From the Institut für Genetik,
Universität Bonn, 53117 Bonn, Germany, the
§ Department of Neuroscience, Albert Einstein College of
Medicine, New York 10461, the ¶ Laboratory of Excitable
Structures, Kaunas Medical University, Kaunas, Lithuania, and the
Laboratory of Molecular Microbiology, NIAID, National Institutes
of Health, Bethesda, Maryland 20892
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ABSTRACT |
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A new mouse connexin gene has been isolated that
codes for a connexin protein of 505 amino acid residues. Based on the
predicted molecular mass of 57.115 kDa, it has been designated
connexin-57. Similar to most other mouse connexin genes, the coding
region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this
new gene shows highest sequence identity to porcine connexin-60 in the
Gap junctions in vertebrates are formed by subunit proteins of the
connexin gene family. Six connexin proteins oligomerize and form a
hemichannel (connexon). Between apposed membranes of adjacent cells,
connexons interact with the opposite connexons to form gap junction
channels (cf. Refs. 1 and 2). Functional gap junction
channels in vertebrates are permeable to small molecules (<1 kDa),
i.e. ions, metabolites, and second messenger molecules. Gap
junctional communication has been suggested to contribute to metabolic
cooperation, synchronization of cellular physiological activities,
growth control, and regulation of development.
To date, 14 different connexin genes in the mouse and rat genome have
been described (1, 3, 4). Many of these genes have been located on
different chromosomes (5), but some of them have been assigned to the
same chromosome (cf. Refs. 6 and 7). Individual members of
the connexin (Cx)1 family are
designated according to the theoretical molecular mass of the protein
(8) or according to a Greek nomenclature that is based on differences
in the cytoplasmic loop (2, 4, 9). The similar membrane-spanning
topology of connexin proteins has been revealed by limited proteolysis
and site-directed antibodies to Cx32, Cx43, and Cx26 (10-12). By these
methods, together with hydropathy plots, we deduced that connexin
proteins span the plasma membrane four times (M1-M4), with two
extracellular loops (E1, E2) and three cytoplasmic regions (amino- and
carboxyl-terminal regions, cytoplasmic loop). Within the connexin gene
family, the four membrane-spanning regions, the amino-terminal region,
and the first extracellular loop show the highest sequence identities. Major differences in sequence and length were found in the
carboxyl-terminal region and the cytoplasmic loop. The transmembrane
regions of a connexon are assumed to be involved in forming the channel
pore. Furthermore, the two extracellular loops mediate docking of two connexin hemichannels, and the cytoplasmic domains of the connexins are
thought to regulate channel voltage and chemical gating (13-16). Most
mammalian cells express one or more connexin genes in a cell type-specific manner. For functional characterization, the various cloned connexins have been expressed in Xenopus oocytes
and/or cultured mammalian cells (cf. Ref. 17). These
reconstitution experiments have shown that different connexin channels
exhibit different permeabilities to tracer molecules (18) and show
different unitary conductances (19). Reconstitution experiments with
hemichannels composed of different connexins, leading to heterotypic
gap junction channels, made it possible to distinguish compatible
(functional) and incompatible (non-functional) combinations of
connexons (18, 19). Evidence for the existence of functional
heteromeric channels (different connexin proteins in the same
hemichannel) has been reported in chicken lens and for the
liver-derived connexin-26 and -32 (20, 21).
Several of the mouse connexin genes have now been deleted by homologous
recombination (for reviews, see Refs. 1, 23, and 24). So far, the
phenotypic abnormalities observed in these connexin-deficient mice were
different from each other, although in most cases residual
intercellular communication in the connexin-deficient cells could be
demonstrated, probably due to the expression of other functional
connexin channels. Thus, a more complete understanding of gap junction
function in mammalian tissues requires the identification and
characterization of the remaining connexin genes. Therefore, we
searched for novel connexin genes in a mouse genomic library and found
a new reading frame, which coded for a previously unknown connexin
protein of 57.1 kDa molecular mass and, thus, was called mouse
connexin-57. Here we describe the relationship of this gene to other
connexins, its expression pattern in embryo and adult tissues, the
chromosomal location of this gene, and its functional activity
following transfection into human HeLa cells.
Isolation of Genomic Mouse Cx57 DNA--
We isolated 25 connexin
homologous recombinant Southern and Northern Blot Analyses--
Genomic DNA from livers
of BALB/c mice was prepared according to a standard procedure (27).
Restriction endonuclease-digested DNA (10 µg) was electrophoresed in
0.7% agarose and blotted by alkaline transfer onto Hybond N+ membrane,
following the manufacturer's directions (Amersham Pharmacia Biotech,
Braunschweig, Germany). High stringency hybridization of the Southern
blot was carried out overnight using a 531-bp BstXI Cx57
fragment (representing nucleotides 59-590 in Fig. 2). The fragment was
labeled with [
Total RNA from mouse tissues was isolated with the TRIzol reagent
according to the manufacturer's procedure (Life Technologies, Inc.,
Eggenstein, Germany). Total RNA from HeLa cells was prepared with the
Qiagen RNeasy kit, as described by the company (Qiagen, Hilden, Germany).
Radioactive Northern blot analysis with total RNA (20 µg) was carried
out as described previously (6) with the mouse Cx57 XhoI/XbaI (1318 bp, positions 412-1745) fragment
as probe. Non-radioactive Northern blot analysis with total RNA (10 µg) was carried out with the RNA-labeling kit, as described by Roche
Molecular Biochemicals (Mannheim, Germany). A mouse Cx57 DIG
(digoxygenin)-labeled antisense cRNA as hybridization probe was
generated by cloning the mouse Cx57 XhoI/ClaI DNA
fragment (1023 bp, positions 412-1435) into XhoI/ClaI-linearized pBluescript II SK+ DNA.
Plasmid DNA was linearized with the restriction enzyme XhoI,
and the cRNA DIG-labeled antisense probe was prepared by in
vitro transcription using the T3 RNA polymerase in the presence of
digoxygenin-UTP. The amount of total RNA in different samples on
Northern blots was standardized by hybridization to a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (29). The
hybridization signals were quantified by densitometric evaluation using
Scan Pack version 14.1A27 (Biometra, Göttingen, Germany)
RT-PCR Analysis--
Reverse transcription (RT) and
amplification of cDNA by polymerase chain reaction (PCR) were based
with slight modifications on the Access RT-PCR system (Promega,
Madison, WI). In order to avoid genomic DNA contamination in total RNA
preparations, they were treated with RQ-DNase (Promega). Aliquots of 2 µg of total RNA were incubated with 2 µl of AMV buffer (5×,
Promega), 1 µl of 25 mM MgSO4, 1 µl of
RQ-DNase (1 unit/µl, Promega), 1 µl of RNasin (40 units/µl,
Promega, RNase inhibitor) and diethyl pyrocarbonate (DEPC)-treated
H2O to a total volume of 10 µl and incubated at 37 °C
for 30 min. Then, RQ-DNase was inactivated at 75 °C for 5 min. The
RT reactions were performed with 10 µl of DNA-free RNA solution by
adding 3 µl of AMV buffer (5×, Promega), 2.5 µl dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP), 1 µl of RNasin, 3 µl
of AMV-reverse transcriptase (10 units/µl, Promega), 1 µl of
oligo(dT)18 primer (50 pmol), and 5.5 µl of
DEPC-H2O for 90 min at 42 °C. The RT reaction mixtures
were stored at Genetic Mapping--
Connexin genes were mapped by analysis of
two sets of mouse crosses: (NFS/N or C58/J × Mus
musculus) × M. musculus (31) and (NFS/N × Mus spretus) × M. spretus or C58/J (32). Progeny of these crosses was typed for over 1200 markers including the chromosome 4 loci Mtv14 (mammary tumor virus 14) and
Ggtb (glycoprotein 4 Cells and Culture Conditions--
Experiments were performed
with HeLa cells (human cervix carcinoma cells, ATCC CCL2) and C3H mouse
keratinocyte-derived cell lines Hel-30 and Hel-37 (34, 35). These cells
and their transfectants were grown in Dulbecco's modified Eagle's
medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with
10% fetal calf serum (Life Technologies, Inc.), 100 µg/ml
streptomycin, and 100 µg/ml penicillin (cf. Ref. 36). The
medium for the HeLa transfectants contained, in addition 1 µg/ml
puromycin (Sigma, Dreisenhofen, Germany). The cells were passaged
weekly, diluted 1:10, and maintained in a 37 °C incubator in a moist
atmosphere of 10% CO2.
Transfection--
For transfection of HeLa cells, an
NcoI/XbaI mouse Cx57 fragment (positions 1-1745)
was cloned into the SmaI site of pBluescriptII SK+
(Stratagene). From this plasmid, a KpnI/BamHI
fragment containing the coding region of mouse Cx57 was cloned into the
KpnI/BamHI site of the transfection vector
pBEHpac18 (37), which contained the SV40 early promoter, a
polyadenylation signal, and a gene that conferred resistance to puromycin.
HeLa cells were transfected with 20 µg of DNA of the mouse Cx57
coding region in pBEHpac18, using the calcium phosphate transfection protocol of Chen and Okayama (38). Forty-eight hours after incubation with the DNA/calcium phosphate precipitate, 1 µg/ml puromycin was
added to the medium. Clones were picked after 3 weeks and grown under
selective conditions. The clones were checked by Northern blot analysis.
Microinjection of Tracers--
Glass micropipettes were pulled
from capillary glass (World Precision Instruments Inc., Berlin,
Germany) with a horizontal pipette puller (PD-5, Narashige, Tokyo,
Japan) and back-filled with tracer solution. Tracers were injected
iontophoretically (Iontophoresis Programmer model 160; World Precision
Instruments Inc.). Dye transfer was examined, using an inverse
microscope (IM35; Zeiss, Oberkochen, Germany) with fluorescent
illumination (HBO100; Zeiss). During injection, cell culture dishes
were kept on a heated block at 37 °C.
Lucifer yellow CH (Molecular Probes, Eugene, OR) as 4% (w/v) in 1 M LiCl was injected by applying negative voltage for
10 s (I = 20 nA). Cell-to-cell transfer was
evaluated by fluorescent microscopy (Zeiss IM-35, filter set 9) 5-30
min after dye injection. Neurobiotin
(N-2(2-aminoethyl)-biotinamide hydrochloride; Vector Lab,
Burlingame, CA) and rhodamine 3-isothiocyanate dextran 10S (Sigma) at
concentrations of 6% and 0.4% (w/v) in 0.1 M Tris-Cl (pH
7.6) were iontophoretically injected by application of positive voltage
for 10 s (I = 20 nA). The transfer of tracer
molecules was observed using filter set 15 (Zeiss) in the microscope.
Five to thirty min after injection, cells were washed twice with
phosphate-buffered saline (PBS), fixed for 10 min in 1% glutaraldehyde
in PBS, washed twice with PBS, incubated in 2% Triton X-100/PBS for
2 h, washed three times with PBS, incubated with horseradish
peroxidase-avidin D diluted 1:1000 in PBS (Vector Lab), for 90 min,
washed three times with PBS, and incubated in 0.05% diaminobenzidine
(Sigma), 0.003% hydrogen peroxide solution for 30 s to 2 min. The
staining reaction was stopped by washing three times with PBS. The
cell-to-cell transfer was quantified by counting the number of stained
neighboring cells around the microinjected cell.
For assay of heterotypic coupling, one cell type was stained with DiI
as described by Goldberg et al. (39) and co-cultivated with
a 1000-fold excess of unstained cells expressing a different connexin
gene. The cells were incubated 18 h before microinjection of
neurobiotin tracer.
Electrophysiology: Cells and Culture Conditions--
In order to
study the electrical properties of gap junction channels, a dual
voltage-clamp method was used, which has previously been described in
detail (40). For the experiments, the cells were seeded at a density of
~104 cells/cm2 onto sterile glass coverslips
placed in culture dishes. Electrical measurements were performed 1-3
days after plating. The experimental chamber was mounted on the stage
of an inverted microscope equipped with phase-contrast optics. It was
perfused with Krebs-Ringer solution at room temperature. Patch pipettes
were pulled from glass capillary tubes with filaments using a
horizontal puller (model P-97; Sutter Instruments Co.). Experiments
were performed in modified Krebs-Ringer solution (in mM):
NaCl, 140; KCl, 4; CaCl2, 2; MgCl2, 1; glucose,
5; pyruvate, 2; HEPES, 5 (pH 7.4). Patch pipettes were filled with
saline containing (in mM): KCl, 140; sodium aspartate, 10;
MgATP, 3; MgCl2, 1; CaCl2, 1.4; EGTA, 5 (pCa
~7.5); HEPES, 5 (pH 7.2), filtered through 0.22-mm pores.
The dual voltage-clamp method was used to control the membrane
potential of both cells individually and to measure the associated membrane and junctional currents (41, 42). After the establishment of
whole-cell patch-clamp conditions, voltage of the same amplitude was
clamped in both cells (V1 = V2), and non-junctional membrane currents
(Im1, Im2) were recorded
individually for each cell. Changing the membrane potential in one cell
induced transjunctional potential (Vj = V2 Cloning of the Mouse Cx57 Gene--
After screening a mouse
genomic library of Analysis of Mouse Cx57 Amino Acid Sequence--
The deduced amino
acid sequence of Cx57 showed all the typical features of a connexin
protein, i.e. four potential transmembrane regions (the
third one has amphipathic features) predicted by the algorithm of Kyte
and Doolittle (43), and could be aligned to topological domains of Cx32
and Cx26 that were previously deduced from studies with site-specific
antibodies and limited proteolysis of the membrane embedded proteins
(12, 18). The pattern of cysteine residues in the two putative
extracellular loops of the Cx57 protein included three cysteine
residues in the sequence CX6CX3C and
CX4CX5C, like in many
other murine connexins.
The comparison of the overall amino acid identities between Cx57 and
all other known mouse or rat connexins showed that Cx57 shared no
significant relationship to any of these connexins (48-32% identity
range), but exhibited relatively high amino acid identity (i.e. 74%) to porcine Cx60 (44). By comparison of the amino acid sequence of the putative extracellular region 1, Cx57 was classified into the Genomic Organization and Chromosomal Localization--
Southern
blot analysis of mouse genomic DNA showed that the Cx57 gene is present
in the mouse genome as a single-copy gene (Fig.
4A). Under these conditions,
single DNA fragments of 1.4, 1.1, and 1.0 kb were detected after
digestion with HindIII, XhoI, and
EcoRI. DNAs of the parental mice after two sets of genetic crosses were typed for restriction enzyme polymorphisms in the Cx57
sequence. For the M. spretus crosses, PstI
produced fragments of 12.9 kb in DNA from parental NFS/N and C57/J mice
and 9.7 kb in M. spretus, whereas no polymorphisms were
detected in the M. musculus × M. musculus crosses.
Inheritance of the variant fragment in the M. spretus
crosses placed this gene on the proximal chromosome 4 (Fig.
4B). We suggest the genetic symbol Gja-9 for
designation of the mouse Cx57 gene, in accordance with the nomenclature
used for connexin genes (cf. Refs. 45 and 46).
Expression of Mouse Cx57 mRNA in Human HeLa Cells--
The
expression pattern of Cx57 mRNA in total RNA from different mouse
tissues was analyzed by Northern blot hybridization (Fig.
5, A and B) and by
RT-PCR (Fig. 5, C-E). Cx57 mRNA was found in 10 dpc
mouse embryos and in heart, intestine, testis, kidney, lung, and skin
(adult and embryo), as well as in the keratinocyte-derived cell lines
Hel30 and Hel37. The transcript signals on the autoradiographs were
detected only after 5 weeks of exposure, suggesting low levels of Cx57
mRNA in these tissues and cells. For further examination we used
the more sensitive RT-PCR technique and found, in addition, that Cx57
mRNA was also expressed in ovary and mouse embryos between 8.5 and
18.5 dpc (Fig. 5, C and D).
Functional Expression of Mouse Cx57 in Human HeLa Cells--
For
functional characterization of the Cx57 channel, we transfected human
HeLa cells deficient in gap junctional communication (47) with Cx57 DNA
(see "Materials and Methods"). The isolated stable Cx57 transfected
HeLa cells were characterized by Northern blot analysis using total RNA
and a Cx57 probe. Fig. 6 shows results of
the nonradioactive Northern blot hybridization and illustrates that
several of the HeLa Cx57 transfectants expressed high levels of Cx57
mRNA, in contrast to wild type HeLa cells.
Tracer Transfer through Homotypic and Heterotypic Cx57 Gap Junction
Channels--
Permeability of homotypic and heterotypic junctions was
examined by using neurobiotin (Mr 287, net
charge +1) and Lucifer yellow (Mr 448, net
charge Vj and Vm-sensitive
Gating--
Experiments were performed on 16 spontaneously preformed
cell pairs by using the double-voltage clamp method. Initially, the voltage in both cells was clamped to the same holding potential (0
Dependence of junctional conductance on Vj was
measured by depolarizing or hyperpolarizing one cell long enough to
establish steady state of gj,
gj(ss). Fig.
8A illustrates an example of junctional current record and voltage protocol in cell 1, V1, and cell 2, V2.
During depolarization of cell 1 with a voltage step of 50 mV,
gj decreased about 10 times, exposing
gmin, and slowly recovered after
hyperpolarization to the holding potential. Summarized data of
gj(ss)-Vj dependence are
presented in Fig. 8B. Individual
gj(ss) data were normalized to instantaneous
gj, gj(ss)/gj(inst).
Continuous lines show fitting of the experimental data to the Boltzmann
equation, gj = {(1
Surprisingly, we found that Cx57 exhibited gj
dependence on the membrane potential, Vm. In all
four cell pairs we tested for gj Single Gap Junction Channel Conductance--
Measurements were
performed either by applying rectangular pulses or ramps with amplitude
up to 120 mV to one of the cells. Fig.
9A illustrates an example of
single-channel Ij record during voltage steps in
cell 2, V2, from the holding potential of
Ij
Based on single-channel records, we estimated the number of gap
junctions in HeLa-Cx57 transfectants. Cell pairs, exhibiting coupling
conductance of ~1.5 nS, contained approximately 60 functional channels. This number is significantly higher than the number of
functional channels between HeLa parental cell pairs, which varied
between 0 and 10, but in most cases cell pairs were
uncoupled.2 Additionally,
single-channel conductance in HeLa-57 transfectants was lower than
Effect of Chemical Factors on gj--
In 6 cell pairs
from 16, we have tested the effect of heptanol (2 mM) on
gj. Fig. 10
demonstrates typical uncoupling effect of heptanol in one of three cell
pairs from three coupled via gap junctions. Three cell pairs with
gj = 15, 20, and 25 nS exhibited no
Vj-sensitive gating and heptanol had no significant effect on gj. These data indicate the presence in these
cell pairs of cytoplasmic bridges (48). Arachidonic acid
(10 The new mouse Cx57 gene and its derived protein show the
structural features of Cx57 mRNA was found in the mouse embryo after 8 dpc and, at low
level, in several tissues: heart, skin, kidney, lung, ovary, testis,
but not in brain and sciatic nerve. Based on its relatively high amino
acid identity and its similar expression in ovary, heart, kidney, lung,
and intestine, at low mRNA abundance in total RNA, it is likely
that mouse Cx57 and porcine Cx60 (44) are analogous genes in the two
species. This expression pattern, however, does not lead to insights
into the functional role of Cx57 in the tissues in which it is
expressed. Expression of Cx43, Cx30.3, and Cx37 in ovaries has been
previously reported (44, 49, 50). Simon et al. (50) had
reported that Cx37-deficient mice suffer from female infertility, due
to inhibited development of oocytes. Transfer of microinjected
neurobiotin has been shown to occur between the oocyte and surrounding
granulosa cells in wild type mice, but not in Cx37-deficient mice. Cx37
is expressed in wild type oocytes (50). It is not known to which
connexin hemichannels the Cx37 hemichannels in oocytes dock for
functional communication with granulosa cells. Cx57 could be a
candidate, since we have shown that it can form heterotypic gap
junctions with Cx37 hemichannels expressed in transfected HeLa cells.
So far, it is not known in which cell type(s) Cx57 protein is expressed in mouse ovaries.
Are there peculiar features of the mouse Cx57 gene and its
channel-forming protein? Compared with other connexin genes, the level
of Cx57 mRNA was rather low in all tissues where we found a
positive signal. It is possible that we have not identified the cell
type that may express Cx57 mRNA at high abundance. Alternatively, coexpression of Cx57 with other connexins in the same cell type could
lead to heterotypic channels (for example with Cx37 (Gja-4), Cx43 (Gja-1), Cx30.3 (Gja-5), as shown in this
paper) or heteromeric channels (not studied in this paper) and,
thereby, influence the pattern of functional connexin channels between
the same or different cell types.
Our electrophysiological analysis revealed that Cx57 channels,
expressed between transfected HeLa cells, exhibited low single-channel conductance, Cx57 demonstrated unique
gj-Vm dependence and
gj rise during depolarization of cells. This
property is well expressed in insect cells, where
gj strongly decreased with depolarization (48, 51, 52). gj-Vm dependence
was unusual for the members of the connexin family and was reported
only for Cx45 (53). The resting potential of cells during different
periods of the cell cycle and during development might change
significantly. This would potentially involve
Vm-sensitive gating in the regulation of
cell-cell coupling.
The Cx57-expressing HeLa-transfected cells showed low intercellular
permeability to neurobiotin and were not permeable to Lucifer yellow.
Thus, Cx57 channels appear to be penetrable preferentially by small
molecules. At present, it is not known why connexin channels of low
single-channel conductance may be coexpressed between cells that are
connected by other channels of higher unitary conductance.
In order to further analyze these questions, antibodies to the Cx57
protein need to be raised and used to study the cell type-specific expression pattern in relation to other connexins. Furthermore, mouse
mutants with defects in the Cx57 gene should allow one to decipher the
peculiar functional contribution of this new connexin gene to molecular
physiology and/or development of the ovary and other tissues.
group of connexins. The connexin-57 gene was mapped to a position
on mouse chromosome 4, 30 centimorgans proximal to a cluster of
previously mapped connexin genes. Low levels of connexin-57 mRNA
were detected in skin, heart, kidney, testis, ovary, intestine, and in
the mouse embryo after 8 days post coitum, but expression was not
detected in brain, sciatic nerve or liver. In order to analyze gene
function, the connexin-57 coding region was expressed by transfection
in human HeLa cells, where it restored homotypic intercellular transfer
of microinjected neurobiotin. Heterotypic transfer was observed between
HeLa connexin-57 transfectants and HeLa cells, expressing murine
connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses
revealed that HeLa-connexin-57 transfectants expressed about 10 times
more channels than parental HeLa cells. Voltage gating by
transjunctional and transmembrane voltages as well as unitary
conductance (~27 picosiemens) were different from intrinsic connexin
channels in parental HeLa cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FixII phage clones by screening a 129/SvJ
mouse genomic library (Stratagene, La Jolla, CA) with a mouse Cx26
probe (25). We used the procedure described by Hennemann et
al. (26), to identify connexin genes in recombinant phages.
Several of these phage clones subsequently failed to hybridize to any
of the known connexin genes under stringent conditions of hybridization
(50% formamide, 5× SSC at 42 °C). DNA of these latter phages was
isolated using standard protocols (27). Southern blot hybridization of
restricted phage DNA was performed under low stringency conditions
(40% formamide, 5× SSC at 38 °C) using the mouse Cx26 probe. It
was concluded that a 2.2-kb XbaI fragment contained
sequences homologous to the mouse Cx26 probe. These fragments were
subcloned in the pBluescriptII SK+ vector (Stratagene, La Jolla, CA).
Sequencing was performed on both strands by the modified chain
termination method (28), using either vector-derived primers or
appropriate primers derived from previous sequencing results. The amino
acid sequence deduced from the longest open reading frame
(i.e. mouse Cx57) was aligned with different connexin
sequences, using the PCGene sequence analysis program (PCGene 6.8, IntelliGenetics, Mountain View, CA) and the HUSAR program (German
Cancer Research Center, Heidelberg, Germany).
-32P]dCTP by random priming (Megaprime
Labeling Kit, Amersham Pharmacia Biotech) to a specific activity of
0.2-1 × 109 cpm/µg of DNA. Filters were washed at
high stringency (0.2× SSC, 0.1% SDS at 60 °C) and exposed to XAR-5
film for 1 day to 5 weeks.
70 °C. PCR reaction was performed with 5-µl
aliquots of the RT reaction mixtures, 4 µl of AMV/Tfl
buffer (5×, Promega), 0.5 µl of dNTP mix, 0.5 µl of upstream
primer (50 pmol) (57 RT F1: 5'-GGC CCC AAG AAT GCA ATG TCT CAG-3'), 0.5 µl of downstream primer (50 pmol) (57 RT R1: 5'-GCT TTT ACT TAC CAT
CGA TGC TC-3'), 0.5 µl of 25 mM MgSO4, 0.5 µl of Tfl polymerase (5 units/µl, Promega), and DEPC-H2O. Amplification reactions were carried out for 40 cycles in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA) (30 s at 94 °C, 30 s at 62 °C, and 1 min at 68 °C). The use of
oligonucleotide primers for mouse Cx57 led to amplification of a 377-bp
segment (positions 1075-1452). The PCR products were analyzed by
Southern blot hybridization to a mouse Cx57 probe
(XhoI/XbaI fragment, see above). Using the mouse
Cx57 RT-PCR protocol described above, we checked the RT reaction
mixtures for genomic DNA contaminations by RT-PCR with
-actin
primers. The mouse
-actin primers were (5'-primer) 5'-CGT GGG CCG
CCC TAG GCA ACC-3', and (3'-primer) 5'-TTG GCC TTA GGG TTC AGG GGG-3'
(30). These primers led to amplification of a 243-bp segment of the
cDNA and a 330-bp fragment of the genomic sequence.
-galactosyltransferase), as described
previously (33).
V1) and associated
junctional current, Ij, equal in amplitude but
opposite in polarity for both cells. The junctional conductance is
determined as gj=
Ij/(V2
V1). Signals were recorded in parallel on
videotape (Digital Data Recorder VR-100, Instrutech Corp.) and computer
(A/D converter TL-1, Axon Instruments). Data acquisition and analysis
were performed using Pclamp software (Axon Instruments) and Sigma Plot
(Jandel Corp.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-FixII phages (Stratagene) with a mouse Cx26
probe using low stringency hybridization conditions, 3 of 25 independently isolated positive recombinant phage clones did not
hybridize under stringent conditions to any of the known rodent
connexin genes. Restriction and Southern blot analyses suggested that
all three phage clones contained the same novel, potentially
connexin-related gene. A 2.1-kb XbaI DNA subfragment of the
recombinant phage clone, which hybridized to the Cx26 probe under low
stringency conditions, was ligated in XbaI-linearized pBluesript II SK+, as indicated in Fig.
1. This XbaI fragment contained the complete reading frame of a new mouse connexin (Fig. 2). The open reading frame coded for a
protein of 505 amino acids with a theoretical molecular mass of 57.115 Da. Following the nomenclature proposed by Beyer et al. (8),
we designated this new connexin as mouse Cx57.
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Fig. 1.
Restriction map of the genomic 2124-bp
XbaI DNA fragment containing the mouse Cx57 coding
region (black arrow).
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Fig. 2.
Genomic nucleotide and deduced amino acid
sequences of mouse Cx57. Potential transmembrane regions according
to the algorithm of Kyte and Doolittle (41) are underlined.
Connexin-specific conserved cysteine residues in the putative
extracellular domains are shown on black
background.
-group of connexins (9). Table
I lists the amino acid identities,
according to predicted topological domains, between mouse Cx57 and
porcine Cx60 (44), in comparison to mouse connexins Cx43, Cx40 (36),
Cx37 (26), and Cx26 (6). Cx57 showed greater similarity to porcine Cx60
than to any other murine connexin protein sequence. A phylogenetic tree
of all known murine connexins and porcine Cx60 supports this conclusion
(Fig. 3).
Amino acid identifies of the putative topological domains of mouse Cx57
compared with those of porcine Cx60 and mouse connexin-43, -40, -37, or -26
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Fig. 3.
Phylogenetic tree of murine connexins.
The dendrogram was deduced by tree (HUSAR) analysis of the amino acid
sequence of the putative first extracellular domain. It is not clear
yet whether the recently described mouse Cx36 should be classified as
or
connexin (cf. Refs. 3, 4, and 54). m,
mouse; r, rat.
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Fig. 4.
Mouse Cx57 is a single-copy gene located on
chromosome 4. A, Southern blot hybridization of mouse
genomic DNA with a Cx57 probe under high stringency conditions. Mouse
genomic DNA was digested with different restriction endonucleases as
indicated, electrophoresed, blotted, and hybridized to a radioactively
labeled mouse Cx57 DNA probe. B, schematic map of mouse
chromosome 4, showing linkage of the Cx57 gene to the genetic markers
Mtv14 (mammary tumor virus 14) and Ggtb
(glycoprotein 4 -galactosyltransferase). Fractions refer
to recombination events, and numbers in
parentheses represent recombinal distances ± standard
errors.
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Fig. 5.
Detection of Cx57 mRNA transcripts in
several mouse tissues by Northern blot hybridization and RT-PCR.
A and B, Northern blot hybridization of total RNA
from several mouse tissues to a Cx57 probe. In each lane, 20 µg of
total RNA from several mouse tissues had been applied. Percentage
numbers indicate estimations of total amounts of Cx57 mRNA relative
to the 100% level measured in the total 10.5 dpc mouse embryo
(A) or in Hel-37 cells (B), after standardization
by hybridization to a probe of the human glyceraldehyde-3-phosphate
dehydrogenase gene (data not shown). C, agarose gel of
RT-PCR reactions with mRNA in several mouse tissues. The 377-bp
band represents the expected Cx57 PCR product. D, Southern
blot hybridization of the agarose gel (C) to a Cx57 probe.
E, the same agarose gel after RT-PCR reactions with GAPDH
primers. The identity of the Cx57-derived DNA fragment was verified by
hybridization to the Cx57 probe under high stringency conditions, and
the RT reactions were controlled for genomic contaminations by PCR with
GAPDH primers (28). The primers led to amplification of only the 243-bp
segment of the cDNA.
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Fig. 6.
Non-radioactive Northern blot hybridization
of total RNA from different HeLa-Cx57 transfectants to
digoxygenin-labeled Cx57 antisense RNA. wt, wild type HeLa
cells for control.
2), as described under "Materials and Methods." No
cell-cell transfer for any of these dye molecules was observed in
parental HeLa cells. Homotypic transfer of microinjected neurobiotin
between HeLa-Cx57 cells was detected in first order neighboring cells
after 10 min, and in second order cells 30 min after microinjection
(Fig. 7). No cell-cell transfer of
Lucifer yellow was observed. Intercellular transfer of calcein was weak and visible only after 9 h of incubation. Among 13 heterotypic cell cocultures, tested for dye transfer, only 4 showed transfer of
neurobiotin (see Table II). None of these
combinations yielded transfer of microinjected Lucifer yellow or
calcein. Neurobiotin transfer was evident in Cx57-Cx30.3, Cx57-Cx37,
and Cx57-Cx43 heterotypic junctions.
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Fig. 7.
Homotypic neurobiotin transfer in Cx57 HeLa
transfectants. A, no visible transfer of neurobiotin between
HeLa wild type cells. B, spreading of neurobiotin 10 min
after microinjection in a HeLa Cx57-transfected cell. C,
spreading after 30 min of incubation. Bar, 50 µm. *,
microinjected cell.
Neurobiotin transfer between different connexin-transfected HeLa cells
60 mV). Junctional current, Ij, was
measured during periodic positive and/or negative pulses of
transjunctional voltage, Vj. All cell pairs
tested were electrically coupled. In three cell pairs cytoplasmic
bridges were identified (see below). In cell pairs connected by gap
junctions, coupling conductance varied between 0.2 and 2.5 nS, with a
mean value of ~1.5 nS (n = 13). HeLa parental cell
pairs typically demonstrated uncoupling or very weak coupling through
only a few channels (47).
gmin)/[1 + exp[A(Vj
Vo)]}+gmin.
Vo corresponds to Vj with
a half-maximum of gj. Parameter A
characterizes steepness of gj(ss) decay, and
gmin shows residual values of
gj for negative and positive
Vj polarities. Fitting parameters were as
follows: Vo+ = 25 ± 1 mV (mean ± S.E., Vo-=
25 ± 2 mV,
A+ = 0.2 ± 0.3, A
= 0.16 ± 0.03, gmin+ = 0.14 ± 0.02, and gmin
= 0.20 ± 0.03, where subscript + or
corresponds to depolarization or hyperpolarization of cell
1, respectively. Vo is close to that measured in
Cx 31.1 (~20 mV), Cx37 (~17 mV), and Cx45 (~20 mV) but
significantly smaller than in Cx 26 (~85 mV), Cx30.3 (~60 mV), Cx40
(~55 mV), Cx43 (~45 mV), and 46 (~45 mV). Numbers in parentheses
refer to our published (42) and unpublished data for different HeLa
transfectants. The difference among gmin+ and
gmin
indicates that Vj
gating is slightly asymmetric and more sensitive at positive Vj values.
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Fig. 8.
Illustration of
Vj and
Vm-sensitive voltage gating in HeLa-57
cell pairs. A, examples of
gj-Vj dependence record.
Instantaneous junctional conductance, gj(inst),
was measured by applying periodic positive and negative pulses of 25 mV
to cell 2. Voltage in cell 1 was constant and equal to the holding
potential (see dashed line). During
depolarization of cell 2 to 50 mV, gj decreased
about 10 times and upon hyperpolarization to the holding potential
slowly recovered to the control value. B, data summarizing
gj(ss)-Vj dependence,
where individual gj(ss) data are normalized to
gj(inst),
gj(ss)/gj(inst).
Continuous lines show fitting of the experimental
data with the Boltzmann equation. C, example of
Ij, V1, and
V2 records illustrating
Vm dependence. When both cells were depolarized
simultaneously from 20 mV to +60 mV and then to +70 mV,
gj increased. Instantaneous
Ij was measured by applying voltage steps of
±25 mV.
Vm dependence, gj rose in
response to simultaneous depolarization of both cells (see Fig.
8C). When both cells were depolarized from
20 mV to +60 mV
and then to +70 mV, gj increased approximately 1.5 times. During both depolarization periods,
gj rose with slow time constant (in tens of
seconds) and slowly recovered during hyperpolarization to the holding potential.
10 mV (see dotted line) to
130,
110, and
80
mV. All Vj values induced single-channel gating
between open (main) state (continuous lines) and
residual state (dashed lines). Most of the gating
transitions were relatively fast with transition time of approximately
1-2 mS. Single-channel conductance of open state (
(open)), is ~27 pS and residual state (
(open)) of ~5.5 pS.
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Fig. 9.
Single gap junction channel analysis.
A, example of single gap junction channel current record.
V2 was stepped from the holding potential of
~ 10 mV (see dotted line) to
120,
100, and
80 mV; V1 was kept at the holding potential.
All Vj values induced single-channel gating
between open state (continuous lines) and
residual state (dashed lines). Single gap
junction channel conductance of open (main) state is ~28 pS and
residual state of ~5.5 pS. B, experimental record
illustrating linear dependence of single-channel current on
Vj for the open state (see
Ij record during first voltage ramp) and for the
residual state (see Ij record during second
voltage ramp). Periodic voltage pulses of ±20 mV and ramps of ±80 mV
were applied to cell 2, while cell 1 was maintained at a holding
potential of
3 mV. We suggest that, during the second positive step,
the channel closed to the residual state (see arrow) and
remained there during the second ramp. C, frequency
histogram of
(open). Continuous line shows
Gaussian distribution. Mean value of
(open)= 27.4 ± 0.3 pS
(n = 143).
Vj dependence of
the single channel was tested by using a ramp protocol (see Fig.
9B). Both cells were at the same holding potential
(dotted line). Periodic voltage pulses of ±20 mV
and ramps of ±80 mV were applied to cell 2. During the first bi-phasic voltage step and ramp the channel was open, exhibiting ~27 pS conductance. Ij
Vj dependence was
almost linear when V2 rose from
80 mV to +80
mV. During the second positive pre-pulse, the channel was closed,
presumably, to the residual state (see arrow). Ij record during the second ramp shows that the
channel was closed to the substate with conductance of ~4 pS (see
dashed line). The frequency histogram in Fig.
9C summarizes single-channel conductance data measured in
six experiments. Mean value of single-channel conductance was 27.4 ± 0.3 pS (n = 143).
(residual) was in the range of
~4-6 pS.
(open) measured in HeLa parental cells (about 40 pS).2
5 M) tested in one cell pair with
gj = 1 nS produced full uncoupling. During the
washout period of 30 min, we observed very slow coupling recovery with
random periods of single-channel activity of ~27 pS conductance (data
not shown). The CO2 effect was tested in two cell pairs and
yielded full uncoupling during 1-2 min. Recovery of intercellular
conductance during period of CO2 was relatively slow and
took 10-20 min to reach 50% of control gj
level (data not shown). Thus, Cx57 channels exhibited similar
properties as other connexin channels, in response to these well
established uncoupling factors.
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Fig. 10.
Illustration of heptanol (2 mM)
uncoupling effect. Junctional current, Ij,
was measured by applying periodic negative pulses of 20 mV to cell 2. Holding potential of 10 mV was identical in both cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
class connexins, i.e. no intron
in the coding region, three conserved cysteine residues in both of the
putative extracellular loops, and relatively high amino acid sequence
identity to other
class connexins. We have located the Cx57 gene,
Gja9, on mouse chromosome 4, to which also the genes for
Cx30.3 (Gjb5), Cx31 (Gjb3), Cx31.1
(Gjb4), and Cx37 (Gja4) have been previously assigned (36, 45). Although these previously mapped genes apparently
form a cluster, the map location of Gja9 places it more than
30 centimorgans proximal to this cluster. Cx30.3, Cx31, and Cx31.1 are
class connexins, mainly expressed in skin, whereas Cx37 is a member
of the
subgroup and highly expressed in endothelium.
(open)
27 pS. Cx57 channels are relatively strongly gated by transjunctional voltage (Boltzmann
Vo
25 mV) exhibiting fast transitions
between open and residual states (see Fig. 9B).
(residual) was in the range of 4-6 pS and the ratio
(residual)/
(open) = 5/27
0.18. This number is close to
the value of gmin (gmin+ = 0.14 ± 0.02 and gmin
= 0.20 ± 0.03) and
suggest that at Vj values more than ~50 mV,
almost all channels are in the residual state.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284 (Project C1) and a grant from the Fonds der Chemischen Industrie (to K. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ010741.
** To whom correspondence should be addressed: Inst. für Genetik, Universität Bonn, Römerstr. 164, 53117 Bonn, Germany. Tel.: 49-228-734210; Fax: 49-228-734263; E-mail: genetik{at}uni-bonn.de.
2 D. Manthey, F. Bukauskas, C. G. Lee, C. A. Kozak, and K. Willecke, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Cx, connexin; bp, base pair(s); kb, kilobase pair(s); S, siemen(s); dpc, days post coitum; DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; RT, reverse transcriptase; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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