Intercellular Communication through Gap Junctions

418 Pages·1995·24.548 MB·English

by  Y. Kanno, K. Kataoka, Y. Shiba, Y. Shibata and T. Shimazu (Eds.)

Progress in Cell Research
Volume 4
Intercellular Communication
through Gap Junctions
Editors
Y. Kanno
Department of Special Education
Joetsu University of Education
Graduate School of Education
Joetsu, Japan
K. Kataoka
Department of Anatomy
Hiroshima University
School of Medicine
Hiroshima, Japan
Y Shiba
Department of Oral Physiology
Hiroshima University
School of Dentistry
Hiroshima, Japan
Y Shibata
Department of Anatomy
Kyushu University
School of Medicine
Fukuoka, Japan
T. Shimazu
Department of Medical Biochemistry
Ehime University
School of Medicine
Ehime, Japan
Amsterdam - Lausanne - New York - Oxford - Shannon - Tokyo
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V
Preface
This volume is based on the 1993 International Meeting on Gap Junctions held in Hiroshima, Japan, on
August 24-27, 1993. About 150 researchers on gap junctions participated in the meeting. We wish to express our
sincere thanks to all those who attended, especially the younger researchers. All the participants enjoyed the
entire, well-attended meeting, and this resulted in fruitful discussions on gap junctions.
Subsequent to the exceptional reports in 1964 by Dr. W.R. Loewenstein and Dr. Y. Kanno, research on
intercellular communication through gap junctions has continued to expand, and meetings on gap junctions have
been held frequently. The previous meeting on gap junctions at Asilomar in 1991 was organized by Dr. J.E.
Hall, Dr. G.A. Zampighi, and Dr. R. M. Davis, and the proceedings were published by Elsevier Science. The
meetings on gap junctions in 1989 and 1987 were organized by Dr. K. Willecke and Dr. P. Meda, and by Dr. E.
L. Hertzberg and Dr. R. G. Johnson, respectively. We thank them and other members of the international
organizing committee of the 1993 meeting (Dr. M.V.L. Bennett, Dr. R.L. DeHaan, Dr. W.H. Evans, Dr. W.R.
Loewenstein, Dr. B. Weingart, and Dr. H. Yamasaki) for their valuable comments. We also wish to express
sincere thanks to the Ministry of Education, Science and Culture, Japan; Hiroshima Prefectural Government;
Hiroshima Convention Bureau; Chiba-Geigy Foundation (Japan) for the Promotion of Science; Inoue
Foundation for Science; Uehara Memorial Foundation; Ryokufukai Foundation; Osaka Pharmaceutical
Manufactures Association; The Pharmaceutical Manufacturer's Association of Tokyo; Nippon Shinyaku Co.
Ltd.; International Science Foundation (Washington); and others for their generous support.
In line with the objective of the meeting, this volume focuses on the biological meaning of intercellular
communication through gap junctions in various organs. Since we decided on the publication of the proceedings
after the meeting, the most recent, up-to-date findings have also been included in the volume which comprises
90 of the 113 papers presented at the meeting. We hope that this publication will help stimulate further
productive studies on gap junctions.
Yoshinobu Kanno
Katsuko Kataoka
Yoshiki Shiba
Yosaburo Shibata
Takashi Shimazu
4Î' * S
^A, >dBt'.. :«■*
1993 International Meeting on Gap Junctions in Hiroshima August 24-27
Y. Kanno et al. (Eds.)
Progress in Cell Research, Vol. 4
© 1995 Elsevier Science B.V. All rights reserved. 3
THE CONNEXIN FAMILY TREE
M.V.L. Bennett, X. Zheng and M.L. Sogin
Marine Biological Laboratory, Woods Hole, MA02543, and
Albert Einstein College of Medicine, Bronx, NY 10461
INTRODUCTION are evolutionary orthologues. Whether particular
The connexins are the proteins that form gap family members are orthologous or paralogous can
junctions. Cloning of connexin cDNAs has be inferred from molecular phylogenies (with
revealed twelve distinct isoforms in mammals, each varying degrees of confidence, see below).
encoded by a separate gene. Comparison of Orthologous genes should cluster together near the
connexin sequences can generate initial hypotheses terminal branches of the tree, each orthologous
about structure-function relations that are testable group forming a subtree of speciation parallel to the
by site-directed mutagenesis and domain subtrees for paralogous genes. Each subtree
replacement (see Rubin et al., 1992; Bennett et al., describes evolutionary relationships after the
1993; Verseiis et al., 1994). Conserved sequences duplication giving rise to the paralogues and should
and structural motifs are presumed to be functional. be congruent with organism phylogenies inferred
Variable regions are presumed either to be neutral from other genotypic, phenotypic, and
or to confer functional differences. A neutral paleontological evidence. In the absence of
difference in the genes' coding regions may be intergene transfer branching patterns between the
accompanied by a functional difference in a subtrees define the number and order of duplication
regulatory sequence (cf. Li and Noll, 1994). Thus, events that led to the gene families.
a difference in gene regulation may be the only
functional difference between two homologs that SEQUENCE COMPARISONS
differ in amino acid sequence. In the available We used all of the connexin sequences available
genomic sequences of connexins there is no in GenBank, but omitted from the final tree several
indication of alternative splicing in the coding rat sequences that were essentially redundant with
region. However, an intron has been found in the included mouse sequences. Drs. D. Paul, E. Beyer,
5' non-coding region of several connexins, which K. Willecke and their colleagues kindly allowed us
may provide for different pathways of access to several of their sequences prior to
transcriptional and posttranscriptional regulation publication. All twelve mammalian connexins have
(Willecke et al., 1991a). The non-coding exon been identified in rodents, eight in mouse and rat,
provides for a functional difference in post- three in mouse only (Cx30.3, Cx45 and Cx50), and
transcriptional regulation unrelated to differences in one in rat only (Cx33). It is likely that all twelve
coding sequences. occur in both groups (or that one or more have
Phylogenetic trees of gene families from the been lost), since the connexins so far found in only
same and different species can delineate the number one of these rodents are too distinct to have arisen
of gene duplications and sometimes the relative after rat and mouse lines diverged. Sequences of a
timing of these events. The structure of the number of orthologues of the rodent connexins are
connexin family tree is the major subject of this known from other mammals, including dog, cattle
paper. Within a species, members of a gene family and human. Several orthologues are known in
are evolutionary paralogues (genes that have chicken and Xenopus. The nucleic acid sequences
evolved in parallel) and they are generally derived were translated into their putative amino acid
from ancestral gene duplications. In different sequences and aligned using CLUSTAL. The
species, corresponding members of a gene family alignments (available upon request) were further
adjusted using GDE2.0 (Genetic Data Environment equivocal. The variable regions are a major part of
2.0) to ensure the juxtapositioning of coding CL and most of CT shortly after M4. These
regions for functionally equivalent amino acid regions differ in length as well as in sequence; CL
sequences. ranges from 37 amino acids in Cx31.1 to 70 amino
The connexin molecules can be divided into 9 acids in Cx45; CT varies from 18 amino acids in
principal domains: four membrane-spanning regions Cx26 to 211 in Cx50 (and 268 in chicken Cx56).
(M1-M4), two extracellular loops (El, E2), and in The greater variability in length of the C terminus
the cytoplasm the N terminus (NT), the C terminus (CT) may in part result from point mutations
(CT) and a cytoplasmic loop (CL) connecting M2 inserting or removing a stop codon; however the
and M3 (Fig. 1). The general topology is short conserved sequences in the middle and near
supported by antibody and protease studies (cf. the end of the C terminus (see below) indicate that
Bennett et al., 1991). other mechanisms must also be operative.
As has been noted before there are two highly There are two short regions of similarity in the C
variable and two relatively conserved regions. The terminus of the larger connexins (Fig. 1, Table I).
two conserved regions are NT-MI-E1-M2 and M3- The function of the more N terminal sequence is
E2-M4 extending slightly into CL and CT. The unknown. The sequences near the C terminal ends
inter-domain boundaries are generally well contain Ser and basic amino acids and are likely to
conserved and readily identified. The ends of M3 be phosphorylation sites, as has been shown
are not clear, but the alignment in this region is un- directly for (rat) Cx43 (Saez et al., 1993). (An
unrelated phosphorylation site for A kinase is found
in Cx32, Saez et al., 1990).
Sequence comparisons have been useful in
analysis of voltage dependence of the conductance
of gap junctions formed of Cx26 and Cx32 (Rubin
et al., 1991; Bennett et al., 1993, Verseiis et al.,
1994). These two connexins each form gap
junctions closed by transjunctional voltage, with
equal sensitivity for voltages of either sign. The
symmetry is ascribable to there being two
oppositely oriented hemichannels, one in each
xenCx38 -GGHNWSRIQMEQ- 284 -HQTSSKQQYV- 333
musCx37 -TEQNWANLTTEE- 298 -NSSASKKQYV- 334
musCx40 -SRKNPDALATGE- 300 -ASSKARSDDL- 356
dogCx4 0 -SQQNTDNLATEQ- 299 II 355
chiCx42 -SQQNTANFATER- 308 II 367
-100 0 100 200 300 400 500 600 mamCx4 3 -SEQNWANYSAEQ- 317 -ASSRPRPDDL- 380
musCx45 -YKQNKANIAQEQ- 322 -SSKSGDGKTS- 393
Number of Amino Acids ratCx46 -TEQNWASLGAEP- 339 -SSGRARPGDL- 414
xnusCx50 -REEPPIEEAVEP- 351 -ASSRARSDDL- 438
Figure 1. Domain organization of the connexins. divergent conserved
musCx40 -SRKNPDALATGE- 300 -ASSKARSDDL- 356
Each horizontal line represents a connexin with dogCx4 0 -SQQNTDNLATEQ- 299 -ASSKARSDDL- 355
location on the abscissa indicating the predicted Identities _S D-LAT-- -ASSKARSDDL-
molecular weight and length indicating number of conserved divergent
musCx37 -TEQNWANLTTEE- 298 -NSSASKKQYV- 334
amino acids. The interdomain boundaries and the ratCx46 -TEQNWASLGAEP- 339 -SSGRARPGDL- 414
conserved region in the central region of the C Identities -TEQNWA-L--E-- --S
terminus are indicated by filled circles. The Table I. Sequences of the two conserved regions
connexins included are mouse Cx26, Xenopus found in the C termini of most Group II connexins.
Cx30, mouse Cx31, mouse Cx32, rat Cx33, mouse These regions have diverged at different rates in
Cx37, Xenopus Cx38, mouse Cx40, chicken Cx42, different lineages; either region can be more
mouse Cx43, mouse Cx45, rat Cx46, mouse Cx50 divergent. The numbers refer to the last amino acid
and chicken Cx56. in the sequences.
5
membrane so that one closes for each polarity of second positions of codons that could be
voltage. The two amino acids, KE (LysGlu), at the unambiguously aligned were included in the
beginning of El in Cx26 are unique for this sequence comparisons. For phylogenetic inferences
position among the connexins. Mutation of these based upon distance matrix methods, sequence
residues to the consensus sequence for this position, similarities were converted to evolutionary
ES (GluSer), increased the steepness of the distances expressed in nucleotide changes/site by
conductance/voltage relation. The converse change the method of Jukes and Cantor (1969). Trees
in Cx32 increased the steepness of its were constructed using a modification of the least
conductance/voltage relation. The properties of squares distance matrix methods (Elwood et al.,
heterotypic wild type/mutant channels revealed that 1985). Trees based upon the principles of
the polarity of sensitivity of the hemichannels was maximum parsimony were inferred using heuristic
opposite in the two cases, that is closure with the search options in the computer program PAUP3.0
mutant hemichannel properties occurred with the (Swofford, 1991). Initial tree topologies were
mutant side positive in the case of Cx26 and with obtained by addition of randomly selected taxa
the mutant side negative in the case of Cx32. using ten repetitions for each round of parsimony
Changing domains showed that the NT-MI analysis. To assess the fraction of sites that
domains determined the polarity of sensitivity support elements in our distance and parsimony
(Verselis et al., 1994). The only site in the NT-MI analyses, we employed bootstrap procedures
domains in which the two differ in charge is the (Felsenstein, 1985).
third amino acid which is Glu in Cx26 and Asn in The distance matrix tree in Fig. 2 describes
Cx32. Exchange of these amino acids in each case evolutionary relationships between orthologous and
reversed the polarity of voltage sensitivity. The paralogous members of the connexin gene family.
conclusion was that all three amino acids are part It is largely congruent with trees generated using
of the voltage sensor. This study is illustrative of either the first or the second conserved region
how sequence comparisons can guide structure alone, and it is consistent with bootstrap analyses
function analysis. based upon parsimony. The number of bootstrap
It has recently been reported that X-linked replicates displaying corresponding topological
Charcot-Marie-Tooth disease is associated with elements in distance matrix and parsimony
mutations in the Cx32 gene (Bergoffen et al., 1993; procedures (in parentheses) is indicated in the
Fairweather et al., 1994). Fifteen different lineages figure (values below 50 percent are not included).
with distinct mutations in the coding sequence are Rather than interpreting bootstrap values as
known. Several of the mutations are frame shifts measures of statistical confidence in phylogenetic
that would be certain to prevent the gene product tree reconstructions, they should be regarded as
from forming junctions. Other point mutations in measures of relative confidence between topological
M3 and El and E2 are of highly conserved elements in the tree. Bootstrap values are known
residues; it appears likely that these mutant proteins to be influenced by the number of changes that
also would not generate functional gap junctions. define internal branches in phylogenetic trees
These predictions can be and are being tested by (Hillis and Bull, 1993), and are often influenced by
site directed mutagenesis. Two additional lineages use of different taxa or genes in molecular
show no mutations in the coding sequence; reconstructions (Liepe et al., 1993).
disruption of the promoter region or regulatory sites Although the tree is unrooted, separation into
may produce the phenotype in these cases. Groups I and II as indicated appears reasonable.
Branch points that represent duplications are
THE TREE(S) indicated by black ovals. Branch points that
We restricted our phylogenetic analyses to the represent speciation are unmarked. One branch is
two major conserved regions in connexin genes. questionable in nature (Cx37-Cx38, see below).
Because of higher rates of substitution leading to The second and third duplications within the Group
frequent but unseen reversals, only the first and I lineage appear well resolved by the distance
6
Figure 2. Our best connexin
97 (98) Mouse Cx26
89 (50) Human Cx26 family tree obtained by distance
97 (96) Xenopus Cx30
methods. The branch points at
• Mouse Cx32
(5β) Mouse Cx30.3 Group I filled ovals represent gene
97(100)
ioo(ioo) r Mouse Cx31
97(100) duplications. The branch points
LRatCx31
97(100) r-Mouse Cx31.1 without ovals represent
L_RatCx31.1
speciation. One branch point
66 (70)e M ouse Cx37
Rat Cx37 indicated by a question mark is
96 (80) 97(100) Human Cx37
ambiguous (see text). The
Xenopus Cx38
97(100) Rat Cx33 numbers on the horizontal lines
Xenopus Cx43
represent the number of bootstrap
Chicken Cx43
91 (68) Mouse Cx43 replicates of 100 trials displaying
97(100) Rat Cx43
corresponding topological
Bovine Cx43
Human Cx43 Group II elements in distance matrix and
-Chicken Cx42
97(100) i97(ioo)_r-Dog Cx40 (in parentheses) parsimony
Mouse Cx40 procedures. The calibration
100(100) l Rat Cx40 represents a difference of 0.1
97 (73) Chicken Cx56
Utt — RatCx46 of a possible 1.0, i.e., 10 %.
— Mouse Cx50
97(100) ■ Chicken Cx45
j Mouse Cx45
97(87) ' Dog Cx45
.10
method. The later node is less probable according ly, mammalian Cx43s are more like each other than
to parsimony analysis, but this uncertainty may be they are like Xenopus Cx43 and mammalian Cx32s
an artifact of the long branch going to the Cx30.3, are more like each other than they are like Xenopus
Cx31, Cx31.1 group. Relations among Cx30.3, Cx30. The rate of divergence differs among
Cx31 and Cx31.3 are uncertain, although they are connexins. Cx43s of mammals and Xenopus are
a group and their genes are on the same less divergent than are the orthologues, Xenopus
chromosome (Schwarz et al., 1992). The Cx30 and the mammalian Cx32s. Xenopus Cx38
duplications giving rise to this group may be and mouse Cx37 are more closely related to each
relatively recent, and intergene transfer may be other than to any other connexin but considerably
responsible for the ambiguity in the relations more divergent than Cx30 and Cx32. These
between them. There is some conservation in the differences in rates of divergence are true of the
CL domain in these connexins, but inclusion of this less conserved regions of the sequences as well as
information in the tree construction was not helpful. those used to make the tree. Comparing mouse and
Their C termini are very divergent and are not Xenopus CL and CT domains are 85% and 78%
informative with respect to their phylogeny. In identical for Cx43; 68% and 36% identical for
Group II the affiliations of Cx43s and Cx33 and of Cx30, Cx32 and 32% and 22% identical for Cx37,
Cx37s and Cx38 are well resolved. Deep branches Cx38. Although Cx37 and Cx38 are functionally
involving these groups, Cx50 and the presumably related by their pronounced voltage dependence,
orthologous Cx40, Cx42 group, the Cx45 group and their very disparate tissue distribution and CT
the Cx46, Cx56 group are ambiguous. Resolution sequences suggest that they are distinct connexins
of these branches may be possible when sequences (Willecke et al., 1991b). There may be a Cx38
from lower forms become available. orthologue in mammals and /or a Cx37 orthologue
The connexin trees are consistent with vertebrate in Xenopus.
evolution in that mammalian orthologues are closer As noted above in relation to the El and E2
to each other than to their chicken orthologues (see domains, different conserved regions of the
Cx43s, Cx45s and the Cx40, Cx42 group). Similar-
7
connexin molecules have diverged at different rates. tree shows that most gene duplications giving rise
Differences in rates of change are also evident in to the family occurred early in or prior to vertebrate
the two regions of conservation in the C terminal divergence. The topology of most deep branches of
domain, either of which can be more divergent. In the tree is uncertain. Evolutionary rates vary for
Cx43s both regions are completely conserved in all different paralogous connexin genes.
the sequences, those of Xenopus, chicken and
mammal, again indicating the relative stability of ACKNOWLEDGEMENTS
this gene. In the Group II connexins the distal C This paper is abridged from Bennett et al., 1994.
terminal sequences that contain putative We are indebted to J.Y. Liu, who helped with the
phosphorylation sites give two clusters, the Cx37, initial alignments. The work was supported in part
Cx38 group and the others with Cx45 as an outlier. by grants from the National Institutes of Health
The more central conserved region shows closer NS-07512 and HD-04248 to MVLB and GM-32964
affiliation between Cx37, Cx43 and Cx46 than to MLS. MVLB is the Sylvia and Robert S. Olnick
between Cx37 and Cx38. In the Cx40, Cx42 Professor of Neuroscience.
orthologous group the dog and chicken orthologues
are more closely related in this region than the dog REFERENCES
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