Download Cardiac myocyte gap junctions

Cardiac myocyte gap junctions: evidence for a major connexon protein with
an apparent relative molecular mass of 70000
E. HARFST, N. J. SEVERS
Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, England
C. R. GREEN*
Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, England
* Author for correspondence
Summary
It is widely accepted that there is a family of gap
junction connexon proteins, their distribution appearing to vary with tissue type and species. In
cardiac tissues the major junctional channel component identified is a 43K (K=103Mr) polypeptide.
Using a gap junction isolation protocol in which low
temperatures are maintained, and which is detergent-free, we have identified a second gap junctionrelated protein in cardiac tissues with an apparent
relative molecular mass of 70 000. Antibodies raised
to three synthetic peptides matching portions of the
43K gap junction protein cDNA sequence cross-react
with the 70K protein, but biochemical studies indicate that these proteins are distinct from one
another. The structures that contain the 70K protein
are susceptible to fragmentation at warm temperatures, and by electron microscopy this can be correlated with loss of 'minidomains' within the junctional
plaque. Using a gap junction enriched-fraction prepared from purified isolated adult myocytes we show
that both the 43K and 70K gap junction proteins are
present in ventricular cardiac myocytes. In such
preparations, and those from whole heart, the 70K
protein appears to be the major gap junction-related
protein present
Introduction
also been described in Xenopus embryonic tissues, a 30K
form (Gimlich et al. 1988) and a 38K form (Ebihara et al.
1989). In only one example have two protein types been
shown to coexist within the same junctional plaque; the
26K and 32K types of rodent liver (Nicholson et al. 1987).
Where full sequence data are available, the proteins all
show a high degree of homology with the extracellular and
transmembrane regions, in particular, highly conserved.
In the heart the 43K connexon polypeptide is considered
to be the major gap junction protein present (Beyer et al.
1988; Manjunath et al. 1982,1984,1985), although there is
some molecular evidence for a second gap junction protein
gene (Beyer et al. 1988, 1989; Fishman and Leinwand,
1989). However, using a cardiac gap junction isolation
protocol that we have developed (Gourdie et al. 1988),
which permits the enrichment of junctions while maintaining preparations at low temperatures and, in addition,
avoids the use of detergents, we have identified a major
gap junction-related polypeptide with an apparent molecular mass of 70000. Three antibodies to synthetic peptides
have been raised to putative cytoplasmic portions of the
cardiac 43K protein, all of which recognise the 70K protein
on Western blots, indicating a high degree of sequence
homology between the two protein types. A series of
biochemical experiments indicates that they are, however,
separate entities. Furthermore, by isolating ventricular
myocytes and using these as a starting material, we have
Gap junctions are the membrane specialisations that
permit the exchange of small metabolites and ions between neighbouring cells. In the heart, their major role
appears to be in electrically coupling the myocytes, and
coordinating their contractions (for review, see Page and
Manjunath, 1986; Severs, 1990), but gap junctions also
play an important role during development (Warner et al.
1984), tissue patterning (Fraser et al. 1987), regulation of
cell growth (Mehta et al. 1986) and metabolic coupling
(Lawrence et al. 1978).
Gap junctions are composed of aggregates of channels
formed where connexons in one cell membrane become
aligned with those of the adjacent cell. It is now widely
accepted that there is a family of related gap junction
proteins that make up the connexons, with six protein
subunits to each connexon (Zimmer et al. 1987; Milks et al.
1988). The occurrence of the different proteins varies with
species and tissue type. In mammalian tissues, the major
gap junction proteins identified are a 32K (K=10 3 M r )
liver type (Kumar, and Gilula, 1986; Paul, 1986), a 26K
type in rodent tissues (Nicholson et al. 1987; Zhang and
Nicholson, 1989), an eye lens protein of 70K with in vivo
breakdown to 38K (Kistler et al. 1988), and a cardiac gap
junction protein of 43K (Beyer et al. 1987; Manjunath et al.
1982, 1984, 1986). Two other gap junction proteins have
Journal of Cell Science 96, 591-604 (1990)
Printed in Great Britain © The Company of BiologiBts Limited 1990
Key words: gap junction proteins, connexin43, ventricular
myocyte junctions, cell communication.
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been able to demonstrate that the two gap junction-related
proteins are both present within this specific cell type. The
70K protein is rapidly lost from membrane fractions when
preparations are warmed, offering an explanation as to
why it has not been recognised by workers using detergent-extraction junction isolation procedures that require
warmer temperatures. This loss is not by protein solubilization, but by fragmentation of the structures in which it is
contained, and in structural studies we show that cardiac
gap junctions are susceptible to such fragmentation.
The 70K gap junction-related protein identified here
appears to be the major gap junction protein in the heart.
Materials and methods
Synthesis ofpeptides
Three peptides were obtained from Dr N. B. Gilula (Research
Institute of Scripps Clinic, La Jolla, CA). These had been synthesised to match portions of the amino acid sequence of the cardiac
43K gap junction protein predicted from the nucleotide sequence
of a cDNA clone (Beyer et al. 1987). The peptides, each 12 amino
acids long with a cysteine added to the carboxyl terminus to
facilitate carrier protein attachment, were synthesised using the
simultaneous multiple-peptide synthesis procedure of Houghten
(1986). They were made to match residues 101-112 (termed HH),
131-142 (HJ) and 237-248 (HQ) of the 382 amino acid rat heart
protein (Table 1).
Coupling of synthetic peptides to carrier protein
Initially peptides were coupled to the carrier protein keyhole
limpet haemocyanin (KLH) at their carboxyl- or amino-terminal
ends with l-ethyl-3-(3-dimethylaminopropyl)carbodiimid (Sigma),
using the method of Tamura et al. (1983). These conjugates did not
prove immunogenic in our hands. Following glutaraldehyde
crosslinking of the antigen to KLH, however, antibodies were
subsequently obtained. KLH was prepared aa a lOmgml" 1
solution in phosphate-buffered saline (PBS) (pH7.2), each of the
peptides in a 2mgml~ 1 solution in PBS, and glutaraldehyde as a
5 % (v/v) solution in PBS. Each peptide was mixed at a ratio of
1:20 (w/w) with the solubilised KLH. Glutaraldehyde was added
(50/d ml" 1 ) and the mixture vortexed periodically over 30min.
The conjugates were dialysed against PBS (12 000-14 000 Mr cut
off) for 4-5 days at 4°C before use.
Preparation and screening of peptide antisera
Antibodies were raised in Sandy half-lop rabbits. Initial injections were with 0.4 mg peptide (linked to KLH) in Freund's
complete adjuvant (Sigma) with 0.2 mg boosts in Freund's incomplete adjuvant. Injections were made subcutaneously, with bleeds
taken from ear vessels 2 weeks after the initial injections, and 10
days after each of three boosts. Rabbits were administered
Table 1. Sequences of the synthetic peptides used for
rabbit immunizations
Peptide
Residue
assignment
HH
101-112
HJ
131-142
HQ
237-248
Sequence
Arg-Lys-Glu-Glu-Lys-Leu-Asn-Lys-Lys-GluGlu-Glu-<Cys)
Glu-Ile-Lys-Lys-Phe-LyB-Tyr-Gly-Ile-GluGlu-HMCys)
Lye-Asp-Arg-Val-Lys-Gly-Arg-Ser-Asp-ProTyr-His-<Cys)
Column 1 gives the terminology used, H referring to heart, and the
second letter to the approximate position of the sequence along the
length of the 43K connexin protein measured from the amino-terminal
end (see Milks et al. 1988). The second column gives the amino acid
positions along the protein coded for by the cDNA, and the third, the
sequences of the peptides Bynthesised. The cysteine on the end of each
peptide was added to allow for carrier peptide coupling.
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E. Harfst et al.
0.5 ml kg x Hypnorm (Janssen Pharmaceutical) 15min before
bleeding to help with serum collection. Following the final boosts,
rabbits were bled regularly at 1- to 2-week intervals.
Anti-peptide antibodies were screened for reactivity and specificity using dot blot assays (modified from Hawkes et al. 1982).
Screening was carried out against each peptide, KLH, Trisbuffered saline (TBS) (used as buffer solution for blot studies) and
fibrinogen (selected to provide a specificity test against an
unrelated protein). Solubilised peptides, KLH or fibrinogen
(1-2 fig in l m g m T 1 solution) were dotted onto nitrocellulose
strips (0.2/an pore, Schleicher and Schuell, BA83), which were
then allowed to dry before blocking with bovine serum albumin
(BSA) (essentially fatty acid free, Sigma), or non-fat milk powder
(BLOTTO, Johnson et al. 1984) in TBS (pH7.4), for 2h at room
temperature or overnight at 4°C. Primary antibody incubations
were carried out at room temperature for 1-2 h, with the crude
serum diluted 1:5 or 1:10 in TBS. The secondary antibody used
was 126I-radiolabelled anti-rabbit IgG or protein A with subsequent exposure on Fuji RX or RX-G X-ray film.
Affinity purification of anti-peptide antibodies
Glutaraldehyde-activated GTA-Prodisks (FMC, New Jersey) were
used to construct KLH and peptide HH affinity purification
substrates. In each case 5-10 mg of protein at a concentration of
O.Smgml"1 in disodium phosphate buffer was filtered (0.2/im
pore) and cycled over the activated disc for 1 h. The system was
then flushed with buffer and 0.4 M glycine pumped through for 1 h
to block any remaining active sites. Efficiency of the immobilization was assessed by A280 spectrophotometry of the starting and
end protein solutions. Discs were stored after flushing with 0.02 %
sodium azide in buffer. Antibody purification was achieved by
passing filtered, diluted (1:2 or 1:5) serum over the disc (prewashed to remove the sodium azide present) at a rate of
Smlmin" 1 for l h at room temperature. The system was then
flushed with 1 M NaCl to remove non-specific IgGs, followed by
elutdon of antibodies with 0.1 M glycine, pH 2.5, pumped through
at 4 ml man"1. Antibodies were collected and the glycine solution
neutralised with Tris buffer. The process was monitored using a
fraction collector with absorbance measurement and chart
recorder facilities (Pharmacia). Antibody fractions were dialysed
for 3 days against 76 mM KC1, 5mM Trizma base, pH7.4, and
concentrated in a GyroVap centifugal evaporator to l . S 1
for storage. They were diluted 1:100 in TBS before use.
Protein gel electrophoresis and Western blot analysis
SDS—polyacrylamide gel electrophoresis was carried out using
the procedure of Laemmli (1970) with modifications as outlined by
Zimmer et al. (1987). Gels were routinely run with 6% (w/v)
stacking gels and 12.5 % (w/v) separating gels. Relative molecular mass markers (Sigma SDS-7) were bovine albumin (66K), egg
albumin (45K), glyceraldehyde-3-phosphate dehydrogenase
(36K), carbonic anhydrase (29K), trypsinogen (24-28K), trypsin
inhibitor (20.1K) and lactalbumin (14.2K). Gels were stained with
Coomassie Brilliant Blue or transferred for Western blotting as
appropriate.
Samples were routinely solubilised in SDS—mercaptoethanol
sample buffer, but for some gap junction protein studies membrane preparations were solubilised with a solution containing an
alternative reducing agent, dithiothreitol (40 mM DTT, 1 % SDS,
5% sucrose, 100mM Trizma base (pH8.0), lmM EDTA, room
temperature), or alkylated by boiling for 4 min in 20 mM dithiothreitol (DTT), 1% SDS, 100 mM Trizma base (pH8.0) and then
adding 45 mM iodoacetamide (2.25-fold molar excess over the
reducing agent) with a further 2 min of boiling. These samples
were then loaded directly onto separating gels. Western blotting
was carried out according to the method of Towbin et al. (1979).
Transfer to nitrocellulose paper was at 110 V (constant voltage)
for 45 min at room temperature. Immunoblots were blocked in
essentially fatty acid-free BSA (Sigma) or BLOTTO (Johnson et
al. 1984) and incubations carried out as outlined above for dot blot
assays. Antibodies were used separately, or combined into a
'polyclonal' cocktail.
Cardiac sarcolemma and gap junction enrichment
Whole rat-heart homogenates for gel electrophoresis were prepared by freezing fresh material in liquid nitrogen, grinding with
a mortar and pestle and thawing directly into SDS-mercaptoethanol sample buffer.
Membrane and gap junction enrichment was carried out using
the detergent-free method of Gourdie et al. (1988). Briefly, hearts
from stunned and neck-dislocated rate (200-250 g) were diced and
homogenised at moderately low speed in a Polytron (25 mm blade,
speed setting 2, 60s for 10 hearts in 150ml buffer). The buffer
used contained 5 mM Trizma base, 3 mM EGTA, 3 HIM EDTA, 0.5 %
BSA and 250 mM sucrose (pH8.3) and was designed to maintain
the integrity of the mitochondria (which make up ~35 % of the
volume of a cardiac myocyte), permitting subsequent separation
from the membrane fraction. The homogenate was filtered and
pelleted at 28 000 # for lOmin, resuspended in buffer and
1 mgml" 1 DNase I (2500 units mg" 1 ; Sigma) was added to disrupt
the contractile apparatus by depolymerisation of actin (Zimmer
and Goldstein, 1987). The mixture was incubated for 45min, and
the efficiency of the DNase action was followed by phase-contrast
microscopy. The homogenate was pelleted (40000g) for lOmin,
resuspended in buffer containing 0.6 M KI, 6mM NaS2O3 and
stirred for 10 min to 1 h. The suspension was pelleted as above and
the KI wash repeated if necessary. The washed pellet was
resuspended through a syringe and needle into the original buffer
containing 200 mM KC1 and layered onto a discontinuous sucrose
gradient (sample plus sucrose at final concentration of 30 % (w/w)
overlayed with buffer/KCl solution). The gradients were centrifuged at 115 000 g for 2.5 h and the interface was collected and
washed in 1 mM NaHCOa. Mitochondria and most non-membrane
material were trapped in the sucrose layer or pelleted. The entire
preparation was carried out at 4°C, and lmM phenylmethylsulphonyl fluoride (PMSF) (final concentration) was included in each
solution prior to use. In preparations carried out specifically to
obtain gap junction protein breakdown products, the PMSF was
omitted.
A further enrichment of gap junctions was achieved by sonicating the membrane fraction for 30 s in 20 mM NaOH, 250 mM KC1,
pelleting the suspension, resuspending in bicarbonate buffer and
adding deoxycholate to a final concentration of 0.3 % (w/v). The
preparation was resonicated for 10 s in a 1.5 ml microfuge tube,
pelleted and washed. Gap junctional purity could be improved by
carrying out a brief sonication in 0.3 % deoxycholate at 4°C, but in
the majority of our work, and in all the biochemical studies, no
detergents were used.
All steps were monitored by electron microscopy.
Cardiac myocyte dissociation and myocyte gap junction
enrichment
Myocytes were isolated from hearts of 2-2.5 kg New Zealand
White rabbits using the method of Powell et al. (1980). All
glassware was acid-washed and siliconised (Repelcote; Hopkin
and Williams) before use. Rabbits were injected with 2500 units of
heparin before killing, and the heart and lungs were removed and
placed into ice-cold cardioplegic Krebs-Henseleit solution containing 20 units ml" 1 heparin. Other organs and tissues were
trimmed from the heart, which was then cannulated via the aorta
for perfusion. Cannulation was routinely completed within
2-4 min of animal death. The heart was perfused for 5 min at a
rate of 30-40 ml min" 1 with nominally calcium-free Krebs-Henseleit solution followed by a 10-min perfusion (with recirculation)
with collagenase (lmgml" 1 crude bacterial type 1 collagenase
(Lome Laboratories, Reading), in Krebs-Henseleit buffer containing 0.15% fatty acid-free BSA (Sigma) and 25/iM CaClj).
After this time the heart was removed from the cannula, trimmed
of connective tissue and aorta, and the ventricles were cut into
four pieces vertically from the apex. The pieces were agitated in
sealed Erlenmeyer flasks (rotary agitation, 60 cycles min" 1 ) at
37 °C in four consecutive 5-min changes of the collagenase
solution above, but now containing 2 % BSA (7.5 ml solution each
change). Following each digest (except the first, which was
discarded) the tissue was filtered through 250 /an nylon mesh (R.
Cadish and Sons, London), and the filtered cells were carefully
layered onto two volumes of a wash medium (Krebs-Henseleit
buffer, 2% Fraction V BSA, 25/u* CaCy and spun at 37 g in a
bench-top centrifuge (swing-out buckets, 3-min spins, 3x10 ml
tubes per digest). This wash step was repeated three times, and
the final pellet resuspended into Krebs-Henseleit, 2 % Fraction V
BSA, 0.5 mM CaClj. Cell purity and yield were assessed by light
microscopy. Further purification of the myocytes was obtained by
centrifuging on a preformed Percoll gradient consisting of 40 ml
Percoll (Pharmacia, Sweden), 60 ml 1.5x strength Krebs-Henseleit buffer containing 2.5mM CaClj, 0.1% Fraction V BSA.
Gradients were preformed by centrifuging them for 45 min at
20 000 g. Myocytes in buffer were layered on top of the gradients,
which were then spun at 1500 revs min" 1 for 2 min at room
temperature in swing-out buckets. Purified myocytes were harvested from a band about three-quarters of the way down the
gradient. The cells were washed twice in Krebs-Henseleit containing 2mM CaCl2 (2-3 min spins at 31 g).
All solutions used throughout the isolation protocol were
gassed with 95 % O2/5 % CO2. For perfusion of the heart, the
solutions were gassed continuously at a level low enough to
prevent frothing. Other solutions were gassed immediately before
use and intermittently during use, where possible.
The partial enrichment of gap junctions from purified dissociated myocytes was achieved by modifying the method of
Gourdie et al. (1988) outlined above for whole-heart junctions.
This modification, eliminating sucrose-gradient steps, is necessary as the gap junctions become vesiculated during myocyte
isolation and contain trapped cytoplasmic material (Severs et al.
1989), substantially altering their separation characteristics. All
subsequent steps were at 4°C and solutions contained lmM
PMSF. Following their final wash, myocytes resuspended in the
cold Krebs solution were allowed to settle in round-bottom tubes
and the supernatant was removed. A 3-ml sample of the isolation
buffer used for whole hearts was added and the cells were
homogenised in a Polytron (10 mm blade, 2 x 20 s speed setting 5).
The homogenate was immediately diluted to 50 ml and centrifuged 10 min, 20 000 g. The pellet was then resuspended through a
19-gauge needle into the same buffer containing 1 mg ml" * DNase
I and incubated with slow stirring for 45 min. The homogenate
was again pelleted as above and resuspended into 150 ml isolation
buffer containing 0.6 M KI, 6 mM Na2Sa03, and stirred for 10 min
before repelleting. This step was repeated, with the pellet from
the second KI wash resuspended to 5 ml in 1 mM NaHCO3, frozen
in liquid nitrogen and thawed. This freeze—thaw step is to aid in
the disruption of vesicles, allowing release of their contents. The
membrane suspension was then sonicated in 20 mM NaOH,
250 mM KC1, 0.3 % deoxycholate, and washed as above for wholeheart junction preparations before storing at —80°C until
required for electrophoretic and Western blotting experiments.
Gap junction enrichment was monitored by electron microscopy.
Electron microscopy, immunocytochemistry and
immunohistochemistry
Samples taken to monitor gap junction enrichment during the
isolation protocols, or following antibody labelling of enriched
junction fractions, were fixed in 2.5% glutaraldehyde in sodium
cacodylate buffer. The fixed pellets were post-fixed in 1 % OsO4,
dehydrated in ethanol and embedded in Emix (Emscope Laboratories) as previously described (Severs et al. 1986). For immunocytochemistry extra en bloc and tannic acid staining steps as
outlined by Zimmer et al. (1987) were introduced to enable
visualisation of antibody labelling.
For immunocytochemistry, junction-enriched preparations
were incubated with primary antibody (1:10 dilution in PBS) and
gold-labelled secondary antibody (Jannsen or BioCell) using the
suppliers' recommended protocols, before fixation and embedding
as above. Immunohistochemical localisation was carried out on
frozen sections or sections of material fixed in Zamboni's fixative
(Toshimori et al. 1987) and wax embedded. Wax sections were
dewaxed in xylene, rehydrated and incubated with 0.1 % trypsin
to re-expose antigenic sites before antibody localisation (for
details see Gourdie et al. 1990). Localisation was carried out with
antiserum diluted 1! 10 in PBS and FITC-conjugated swine anti-
Cardiac gap junction proteins
593
Fig. 1. Isolation of gap junctions from whole heart. A. A thin-section electron micrograph showing a membrane fraction from the
buffer/30 % interface of the KCl/sucrose gradient. The proportion of gap junctions (arrow) to the total material from this interface is
approximately the same as that obtained at the equivalent stage of the standard liver gap junction preparation. Non-junctional
material consists predominantly of membrane vesicles. B. An indication of the level of gap junction enrichment possible after
NaOH/KCl washing and sonication of the fraction shown in A. In this case the preparation was also sonicated briefly in 0.3 %
deoxycholate. This is a selected area, and, although such areas are common and junctions were highly enriched in the preparation,
they do not form strata in the pellet and further purification is difficult. C. A freeze-fracture image of a preparation equivalent to
that shown in B. Several junctions are visible in both face-on and cross-fracture view (arrows), and the connexons in the membrane
(viewed as E-face pits) show remnants of the clumped hexagonal pattern typical of cardiac gap junctions in vivo. A, x l l 200;
B, x72 8O0;C, x 133 000.
rabbit secondary antibodies (Dako). Sections were viewed using a
laser scanning confocal microscope (BioRad MRC 500).
Freeze-fracture of propane plunge-frozen (non-pretreated) junc-
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E. Harfst et al.
tion preparations was carried out using copper sandwich holders
and a complementary fracture device in a Balzers BAT 4O0T
freeze-fracture apparatus (for details see Severs and Green, 1983).
Results
Gap junction enrichment from whole heart
Gap junction enrichment in the cardiac sarcolemmal
fractions was comparable to that achieved at the plasma
membrane stage of gap junction isolation from rat liver
using the protocol of Hertzberg (1984), (Fig. 1A). The
junctions were seen dispersed in the buffer/30% sucrose
membrane fraction with no sign of stratification when the
material was pelleted. Treatments of these fractions with
NaOH/KCl, or NaOH/KCl and detergent gave a substantially increased gap junction enrichment (Fig. IB), the
KC1 being essential to , prevent junction splitting and
hence loss of the morphological marker for purity. Further
separation of junctions from the remaining membrane
fragments was not possible, however, because there was no
stratification in the pellet following this treatment as
there is in liver junction preparations after the NaOH
wash step. Fig. IB shows a selected area of enrichment,
but such aggregations of junctions can occur throughout
the final pellet obtained. While the treatment with deoxycholate can give further enrichment over NaOH/KCl
alone, this is limited and the yield is greatly reduced.
Freeze-fracture of the final preparations confirmed the
presence of true gap junctions. In freeze-fracture, the
junctions were commonly seen in cross-fracture, but en
face views were also detected, scattered within a background of non-junctional membrane and amorphous material (Fig. 1C). The connexon arrays had become compressed, but remnants of the multiple mini-array pattern
characteristic of cardiac myocyte gap junctions (Green and
Severs, 1984; Page et al. 1983), were still recognisable.
Isolation of ventricular myocytes and enrichment of gap
junctions from isolated myocytes
Isolation of ventricular myocytes from adult rabbit hearts
routinely gave 50-60% intact 'rod-shaped' cells (Fig. 2A),
with no significant decrease in the percentage of these
viable cells over 2—3 h. The remainder of the cell population comprised rounded-up myocytes. Typically,
0.5-0.75 ml of myocytes were obtained from one rabbit
heart. Following Percoll gradient separation, the percentage of'rod-shaped' myocytes was increased by 5-10% and
the background comprising small vesicles and particles (as
seen in the light microscope) was greatly reduced. Electron microscopy confirmed that, after this final purification step, no other cell type apart from myocytes, and no
non-myocyte cell remnants containing gap junctions, were
present in the preparation. Gap junctions in the isolated
myocytes are seen either as surface-located structures
with a loop of membrane covering the extracellular face of
the junction, or more commonly as internalised vesicles
(Fig. 2B). The cytoplasmic junctional vesicles contain cellular material, in the form of cytoplasmic matrix or small
organelles such as mitochondria, trapped during the myocyte separation process (for a full description of the
formation and the fate of these structures, see Severs et al.
1989). It is the presence of this trapped material that
necessitated the modification to the junction enrichment
protocol that had been developed for whole heart tissue.
Following homogenisation and washing to remove the
contractile components of the myocytes, the junctions in
the fraction remaining still contained much of this trapped
cellular material (Fig. 2C). Subsequent freeze-thawing
and sonication in NaOH/KCl, however, gave an enriched
gap junction-membrane fraction (Fig. 2D) suitable for gel
and Western blotting studies, allowing results to be
obtained for this specific cell population from the heart.
Antibody characterisation
The three peptides to which antibodies were raised are
termed HH, HJ and HQ (see Table 1). All three peptides
were designed to match regions of the gap junction protein
thought to be exposed on the cytoplasmic surface of the
junction; HH and HJ in the loop region of the protein
between the second and third trans-membrane crossings
and HQ toward the carboxy-terminal end where the
protein exits the membrane after its fourth crossing
(Beyer et al. 1987; Milks et al. 1988; Zimmer et al. 1987).
Screening of serum by dot blot assay showed a clear
Cardiac gap junction proteins
595
Fig. 2. The progressive enrichment of cardiac ventricular myocyte gap junctions. A. A light micrograph showing the dissociated
adult myocytes obtained by collagenase perfusion of rabbit heart. The myocytes are 'rod-shaped', and their sarcomeric banding is
clearly visible. A typical gap junction in an isolated myocyte, internalised as a vesicle during cell separation is shown in B. Note
dense cytoplasmic matrix components trapped in the vesicle interior. The muscle fibres of the myocyte are seen inserting into the
remnants of the fascia adherens junction at the top of the field. After homogenisation of the myocytes, and incubation in DNase and
KI to remove the contractile proteins, gap junctions remain in vesicular form (C), still containing cytoplasmic material in their
interior (compare with B). Finally, following freeze-thaw and NaOH/KCl wash steps, a gap junction-membrane fraction is obtained
from the isolated myocytes (D). While the final preparation is by no means pure, gap junctions (arrows) are enriched in the
preparation and are known to be derived from one cell type only - the ventricular myocyte. A, x350; B, x73 000;C, X79OOO;D,
X68000.
response to the peptide antigens and to their carrier
protein, keyhole limpet haemocyanin. Following boosts
the titre went up markedly (results not shown) but their
specificity was retained. Fig. 3 shows a dot blot matrix
screen with each anti-peptide antibody screened separately against each of the three peptides, the carrier
protein, fibrinogen (an unrelated protein used to test for
non-specific binding) and TBS, the buffer used for all blot
studies. A strong response to KLH was observed and some
very limited non-specific binding was detectable using the
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E. Harfst et al.
crude serum against fibrinogen. However, the specificity of
each of the sera to its respective immunogen peptide is
clear. Western blotting with the sera against liver gap
junctions showed no cross-reactivity with the 32K connexin protein (results not shown), as would be expected
from the lack of sequence homology in the regions selected
for antigen synthesis.
Immunofluorescence localisation of all three antibodies
in mammalian heart showed specific binding to gap
junctions. Fig. 4A shows an example using antibodies to
12
3
HH
HJ
HQ
FIB * t
•
§
•
Fig. 3. A dot blot screening matrix showing the specificity of
each antibody to its respective peptide antigen, and to the
haemocyanin carrier protein used for immunisations. Each of
the antisera has been screened against TBS, peptide HH,
peptide HJ, peptide HQ, fibrinogen (FIB, an unrelated protein
used to test for non-specific sticking), and keyhole limpet
haemocyanin (KLH), respectively. Lane 1 was incubated with
HH antiserum, lane 2 HJ antiserum and lane 3 HQ antiserum.
While there is some non-specific binding to fibrinogen it is
minimal and the specificity of the antibodies to their respective
immunogens is clear.
peptide HJ on wax sections of rat right ventricle. A
punctate staining pattern confined to the intercalated
disks is consistently obtained, as would be expected on the
basis of gap junction distribution determined by electronmicroscopical studies (for review, see Severs, 1990). The
use of laser scanning confocal microscopy has allowed
individual junctions to be clearly discerned, especially
where the disks are seen in oblique view. Antibodies to
both HH and HJ also gave similar staining patterns on
frozen sections, but HH failed to localise gap junctions in
wax sections. Antibodies to HQ gave very faint localisation on wax sections and on frozen sections only after they
had been detergent extracted. These results are consistent
with the putative position of the binding sites on the
protein and have been described and discussed in full
elsewhere (Gourdie et al. 1990). Immunogold labelling of
gap junction-enriched fractions also demonstrated the
antibody specificity. Examples are shown following incubation with PBS in place of the primary antibody (Fig. 4B)
or with antibodies to peptide HJ (Figs 4C,D). No binding
was apparent with PBS alone, but specific binding of the
antibody is clear, even when there is no gold labelling
present (Fig. 4C). In all cases where peptide antibody
serum was used a thick 'hairy' coat is formed on the gap
junctions by the binding antibodies. Non-junctional membranes remained uncoated. PBS-treated junctions always
appeared identical to those in the original preparations
prior to antibody incubation.
Western blot analysis
The bulk of the Western blotting was carried out using
diluted serum with each anti-peptide antibody being used
independently. Antibodies to peptides HH, HJ and HQ
gave similar results, though none gave a particularly
strong signal. A cocktail of all three peptide antibodies
forming a 'polyclonal' serum gave identical binding patterns to those obtained with the separate antibodies, but
with a much stronger signal and greater sensitivity.
Western blotting of preparations of homogenised whole
rat heart, even when using an antibody cocktail, did not
show any specific binding (Fig. 5A). The large amount of
actin and myosin in the whole heart homogenates limits
the amount of material that can be loaded on a gel and the
amount of gap junction protein then present was too low to
be detected. Membrane fractions taken from the buffer/
30 % sucrose gradient interface were sufficiently enriched
in gap junctions to permit blotting with all three antibodies, even without NaOH/KCl extraction. Blots were
carried out in which the antibodies to HH, HJ and HQ are
used separately and as a cocktail (Fig. 5B). In all cases a
major band is seen at 43-45K, matching the 43K protein
coded for by the cDNA used to predict the peptide sequences. In some cases breakdown products with molecular masses of approximately 30, 32 and 34K are seen,
similar to those described by Manjunath et al. (1985).
Preimmune serum recognised none of these proteins.
A consistent feature of the Western blots was a major
band at 70K, which in the majority of our preparations
was by far the most conspicious band present (Fig. 5B). It
was not recognised by preimmune serum.
After further enrichment for gap junctions using
NaOH/KCl washing and detergent treatments the blot
profiles obtained did not change significantly (Fig. 5C).
The major proteins recognised had molecular masses of 43
and 70K; the 70K band again being the most prominent.
Again preimmune serum showed no binding to any proteins.
Western blotting of gap junction preparations from
isolated rabbit myocytes (Fig. 5D) gave a similar pattern
of results to those obtained using gap junction fractions
isolated from whole heart, although several proteins
became apparent between the 70 and 43K bands. There
was little evidence of the lower molecular mass breakdown
products, and preimmune serum failed to bind to any of
the proteins present.
Affinity purification of antibodies and Western blotting
Dot blot assays carried out with 0.3 % KLH added to the
HH antibody serum prior to incubation showed a reduced
binding to the KLH dotted onto the nitrocellulose (Fig. 6).
As expected no significant change in the binding of the
antibodies to the HH peptide was observed. However,
Prodisk affinity-purified antibodies to HH showed markedly increased binding to the peptide, and with 0.3 % KLH
added to the primary antibody prior to incubation relatively little binding to the KLH dotted onto the nitrocellulose was evident (Fig. 6). Western blotting with these
affinity-purified antibodies (containing 0.3% KLH)
against a heart membrane preparation showed increased
binding to the same proteins recognised in the diluted
serum blots. Both the 43 and 70K bands were now strongly
labelled, as were breakdown products present in the
preparation (Fig. 6). No additional proteins were recognised.
Protein studies
Coomassie blue staining of the plasma membrane fractions prepared with PMSF in all solutions showed that the
two major bands present match those recognised by the
antibodies, 43K and 70K, respectively (Fig. 7). On many
gels the 70K protein band was by far the more prominent.
Cardiac gap junction proteins
597
Fig. 4. Imnjunohistochemical and immunocytochemical localisation of cardiac gap junctions using the antibodies to peptide HJ.
A. Immunofluorescence localisation to gap junctions in a section of Zamboni-fixed, wax-embedded rat ventricle. The stained section
was viewed using a laser scanning confocal microscope, this image being derived from a single optical section. Staining is clearly
confined to the intercalated disks, which are seen in cross-section (arrow) and slightly oblique view (double arrow). The use of the
confocal microscope to eliminate out-of-focus blur means each separate junction is clearly discernible. B-D show the electron
microscopical detection of antibody binding to gap junctions in a membrane preparation similar to that shown in Fig. 1. A control is
shown in B; the preparation has been incubated with PBS in place of immune serum and the junctions appear 'clean' with no
labelling. In C and D preparations were incubated with gap junction antibodies alone, or with gap junction antibodies followed by an
immunogold second antibody. In both cases a *hairy' coat, representing directly visualised bound antibody, is seen. In D the use of
the immunogold second label further confirms specific binding of the antibody to gap junctions, with no labelling of non-junctional
material. A, x 800; B and C, x 73 000; D, x 100 000.
When preparations were carried out without PMSF the
43K protein was degraded, and breakdown products were
seen at 30, 32 and 34K (Fig. 7), their gap junction origin
598
E. Harfst et al.
being confirmed by Western blotting. This result is consistent with previous reports showing the tendency for the
43K cardiac gap junction protein to be degraded in prep-
C
A
D
1 2 3 4
123
1 2 3 4
66
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45
29*- —
2 4
••-
Fig. 5. Immunoblot analysis of whole rat heart (A), a rat heart membrane fraction (B), a NaOH/KCl and detergent-washed
membrane fraction from rat heart (C), and an enriched gap junction fraction from isolated rabbit myocytes (D). In each case lane 1
shows the relative molecular mass standards (xlO~3) and lane 2 the appropriate Coomassie Blue-stained protein profiles. Lane A3
shows there was no binding apparent to whole heart homogenates even when all three antibodies were used together as a cocktail.
The contractile proteins (the major band in lane A2 is actin) limit the amount of whole heart homogenate that can be loaded. Lanes
B3-B6 show Western blot results of antibodies to HH, HJ, HQ, and all three in a cocktail, respectively, blotted against the cardiac
membrane preparation. Lane B7 was blotted with preimmune serum. Lane C3 shows Western blotting of the detergent-extracted
preparation with the antibody cocktail, lane C4 with preimmune and lane D3 shows the cocktail blotted against an isolated myocyte
membrane fraction, D4 with preimmune serum. In all positive blots the major bands identified by the antibodies are at 43K and
70K, with the latter band by far the most dominant in most cases. In the isolated myocyte blot (lane D3) there are additional bands
recognised between the 43K and 70K bands. In some lanes signs of the cardiac gap junction protein breakdown products (see Pig. 7)
are seen.
arations produced in the absence of protease inhibitors
(Manjunath et al. 1985). The 70K protein was, however,
unaffected (Fig. 7). In these preparations without PMSF a
higher molecular weight protein, around 150K, was also
recognised by the antibodies, but its origin is unclear.
Boiling of the preparation in sample buffer, known to
induce dimerisation of the liver gap junction protein, had
no effect and the proportion of 70K protein to 43K protein
was unchanged (Fig. 7). Similarly, preparations carried
out using an alternative reducing agent (dithiothreitol) or
under strong reducing conditions using iodoacetamide did
not alter these proportions (Fig. 7).
A major change in the gel profiles was, however, obtained simply by allowing the membrane preparation to
4
12
TBS
HHp
FIB
5
6
3
•
66
45
36
29
24
20
N-
stand at room temperature for 15min or more, with
occasional vortexing, prior to pelleting and adding gel
sample buffer (Fig. 8). It was clear that warming the
preparation caused the 70K protein to remain in the
supernatant during subsequent pelleting in microfuge
tubes (13 000 g, 5-10 min), as shown by its reduction in the
pellet obtained and its recovery after prolonged centrifugation of the supernatant (13 000 g; 15 min). The 43K
protein was always present in the initial short spin pellet
after shorter incubation times, but with increased incubation times (2-6 h) all of the proteins in these preparations could be recovered in part from the supernatant
Fig. 6. Affinity purification and subsequent Western blot
analysis using antibodies to peptide HH. Lane 1 shows a dot
blot of diluted HH antiserum against TBS, peptide HH,
fibrinogen (to test for non-specific binding) and the carrier
protein keyhole limpet haemocyanin (KLH). Lane 2 is a similar
dot blot but with 0.3 % KLH added to the serum prior to
antibody incubation. This has competed out some of the KLH
antibodies, reducing their binding on the blot, but has no effect
on the peptide HH antibody binding. Lane 3 shows binding to
the same proteins of affinity-purified HH antibodies with 0.3 %
KLH added prior to incubation. Binding to peptide HH is
considerably enhanced and remaining KLH binding activity is
now very low. There is no binding to the non-specific protein
(fibrinogen). Western blot analysis with the affinity purified
antibodies against a cardiac membrane preparation is shown in
lanes 4-6. Lane 4 shows relative molecular mass standards
(xlO~3), lane 5 the Coomassie Blue-stained protein profile and
lane 6 the Western blot result. The affinity-purified antibodies
pick up the same bands as in the diluted serum blots (Fig. 5)
although their increased sensitivity shows up the breakdown
products of the 43K protein more clearly. The two major bands
are at 43K and 70K.
Cardiac gap junction proteins
599
12
66—
3 4
5
5
1 2
7 8
G
IlL
66——
45 —
—
36*29*""
—
<4 fT wr-im
36—
29—
24 ™
20i
20—
Fig. 7. SDS-polyacrylamide gel and Western blot studies of the
43K and 70K proteins recognised by the cardiac gap junction
peptide antibodies. Lanes 1-6 are Coomassie Blue-stained
protein profiles, and lanes 7 and 8 Western blot analyses. Lane
1 shows relative molecular mass standards (xlO~3). In lane 2 a
standard cardiac membrane preparation is shown with the two
major bands obtained running with apparent relative molecular
masses of 43K and 70K. In preparations made without protease
inhibitors present (lane 3) the 43K protein breaks down with
the three major breakdown products (approximately 30, 32 and
34K) now becoming major bands. The 70K is unaffected. Lane 4
shows a standard preparation boiled before loading on the gel,
while lanes 5 and 6 show preparations treated with an
alternative reducing agent (dithiothreitol) or a strong reducing
agent (iodoacetamide), respectively, prior to loading. None of
these treatments has altered the gel profiles obtained. Lanes 7
and 8 show Western blot analysis of a protease inhibitorincluded and a protease inhibitor-excluded preparation. The
bands thought to be gap junction-related in the equivalent
Coomassie Blue profiles, lanes 2 and 3, respectively, are
recognised by the peptide antibodies, confirming their
junctional origin. An extra high relative molecular mass band
is recognised in the preparation made in the absence of protease
inhibitors; its origin remains uncertain.
fraction, including the 43K junction protein. That these
were the same proteins as those obtained in standard
preparations was confirmed by Western blotting (Fig. 8).
Morphological studies of warmed gap junction
preparations
The partitioning of the 70K gap junction-related protein
stimulated a closer look at the gap junction structures
remaining in pellets after warming of the membrane
preparations, both in undisturbed pellet form, and resuspended and vortexed during the warming period. Even in
undisturbed pellets disrupted junctions were regularly
observed following room temperature incubation. These
junctions often appeared to be splitting, and an extensive
amount of single-membrane material appeared continuous with the intact junctional structures remaining. In
resuspended pellets, warmed and occasionally vortexed,
and repelleted in a short spin, 25-35 % of the junctions had
discontinuities in their profiles (Fig. 9). These appear as
breaks in the junction in regions where they have been
cross-sectioned, but as distinct holes where the junctions
have been sectioned tangentially. Less than 10% of junctions in unwarmed control samples had this disrupted
appearance.
600
E. Harfst et al.
Fig. 8. SDS-polyacrylamide gel and Western blot analysis of a
cardiac membrane preparation allowed to stand at room
temperature for 1 h. Lane 1 shows relative molecular mass
standards (xlO~3) and lane 2 a normal preparation for
comparison. Following the warming treatment the 70K protein
is considerably reduced in the short spin pellet (lane 3)
although the 43K protein is unaffected. Extended
centrifugation of the supernatant allows recovery of the 70K
protein, indicating it has not been solubilised, but its source
fragmented. Lanes 5 and 6 show Western blot analysis of the
short spin pellet and recovered supernatant fractions,
respectively, confirming that they are the same gap junctionrelated proteins seen in the normal membrane fraction.
Discussion
It is now widely accepted that there is a family of related
gap junction proteins. In mammalian tissues the major
types identified are a 32K liver type protein (Kumar and
Gilula, 1986; Paul, 1986), a 26K liver type protein in
rodent tissues (Nicholson et al. 1987; Zhang and Nicholson, 1989), an eye lens protein of 70K with in vivo
breakdown to 38K (Kistler et al. 1988) and a cardiac gap
junction protein of 43K (Beyer et al. 1987; Manjunath et al.
1982, 1984, 1985). The cDNAs of two other gap junction
protein types that occur in Xenopus embryos have been
sequenced, a 30K protein (Gimlich et al. 1988) and a 38K
protein (Ebihara et al. 1989). The naming of the proteins
reflects the tissue in which they were first identified, but
most have been recognised by Western blot or Northern
blot analysis in other tissue types as well (Beyer et al.
1989; Dupont et al. 1988; Hertzberg and Skibbens, 1984) or
down through the animal kingdom (Fraser et al. 1987;
Green, unpublished results). Where sequence data are
available, the proteins all show a high degree of homology,
with their extracellular and transmembrane regions, in
particular, being highly conserved (Beyer et al. 1987;
Milks et al. 1988). Models based on hydropathicity plots,
enzyme digest and antibody studies indicate that the
proteins cross the membrane four times with both the
carboxyl and amino termini on the cytoplasmic side (Beyer
et al. 1987; Milks et al. 1988; Yancey et al. 1989; Zrrnmer et
al. 1987). The major variation between the protein types
appears to be in the length of the carboxyl-terminal tail
region. Our experiments now indicate the presence of
another gap junction- related protein in the heart, in
addition to the 43K protein. This newly identified protein
has an apparent relative molecular mass of 70 000 and our
Fig. 9. These four panels show electron microscopic views of gap junctions in the short-spin pellet following incubation of a cardiac
gap junction-enriched fraction at room temperature for l h (with occasional vortexing). While regions of the junctions have a typical
pentalaminar structure, there are discontinuities in their structure. When junctions are viewed in cross-section (for example, B),
these appear as breaks in the junctions. When junctions are viewed more tangentially, however (for example, A and D), the
discontinuities are clearly holes within the junctional plaque. A and D, x 64 500; B, x 67 000; C, x 45 000.
results suggest it to be the major gap junction protein in
the heart.
Gap junction enrichment and anti-peptide antibodies
Our isolation technique (Gourdie et al. 1988) has enabled
us to obtain enriched gap junction fractions from whole
heart while maintaining a low temperature during the
entire preparation. Previously published protocols have
involved various stages at relatively warm temperatures
and have required the extensive use of detergents (Kensler
and Goodenough, 1980; Manjunath et al. 1982, 1984). We
have also been able to modify the technique, permitting
the enrichment of gap junctions from one specific cell
population in the heart, the ventricular cardiac myocyte.
In the heart, the myocytes make up 12-35 % of the total
cell numbers present (for review, see Severs, 1989) and
although in bulk they make up the major mass of the heart
(~75 % of the myocardium by volume) and they contain
abundant gap junctions, other cell types are nonetheless
potential contributors of gap junctions to preparations
obtained from whole heart material. Gap junctions are
known to be present between the endothelial and smooth
muscle cells of the coronary vasculature, for example, as
well as between myofibroblasts of the valves and between
nerve cells (for review, see Severs, 1989). By using purified
isolated myocytes as a starting material, the possibility of
contamination with gap junctions from non-myocyte cell
types is excluded. The gap junction preparations we have
obtained from both whole heart and myocyte preparations
were enriched, rather than entirely pure, but the presence
of some remaining non-junctional material did not interfere with the immunological and biochemical studies
carried out, and the 70K gap junction-related protein was
preserved throughout.
The three anti-peptide antibodies used in our experiments specifically recognised their corresponding synthetic peptide used as the antigen, and gave similar
Western blotting results on the cardiac membrane preparations. All three localise gap junctions in cardiac tissue
from a variety of mammalian species (unpublished results), although they do have different immunohistochemical characteristics related to the proximity of their
respective epitopes to the membrane (Gourdie et al. 1990).
Western blotting results obtained with diluted serum or
affinity-purified antibodies were similar, although the
sensitivity of the latter, or of a 'polyclonal' cocktail of all
three, was greater.
The 70K gap junction-related protein
All three of the anti-peptide antibodies recognised the 43K
and 70K proteins in Western blot studies, as did the antiHH antibodies following affinity purification. They also
recognised breakdown products derived from the 43K
protein similar to those reported by Manjunath et al.
Cardiac gap junction proteins
601
(1985). The omission of protease inhibitors in our preparations increased the prominence of breakdown products
concomitant with a reduction in the amount of the 43K
cardiac gap junction protein, but had no effect on the 70K
protein. In the isolated myocyte preparations, there was
some evidence that the 70K protein was being slightly
degraded but there was no sign of the lower molecular
mass products derived from 43K protein breakdown. This
is not surprising if the proteases that cause the breakdown
are being released from mast cells (Manjunath et al. 1985).
In the myocyte preparations, the mast cells are removed
prior to homogenisation and so exposure of the gap
junction protein to the serine proteases released from mast
cell granules will not occur.
Despite these differences in their degradation properties, the fact that all three antibodies made to the 43K
protein sequence recognise the 70K protein suggested that
the two proteins are highly homologous, and might even
be aggregation or breakdown products of one another. The
series of gel experiments was designed to examine this
possibility. The 43K protein could not be heat dimerised
(heating in SDS is known to dimerise both liver type gapjunction proteins; Green et al. 1988; Hertzberg, 1984;
Nicholson et al. 1987). Moreover, alternative or strong
reducing conditions did not alter the 70K protein, indicating that it is not itself a dimeric form of a smaller gap
junction protein. These results are consistent with those
previously published, which show that, while the 29K
breakdown product is able to form a dimer of around
50-52K, the intact 43K protein does not appear to be
capable of aggregation (Dupont etal. 1988; Manjunath and
Page, 1986). The major difference between the 43K and
70K proteins was their partitioning into different parts of
the preparations upon warming. The 70K protein was
rapidly partitioned into the supernatant, whereas the 43K
protein only became apparent in the supernatant after
extended warm periods. It is of note, however, that the 70K
protein was not being solubilised (or released from vesicles
in soluble form) as evidenced by the fact that it could be
recovered within a pellet by extended centrifugation. This
suggests that structures containing the protein were being
disrupted and fragmented.
Morphological studies
Mammalian liver gap junctions are resistent to NaOH
extraction, which is used routinely during their isolation
(Hertzberg, 1984). It is clear that the cardiac gap junction
cannot be treated in this manner although the use of KC1
does help stabilise the structure during an NaOH extraction step. The cardiac gap junction is made up of discrete
hexagonal clusters of connexons with lipid aisles between
(Green and Severs, 1984; Page et al. 1983), in contrast to
the liver junctions in which connexons form large and
more homogeneous hexagonal arrays. This structural
difference is possibly a contributory factor in their different isolation characteristics. The differential fragmentation of the structures containing the 70K or 43K proteins, or both, may reflect further junctional plaque
differences.
Our thin-section studies show that fragmentation of gap
junctions was occurring at warmer temperatures. Where
portions of junctions are released, it might be assumed
that the 70K protein coexists in the same junctional
plaques as the 43K connexin protein. However, only
25-35 % of the junctions in warmed preparations appeared
abnormal, yet the 70K protein is a major protein in our
membrane preparations. This would imply that many
602
E. Harfst et al.
junctions may consist only of this polypeptide, and are
being lost entirely during isolation protocols involving
wanner incubation steps during detergent extraction.
Our results here are not unique. Zervos et al. (1985) also
showed evidence for cardiac-type gap junction fragmentation during isolation. In their case, there was clear
evidence for vesicular budding from the junction, not
unlike that which we have observed as a result of warming
unresuspended pellets. Manjunath and Page (1988) have
reported that atrial gap junctions are not 'detergentresistant' and were always lost from their preparations.
When they used a low temperature and non-detergent
protocol, however, they were able to obtain an atrial gap
junction fraction. Unfortunately, they show no biochemical data from this preparation, but it is possible that the
atrium has gap junctions of only the 70K type, while the
ventricular myocytes clearly have both cardiac gap junction-related protein types. Why there should be two
protein types in the one cell is not clear, but neither is this
unique. The two liver connexin proteins (26K and 32K)
can coexist within the same cell and within the same
junction plaque (Traub et al. 1989).
Other evidence for a second cardiac gap junction protein
Our results indicate that the 70K protein may be the
major gap junction-related protein in the heart, yet it does
not appear to have been recognised earlier by other
workers in the field. One reason for this could be the
fragmentation of the 70K-containing structures at the
warmer temperatures used in previously published isolation protocols, which would alter their apparent density
in subsequent centrifugation steps. Furthermore, the
clumped hexagonal array structure of the cardiac gap
junction must also reduce its resistance to detergentextraction techniques.
Further evidence for a second cardiac gap junction
protein comes from careful scrutiny of the literature. Antipeptide antibodies to the 43K cardiac protein have been
shown previously to localise gap junctions in myocardial
tissues in a similar way to those described here (Beyer et
al. 1989). The two antibodies used by Beyer et al. (1989)
were made to synthetic peptides matching amino acids
119-142 and 252-271. The first therefore overlapped with
peptide HH used in this study, the second is to a region of
the protein closer to the carboxyl-terminal end of the
protein than the three used here. Both antibodies recognise the 43K cardiac gap junction protein, but one, that
towards the carboxyl-terminal end, also picks up a higher
molecular mass polypeptide ('approximately 60K') that
looks to be in a similar position on gels to the 70K protein
that we describe here (see Fig. 1 of Beyer et al. 1989).
These Western blots were carried out on a cardiac intercalated disc preparation (Green and Severs, 1983), which
also involves no detergent steps and is carried out entirely
at low temperature. The fact that only one of their
antibodies appeared to recognise this high relative molecular mass protein provides further evidence that while
the 70K protein is highly homologous with the 43K
protein, it is may also have variability within the overlap
regions.
At the molecular level, a cDNA predicting a 46K
polypeptide has been cloned from a rat eye lens cDNA
library (Beyer et al. 1988). Northern blots indicate that a
matching cDNA is present in the heart (Beyer et al. 1985,
1988), and while this does not match the apparent 70K of
the protein we describe here, it is of note that the protein
isolated from eye lens also runs at 70K on polyacrylamide
gels (Kistler et al. 1988). Furthermore, while Fishman and
Leinwand (1989) were able to report only a single transcript in the foetal human cardiac library they screened,
their Southern blot analysis nonetheless indicated the
presence of two genes.
Finally, while we have isolated specifically ventricular
myocytes, this population will consist of both 'working1
cells and cells of the conduction system (Purkinje cells).
Studies by Imanaga (1987) have indicated that these two
cell forms may have different junctional properties, the
permeability of gap junctions between 'working1 ventricular cells exceeding that of Purkinje cells. These physiological differences could result from the presence of different
gap junction proteins. Certainly, in the heart, there is a
need for both electrical and metabolic coupling between
cells, with electrical coupling occurring over short distances (directly from cell to cell), and over longer conduction pathways via the Purkinje fibre system.
In conclusion, all three anti-peptide antibodies used in
this study recognise, in addition to the 43K gap junction
polypeptide, a 70K protein in cardiac gap junction-enriched fractions prepared at low temperature using detergent-free methods. In addition, the 70K protein appears to
be recognised by one of two cardiac gap junction antipeptide antibodies studied independently (Beyer et al.
1989). The 70K protein therefore appears to have a high
homology with the 43K gap junction protein, although the
former appears to be more resistant to proteases during
junction isolation. Our experiments indicate that the 70K
protein is an integral protein, and may in some cases at
least be in the same gap junctional plaques as the 43K gap
junction protein. Both proteins are present in ventricular
cardiac myocytes, but whether they also occur in other cell
types within the heart remains to be determined. With
molecular evidence indicating that at least two genes for
cardiac gap junction proteins exist, it seems likely that the
70K polypeptide may be this second, and major, member of
the gap junction protein family in cardiac tissues.
We are extremely grateful to Dr N. B. Gilula (Research
Institute of Scripps Clinic, La Jolla, California) for providing us
with the synthetic peptides that made this work possible, and for
his encouragement throughout. We acknowledge the help of Dr R.
G. Gourdie with the confocal microscope immunolocalization and
the electron microscopy, and that of Dr T. Powell with myocyte
isolation. We thank Mr Stephen Rothery for photographic assistance, and also Dr W. H. Evans (National Institute for Medical
Research, Mill Hill, London) and Professor Anne E. Warner
(University College London) for stimulating discussion. This
work was funded by project grants from the Medical Research
Council (to C.R.G. and N.J.S.) and the British Heart Foundation
(grant no. 86/39 to N.J.S. and C.R.G.), and an equipment grant
from the Wellcome trust (to C.R.G.). Dr Green is a Royal Society
University Research Fellow. •
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(Received 21 February 1990 - Accepted, in revised form, 14 May 1990)