We
think this unlikely since the lower order aggregates (i.e. dimers, trimers, and tetramers) run equivalently independent of
cross-linking and their mobility is consistent with the linear
regression analysis. Parallel experiments using membrane-bound samples
yielded aggregates which did not enter the resolving gel, probably due
to extensive cross-linking of the close-packed oligomers.
90 Å from the
center of the gap, and it has a radial diameter of
50 Å
within the bilayer and
60 Å on either face (the cytoplasmic
domain may also project an additional 20 Å onto the membrane
plane)(45) . Gel filtration studies indicate that the Stokes
radius of the solubilized hexamer is 41.0 ± 0.6 Å, which
is consistent with these channel dimensions(45) . The molecular
mass for the complex was determined to be 208 kDa. This calculated mass
is the sum of the contributions of protein and detergent in the
complex. The calculated R
, which is
consistent with the channel dimensions, suggests that detergent binding
was not extensive. Given that the
of
gap
junction as calculated from its amino acid composition is 0.748
(proteins typically have
of 0.72-0.76), and LDAO has a
of 1.112, it is calculated that
85% of the mass ratio in the
protein-detergent complex was due to the protein, and hence the
calculated molecular mass for the connexin oligomer is 175 kDa. This
must be considered an estimate, since it is critically dependent on
determination of v for both detergent-bound and detergent free
connexin. Given that the gap junction
polypeptide has properties which cause it to run anomalously on
SDS-PAGE as a 26-28 kDa band, our calculated molecular mass
(which, like SDS-PAGE, is a function of R
) of 175 kDa is consistent with a
hexameric assembly of these units.
-helix
(66% relative to 52% in the membrane bound form). A possible source for
this difference may be the partial refolding of the cytoplasmic domain,
which has been shown to be sensitive to detergent or alkali
treatment(22) . Alternatively, the exposed extramembranous
domain of the solubilized hexamer may be folded differently than this
domain in the membrane-bound samples, which are paired and not exposed
to solvent. It is also possible that some of the differences may be
attributed to the presence of contaminating species in our membrane
preparations, which are removed by subsequent solubilization and
purification. However, the elevated
-helix content in both samples
is more than sufficient for the transmembrane domains to be modeled as
-helical
bundles(14, 45, 46, 47) .
-gap junction
protein from rat liver in detergent solution exists as a hexamer and/or
multiple of this basic unit. Whether other connexins also exist as a
hexamer is not known. However, based on the predicted common topology
of the connexin proteins, it is likely that all gap junction proteins
exist as hexamers. As shown in this study, both
and
-connexin co-elute suggesting that they may be
associated with each other. However, further experiments will be
required to distinguish between heterotypic and heteromeric
associations.
We are grateful to Jessica van Leeuwan for excellent
technical assistance, Larry Xue at the Center for Statistics at the
University of Pittsburgh for help with the error analysis, and Mark
Yeager for helpful discussions throughout the study.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.