1 Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA
2 Department of Dermatology and Cutaneous Biology, and Jefferson Institute of
Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA
¶ Author for correspondence (e-mail: mfalk{at}scripps.edu)
Accepted 9 April 2003
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Summary |
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Key words: Gap junction diseases, Gap junctions, Green fluorescent protein, Membrane channels, Oligomeric proteins, Connexin subunit assembly
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Introduction |
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A gap junction channel is formed by integral membrane proteins, termed
connexins (Cx), which oligomerize into a hexameric hemichannel, termed a
connexon. Two connexons, each provided by one of two neighboring cells, dock
to form a functional intercellular channel. Connexins are polytopic membrane
proteins spanning the membrane four times. The N- and C-termini, as well as
the loop between the second and third transmembrane-spanning domains (TM2,
TM3), are located in the cytoplasm. The two conserved sequence domains that
connect the trans-membrane domains form the extracellular loops E1 and E2.
They were implicated in voltage gating
(Rubin et al., 1992), and
connexon compatibility, which leads to the docking of connexons onto complete
homo- and heterotypic gap junction channels
(Dahl et al., 1992
;
White and Bruzzone, 1996
). The
essential features of the trans-membrane topology of connexins, determined
previously by biochemical approaches (Falk
and Gilula, 1998
; Falk et al.,
1994
; Hertzberg et al.,
1988
; Milks et al.,
1988
; Zimmer et al.,
1987
), was confirmed by the cryo-crystallographic structural
analysis of Cx43 (
1) gap junction channels
(Unger et al., 1999
).
Twenty different connexins have been identified in humans, and many
homologues exist in other species. They have been divided phylogenetically
into subgroups with (also termed group 2) and ß (also termed
group 1) connexins being the two major representatives (six and seven members
in humans, respectively); however, connexin classification and definition of
subgroup-specific criteria is still under debate (reviewed in
Bennett et al., 1994
;
Kumar and Gilula, 1992
;
Willecke, 2002
). Connexins are
expressed in a tissue-specific manner and most cell types express more than
one connexin isoform. Therefore, not only are homo-oligomeric connexons
consisting of one connexin isoform, but also hetero-oligomeric connexons
consisting of different isoforms likely to exist in vivo and considerably
increase the number of different gap junction channel types
(Brink et al., 1997
;
Falk et al., 1997
;
He et al., 1999
;
Jiang and Goodenough, 1996
;
Konig and Zampighi, 1995
;
Stauffer, 1995
;
Wang and Peracchia, 1998
).
When we examined the oligomerization behavior of different connexin isoforms,
we found that not all connexins oligomerized with each other to form
hetero-oligomeric connexons (Falk et al.,
1997
). This observation prompted us to suggest that connexin
isoform interaction is selective and restricts the possible number of
hetero-oligomeric connexons (Falk et al.,
1997
). Indeed, all hetero-oligomeric connexons reported to date
are composed of two members of the same subgroup. For instance, Cx43 has been
shown to hetero-oligomerize with Cx37
(Brink et al., 1997
), Cx40
(Cottrell and Burt, 2001
;
He et al., 1999
;
Valiunas et al., 2001
) and
Cx46 (Berthoud et al., 2001
;
Das Sarma et al., 2001
) (all
-types) but not with Cx32 (Das
Sarma et al., 2001
; Falk et
al., 1997
) (a ß-type). Furthermore, Cx46 has been reported to
hetero-oligomerize with Cx50 (Jiang and
Goodenough, 1996
) (both
-types), whereas Cx32 can
hetero-oligomerize with Cx26 (Locke et
al., 2000
; Stauffer,
1995
) (both ß-types). Connexin composition alters the
properties and specificity of gap junction channels towards size, charge and
characteristics of permeate molecules
(Bevans et al., 1998
;
Goldberg et al., 1999
;
Steinberg et al., 1994
;
Veenstra, 1996
), suggesting
that the different channel subtypes are specifically adapted to precisely
regulate the function of the cells in which they are expressed. In analogy,
many different subunits of vertebrate ligand- and voltage-gated ion channels
have been characterized that also can oligomerize into many different
hetero-oligomeric channel subtypes (reviewed in
Green and Millar, 1995
).
However, as with connexins, the number of possible combinations far exceeds
the actual number of different ion channel subtypes that are assembled in
vivo.
In search of signals that regulate the interaction of connexins, we aligned
and ß connexin protein sequences and compared the
physico-chemical properties at all positions. We identified four
discriminatory residues. To determine whether these four residues convey
connexin-specific compatibility, we exchanged each of these residues in Cx43
(an
type) with the corresponding residue of Cx32 (a ß type). The
Cx43 amino-acid exchange variants were expressed as GFP-tagged fusion proteins
and their assembly, intracellular trafficking and lucifer yellow (LY) dye
transfer capability was analyzed. In addition, we co-expressed each Cx43
variant with wild-type (wt) Cx43 or Cx32 and determined their ability to
interact with and inhibit the function of co-expressed wild-type
connexins.
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Materials and Methods |
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Plasmid construction and site-directed mutagenesis
The plasmid containing the Cx43-GFP fusion protein was constructed by
inserting the cDNA encoding the entire coding sequence except its authentic
stop codon of rat Cx43 into the EcoRI/KpnI cloning sites of
the expression vector pEGFPN1 (Clontech Laboratories, Palo Alto, CA USA). The
resulting construct consisted of the Cx43 sequence in frame with the EGFP
coding sequence separated by a 12 amino acid linker sequence. Four different
single and one double amino-acid substitution were introduced into the Cx43
cDNA using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla,
CA, USA) according to the manufacturer's instructions. The forward primers
corresponding to each mutation were as follows. The exchanged nucleotides are
given in bold, and the mutated codons are underlined.
D12S: 5'-CTTGGGGAAGCTTCTGAGTAAGGTCCAAGCCTAC-3'
K13G: 5'-GGGGAAGCTTCTGGACGGCGTCCAAGCCTACTCC-3'
DK12/13SG: 5'-CTTGGGGAAGCTTCTGAGTGGCGTCCAAGCCTACTCC-3'
L152W: 5'-GAGGGGCGGCTTGTGGAGAACCTACATCATCAGC-3'
R153W: 5'-GAGGGGCGGCTTGCTGTGGACCTACATCATCAGC-3'
All Cx43 constructs were verified by automated DNA sequencing.
Cells, cell culture and transfection conditions
Baby Hamster Kidney cells (BHK-21, ATCC CCL10) were used throughout this
study. Stably transfected BHK cells expressing Cx43 and Cx32 (designated
BHK-Cx43 and BHK-Cx32), respectively, were constructed as described previously
(Kumar et al., 1995). In these
cell lines the Cx cDNAs are under the control of an inducible metallothionine
promotor that allows the expression of variable amounts of Cx protein after
induction with different amounts of zinc acetate. Wild-type BHK cells or
stably expressing BHK cell lines were grown at 37°C in an atmosphere of 5%
CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% (vol/vol) fetal bovine serum, 100 units/ml penicillin/streptomycin and 2
mM glutamine. 70-80% confluent cells grown in 35 mm dishes were split the day
before transfection and transfected with 1 µg of wt or mutated Cx43-GFP
cDNA and Superfect Transfection Reagent (Qiagen, Valencia, CA, USA) according
to the manufacturer's instructions.
SDS-PAGE and immunoblot analysis
Complete cell lysates of transfected BHK cells were obtained by adding
SDS-PAGE sample buffer to the cell monolayers. Samples were incubated for 30
minutes at room temperature (Cx32 expressing cells) or boiled for 3 minutes
(Cx43 expressing cells) and analyzed on 12.5% SDS-PAGE gels. Connexin proteins
were characterized using rabbit antipeptide antibodies directed against the
C-terminal tail of Cx43 (1S) (Nishi
et al., 1991
) or a mouse monoclonal antibody generated against the
C-terminal tail of Cx32 (ß1S) (Falk
et al., 1997
), respectively, and secondary antibodies conjugated
to horseradish peroxidase (Biorad, Hercules, CA). Blots were developed using
the enhanced chemiluminescence system (Pierce, Rockford, IL). Relative
expression levels were determined by comparing pixel intensities of each
protein band by computer assisted analysis.
Immunofluorescence analysis
For immunolocalization, BHK cells were grown on coverslips placed into six
well dishes and transfected with 1 µg of DNA. After 24 hours, cells were
rinsed with phosphate-buffered saline (PBS), fixed in 4% (vol/vol)
paraformaldehyde at room temperature for 15 minutes, permeabilized by placing
them in 0.1% Triton X-100 in PBS for 90 seconds and stained with ß1S
antibodies followed by a secondary antibody conjugated to TRITC
(tetra-methyl-rhodamine isothiocyanate) (Zymed, San Francisco, CA, USA). Cells
were mounted using an antifade kit (Molecular Probes, Eugene, OR) and viewed
with a Biorad 1024 confocal microscope or a Zeiss Axiovert 100 microscope with
attached slide-film camera. GFP was detected by its green autofluorescence
with an FITC filter set.
Dye transfer analysis
The ability of the various connexin variants to form functional gap
junction channels was assayed by microinjecting a mixture of lucifer yellow
[0.3% (wt/vol) in 0.1 M LiCl] and rhodamine B isothiocyanate-dextran (MW 40
kDa) [0.5% (wt/vol) in 0.1 M LiCl] (Sigma, St Louis, MO) into the cytoplasm of
cells with visible connexin-GFP clusters assembled at cell-cell appositions.
Transfer of lucifer yellow to coupled cells indicating functional gap junction
channels was determined by imaging the cells 3 minutes after injection.
Non-transfected BHK cells, and BHK cells transfected with wild-type Cx43-GFP
were injected as controls. Representative cell monolayers before and after
injection are shown in Fig.
3.
|
Statistical analysis
Fischer's exact test was used for statistical analysis of dye-transfer
inhibition using GraphPad InStat software version 3.0a for Macintosh (GraphPad
Software, San Diego, CA, USA,
www.graphpad.com).
Between 43 and 121 cells (functionally impaired variants), and 11 and 146
cells (controls) were injected to obtain significantly relevant
(P≤0.005) results.
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Results |
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Assembly and function of Cx43 amino acid substitution variants
To test the hypothesis whether the identified amino-acid residues are
involved in mediating connexin oligomerization compatibility, we constructed
four single and one double GFP-tagged Cx43 variants, in which the
characteristic Cx43 amino acid (an group member) was substituted with
the corresponding amino acid of Cx32 (a ß group member) (designated D12S,
K13G, DK12/13SG, L152W and R153W) and tested their ability to assemble into
functional gap junctions (Fig.
1B, Table 1). BHK
cells were transfected with the corresponding cDNAs, and successful expression
of all Cx43 variants including their GFP-tags was observed by western blot
analysis 24 hours post transfection (Fig.
2A, lanes 3-7, marked with an arrowhead). The level of wt Cx43-GFP
expressed in parallel as a control was similar to the expression levels of the
variants (Fig. 2A, lane 2). No
Cx43-GFP protein was detected in non-transfected BHK cells
(Fig. 2A, lane 1). A small
amount of Cx43 endogenous to BHK cells was detected on overexposed blots for
all samples (Fig. 2A, lanes
1-7, marked with an asterisk). Additional bands detected correspond to
phosphorylated Cx43-GFP (slower mobility), only partially denatured
polypeptides, degradation products and/or unspecific immunoreaction products
(faster mobility). To examine whether the Cx43 variants would assemble into
gap junctions, variants were expressed in wt BHK cells. All Cx43-GFP variants
appeared to assemble normally into connexons, gap junction channels and
channel clusters as indicated by the appearance of large fluorescent
connexin-GFP domains at cell-cell appositions
(Fig. 2B). Furthermore, no
difference in number and size of the clusters assembled from wt Cx43-GFP and
Cx43 variants was observed (Fig.
2B), indicating that the single, and double amino-acid residue
substitutions did not alter trafficking and assembly of the Cx43 variants.
Similar results were observed in HEK 293 cells (data not shown).
|
To determine the function of channels assembled from Cx43 variants, transfected BHK cells with fluorescent connexin domains visible at cell-cell appositions were microinjected with a mixture of LY and rhodamine dextran (Fig. 3). Only the P4 variant R153W-GFP transferred LY at normal levels (100% dye transfer efficiency), indicating unaltered function (Fig. 3, Fig. 4A). In all other Cx43 variants P1 (D12S-GFP), P2 (K13G-GFP), P1/2 (DK12/13SG-GFP), and P3 (L152W-GFP) LY dye coupling was reduced to background levels (5.2%, 8.1%, 2.8%, 3.2%) observed in untransfected BHK cells (6.9%), indicating that their function was impaired (Fig. 3, Fig. 4A). As expected, only LY (457 Da) was transferred between coupled cells, whereas rhodamine dextran (40 kDa) remained in the microinjected cells (Fig. 3, columns 3 and 4). Collectively, these results indicate that each of the residues present at positions 12 (aspartic acid), 13 (lysine) and 152 (leucine) plays a critical role for the normal function of Cx43 gap junction channels.
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Dominant-negative inhibition of wt Cx43 by Cx43 variants
Assembly of LY-dye-transfer impaired channels by the Cx43 amino-acid
exchange variants prompted us to investigate whether the non-functional
variants exerted a dominant-negative effect on co-expressed wt Cx43 and
disturbed its function. Thus, BHK cells stably transfected with Cx43
(BHK-Cx43) were transiently transfected with the cDNAs of the Cx43 amino-acid
substitution variants, and levels of intercellular dye coupling were assessed.
Experiments were carried out at two different Cx43 expression levels
(approximately 2.5- and sixfold higher than endogenous Cx43 expression levels)
in order to elucidate potential dose-dependent differences. Cx43 expression
levels were modulated by the addition of different amounts of zinc acetate
(Materials and Methods) (Kumar et al.,
1995). At both Cx43 wt expression levels, the number of cells
transferring dye was significantly (P<0.0001) higher (73.2% and
98.4%, Fig. 4B) than observed
for non-transfected BHK cells (6.9%, Fig.
4A). Co-expression of Cx43 variants (P2) K13G, (P1/2) DK12/13SG
and (P3) L152W resulted in a highly significant (P< 0.0001)
decrease in dye coupling (to 24.0%, 22.6% and 32.0% at low Cx43 wt expression
levels, and 64.7%, 49.0% and 43.1% at high Cx43 wt expression levels,
Fig. 4A) consistent with a
dominant inhibitory effect of these Cx43 variants on wt Cx43. The Cx43 variant
(P1) D12S also exerted an inhibitory effect on co-expressed wt Cx43 (71.0% at
low and 81.0% at high Cx43 wt expression levels,
Fig. 4B); however, it was less
pronounced (Fig. 4B). As
expected, LY dye transfer by the functional Cx43 variant (P4) R153W was as
efficient (100%) as in cells transfected with wt Cx43
(Fig. 4B). The more pronounced
reduction of LY dye transfer capabilities at lower Cx43 wt expression levels
demonstrates that the dominant negative inhibitory effect of the P1, P2 and P3
substitution variants was dose dependent
(Fig. 4B).
Trans-dominant-negative inhibition of wt Cx32 by Cx43 variants
To further examine whether the non-functional Cx43 variants might also have
a trans-dominant negative effect on the function of Cx32 (a ß-type
connexin), similar co-expression experiments were performed in BHK cells that
stably express wt Cx32 (BHK-Cx32) at lower (approximately 2.5-fold higher than
endogenous Cx43) or higher (approximately fivefold over endogenous Cx43)
levels. As expected, the percentage of cells transferring dye was
significantly increased at both Cx32 expression levels (94.5% and 98.1%,
P<0.0001, Fig. 4C)
compared with untransfected BHK cells (6.9%,
Fig. 4A). Co-expression of the
Cx43 variants (P1) D12S, (P2) K13G and (P1/2) DK12/13SG in cells expressing
lower levels of Cx32 also revealed a highly significant (P<0.0001)
inhibition of co-expressed wt Cx32 channel function. Dye coupling was reduced
to 58.6%, 45.3% and 53.3%, respectively
(Fig. 4C). This trans-dominant
inhibitory effect was also obvious in BHK cells expressing higher amounts of
wt Cx32; however, again the effect was less pronounced (93.1%, 75.9% and
78.4%, Fig. 4C), also
demonstrating the dose dependency of this trans-dominant inhibitory effect. In
contrast, the Cx43 TM3 variant (P3) L152W reduced coupling only slightly
(82.1% and 94.1%, respectively, Fig.
4C). The functional Cx43 variant (P4) R153W again had no
detectable effect (100% coupling, Fig.
4C). Taken together, these results indicate that the N-terminal,
but not the TM3 Cx43 variants, were able to inhibit the channel function of
co-expressed Cx32.
Colocalization of wt and Cx43 variants within connexin clusters
Dominant and trans-dominant inhibition of co-expressed Cx43 and Cx32
channel activity prompted us to explore whether the Cx43 variants would
co-assemble with co-expressed wt connexins within the same junctional
clusters. Cx43 variants were co-expressed in BHK cells stably transfected
either with wt Cx43 (BHK-Cx43) or Cx32 (BHK-Cx32). After fixation, expressed
Cx43 and Cx32 were labeled with specific monoclonal antibodies and visualized
with a TRITC-coupled secondary antibody. Cx43 variants were visualized by
using their GFP tag. In all instances, co-expressed wt connexin and the Cx43
variant assembled into connexin clusters that contained both connexin
proteins, as shown by the yellow plaque color that resulted from merged red
and green signals (shown for Cx32 wt and Cx43 variants in
Fig. 5). In contrast, clusters
assembled between cells expressing only wt Cx32 were red, whereas clusters
between transfected cells that only expressed Cx43 variants were green
(Fig. 5, marked with asterisks
in the merged images of D12S and R152W).
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Co-oligomerization of wt connexins and Cx43 variants into mixed
connexons
To further analyze whether the dominant- and trans-dominant-negative
inhibition of co-expressed wt Cx43 and Cx32 results from a direct interaction
and oligomerization of variant and wt subunits into mixed connexons, we
studied whether wt and variant connexin polypeptides interact. Thus, we
co-expressed transport-deficient DsRed-tagged Cx43 or DsRed-tagged Cx32,
respectively (Lauf et al.,
2001), with the GFP-tagged substitution variants and assessed the
interaction and assembly of the tagged proteins by the appearance of
DsRed-tagged connexins within connexin clusters at cell-cell appositions (see
Discussion). As expected, all Cx43 variants oligomerized with wt Cx43, as
suggested by the presence of DsRed-tagged Cx43 in connexin clusters that were
visible in the GFP, and the DsRed channels
(Fig. 6A, rows 2-6, connexin
clusters are labeled with arrows). Consistent with the less pronounced
dominant inhibitory effect of the D12S variant described above, only a small
amount of DsRed-tagged Cx43 was present in the clusters assembled by this
variant (Fig. 6A, row 2). Wt
Cx43 tagged with GFP and DsRed, respectively, co-expressed in control, also
assembled into mixed connexons and gap junctions
(Fig. 6A, row 1).
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|
When GFP-tagged Cx43-variants were co-expressed with DsRed-tagged Cx32, only the N-terminal variants (D12S, K13G, and DK12/13SG), but not the TM3 variants (L152W, R153W) were able to rescue DsRed-tagged Cx32 that trafficked and co-assembled with the variants (Fig. 6B, rows 2-6, connexin clusters are labeled with arrows). This result is consistent with the trans-dominant inhibitory effect of the N-terminal Cx43 variants on co-expressed Cx32 described previously (Fig. 4C). As expected, wt Cx43-GFP co-expressed in control did not interact and rescue DsRed-tagged Cx32 (Fig. 6B, row 1).
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Discussion |
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Location of the identified amino acids
Relatively little is known about the role of the N-terminal domain in the
function of connexins. Some evidence suggests that the N-terminal tail is part
of the voltage sensor and plays a fundamental role in ion permeation. Residues
1-10 of ß-connexins are predicted to lie within the channel pore, and the
N-terminal domain might form the channel vestibule
(Purnick et al., 2000). The
substitutions at P1 and P2 of Cx43 described here reduce the net charge of
this domain and, thus, might cause the functional impairment of these
substitution variants. On the other hand, recent experiments by Purnick et al.
suggest that G12 in Cx32 (P2) create a turn in the N-terminal tail that is
necessary for the function of this connexin
(Purnick et al., 2000
).
Substitution of the Cx32 G12 residue with amino acids expected to reduce the
flexibility of this domain (such as serine, valine and tyrosine) abolished
junctional currents. However, when G12 was substituted with proline, which is
often found in turns and expected to maintain flexibility, the expressed Cx32
G12P variant formed functional channels. These findings were further
corroborated by structural analyses of a synthetic peptide of this region that
folded into two
helices connected by a flexible hinge
(Purnick et al., 2000
). Since
no glycine or proline is present at, or close to, the P1/P2 position of
-connexins, the structural conformation of their N-terminal domain is
likely to differ from that of ß connexins. Thus, it is tempting to
speculate whether our experimental introduction of glycine into the N-terminal
tail of Cx43, by analogy, may have created a turn in this domain and therefore
rendered channels composed of these Cx43 variants non-functional (see
below).
The third trans-membrane-spanning domain (TM3) has been hypothesized to
line the aqueous pore of gap junction channels
(Milks et al., 1988). This has
been confirmed recently by systematic cysteine mutagenesis of Cx32
(Skerrett et al., 2002
).
Leucine (L) to tryptophan (W) exchange at position 152 (P3) located at the
cytoplasmic end of TM3 resulted in a LY-dye-transfer-impaired, but apparently
normally assembling, gap junction channel, similar to that observed for the P1
and P2 variants. Exchanging the corresponding amino-acid residue in Cx32
(tryptophan) with the corresponding amino-acid residue of Cx43 (leucine) also
resulted in an impaired extremely short-lived connexin (data not shown).
However, exchange of the directly adjacent arginine 153 (P4) with the
homologue Cx32 residue (also tryptophan) (R153W) did not result in any
detectable negative effect on Cx43, whereas the corresponding Cx32 variant
(W133R) is a naturally occurring Cx32 CMTX mutation
(Bone et al., 1995
), and
changing tryptophan 133 in Cx32 into cysteine (W133C) results in
non-functional channels (Skerrett et al.,
2002
). Results comparable to ours observed for the non-functional
Cx43 variants (P1-P3) were recently reported (e.g.
Skerrett et al., 2002
;
Oshima et al., 2003
), who
found that three of the 48 cysteine mutations in Cx32 (W77C, W133C and T134C)
and mutation of methionine 34 into alanine (M34A) in Cx26 resulted in fully
assembled but non-functional channels.
Interestingly, as mentioned above, W133 in Cx32 is homologous to R153 in
Cx43 that we have identified as critical position P4. It is believed that such
critical residues that interfere with function but allow channel assembly are
located at strategic positions, where they define the position of the
trans-membrane helices (Skerrett et al.,
2002) or interact with adjacent connexin subunits within the
connexon to ensure an open channel structure
(Oshima et al., 2003
).
Dominant and trans-dominant inhibition by Cx43 substitution
variants
All non-functional Cx43 substitution variants (P1-P3) exerted a
dominant-negative effect on the dye-coupling of co-expressed wt Cx43 channels
(Fig. 4B). The N-terminal
variants (P1, P2) exerted, in addition, a trans-dominant-negative inhibitory
effect on the dye-coupling of co-expressed Cx32 (a ß-connexin) channels
(summarized in Table 2). A
similar dominant and trans-dominant-negative effect has been reported for
naturally occurring, disease-causing connexin mutations. For example,
mutations of the ß-connexin Cx26, E42, W44C, D66H and R75W have
been found to inhibit co-expressed wt Cx26 function
(Oshima et al., 2003
;
Richard et al., 1998
;
Rouan et al., 2001
). In
addition, all these mutants except W44C were found to also inhibit the channel
function of the co-expressed
-connexin Cx43
(Rouan et al., 2001
). Dominant
and trans-dominant inhibition might be explained by the interaction of wt and
variant connexin subunits that co-oligomerize into mixed connexons and thus
render the resulting mixed connexons and gap junction channels
non-functional.
|
Evidence for direct interaction and co-oligomerization of wt and variant
connexin subunits was obtained in our study. Previously, we have demonstrated
that DsRed-tagged connexins are transport-deficient and will not assemble into
gap junction plaques in the plasma membrane
(Lauf et al., 2001). However,
co-expression of wt or GFP-tagged connexin-polypeptides together with
transport-deficient DsRed-tagged connexins will recover trafficking of
DsRed-tagged connexin polypeptides that then localize within connexin clusters
at cell-cell appositions. Successful trafficking of DsRed-tagged connexins
appears to result from the co-oligomerization of untagged or GFP-tagged
connexin subunits with DsRed-tagged connexins into mixed connexons
(Lauf et al., 2001
).
Comparable approaches that either deployed transport-deficient connexins
tagged with ß-galactosidase, or connexins tagged with endoplasmic
reticulum retention signals have also been developed recently and have been
used to investigate intracellular interaction and co-assembly of co-expressed
connexin isoforms (Das Sarma et al.,
2001
; Das Sarma et al.,
2002
). Consistent with the dominant-negative effect, P1, P2 and P3
Cx43 variants were able to recover trafficking of co-expressed DsRed-tagged wt
Cx43 as indicated by its colocalization with GFP-tagged variant subunits in
connexin clusters at cell-cell appositions
(Fig. 6A). In addition, the
N-terminal, but not the TM3, variants were able to recover trafficking of
co-expressed DsRed-tagged Cx32 (Fig.
6B), consistent with their ability to inhibit dye transfer of
co-expressed Cx32 channels (Fig.
4C). Co-assembly of wt and variant connexin subunits into
functionally impaired, mixed connexons is further supported by the assembly of
connexins into connexons that is a prerequisite for trafficking and channel
formation (Musil and Goodenough,
1993
) and the dose-dependent dominant and trans-dominant
inhibitory effects that were proportional to the amount of co-expressed wt
connexins (Fig. 4B,C).
By co-expressing full-length connexins together with N-terminally truncated
variants in cell-free translation/membrane translocation assays, we had
previously obtained evidence that a distinct amino-acid signal is located in
the N-terminal portion of the connexin sequence (N-terminal domain, TM1,
and/or first extra-cellular loop) that is involved in regulating recognition
and assembly compatibility of (Cx43) and ß (Cx26, Cx32) connexins
(Falk et al., 1997
). On the
basis of our previous and current results we speculate that connexin
polypeptide recognition and oligomerization compatibility depend on different
structural motifs that include amino-acid residues at positions P1 and P2.
These structural differences might allow connexins with similarly folded
motifs to interact and oligomerize but prevents the interaction and
oligomerization of connexin polypeptides with differently folded motifs. Such
structural motifs have been found previously in the N-terminal region of other
membrane channels, such as acetylcholine receptors, potassium channels, GlyR
receptors, and GABAA receptors
(Griffon et al., 1999
;
Li et al., 1992
;
Shen et al., 1993
;
Taylor et al., 1999
;
Verrall and Hall, 1992
).
However, since additional amino-acid residue mutations located downstream in
the first extracellular domain (E42, W44, D66 and R75) were also found to be
involved in a trans-dominant inhibition of Cx26 variants, different signals,
different structural motifs or, more likely, compound structural motifs, which
consist of different segments of the connexin polypeptides, regulate
recognition and co-oligomerization of connexin isoforms.
Dominant and trans-dominant inhibition and connexin disorders
In both the inner ear and epidermis a number of ß-connexins (such as
Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx32) are expressed in overlapping,
spatial and temporal patterns with -connexins (such as Cx37 and Cx43)
(Liu et al., 2001
;
Rouan et al., 2001
). Mutations
in most of these connexins underlie distinct genetic forms of deafness and
skin disorders (Liu et al.,
2001
; Rouan et al.,
2001
). Interestingly, several missense mutations have recently
been identified in three different ß-connexin genes that also affect
positions P1 and P2 (e.g. G11R (P1) in Cx30
(Lamartine et al., 2000
), G12R
(P2) in Cx26 (Richard et al.,
2002
) and G12D and G12R (P2) in Cx31
(Richard et al., 1998
).
Functional analyses of the Cx31 mutants in mammalian expression systems
revealed that both Cx31 mutations did not obviously interfere with expression,
connexon assembly and targeting to the cell membrane, but significantly
altered channel function (Diestel et al.,
2002
; Rouan et al.,
2003
). On the basis of the trans-dominant inhibitory effect of the
Cx43 P1 and P2 amino acid exchange variants described in this study and the
trans-dominant Cx26 variants described above
(Rouan et al., 2001
), it
appears possible that an aberrant hetero-oligomerization between co-expressed
mutant and wt
and ß-connexins cause disease phenotypes in certain
cases of connexin-related disorders. Further experiments will elucidate
connexin subunit oligomerization under normal and pathological conditions.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: Department of Biology, University of Fribourg, Perolles,
Ch-1700 Fribourg, Switzerland
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References |
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