Direct intercellular communication occurs through specialized channels, which are formed by the interaction of two half-channels, or connexons, contributed by each of the two participating cells. The ability to establish intercellular communication is specified, in part, by the expression of different structural proteins, termed connexins. Connexins can control the establishment of intercellular communication by selectively pairing with some but not other family members. To characterize the protein domains that allow connexins to recognize and discriminate between alternative partners, we have created chimeras composed of selected regions of rat connexin43, which forms channels with Xenopus connexin38, and rat connexin32, which cannot. Pairs of Xenopus oocytes were used to test the ability of the chimeras to form homotypic channels with themselves, and heterotypic channels with the parent connexins or with endogenous Xenopus connexin38. While all hybrid molecules tested were efficiently expressed by oocytes, most were devoid of functional activity. A chimera consisting of connexin32 from the N terminus to the second transmembrane domain, fused to connexin43 from the middle cytoplasmic loop to the C terminus, designated as 3243H4, was able to pair functionally with Xenopus connexin38 and one of its parent connexins, connexin43. Voltage-dependent closure of heterotypic channels containing 3243H4 was asymmetric, exhibited novel characteristics that were not predicted by the behavior of the parent connexins and was dependent on the type of connexin with which 3243H4 was paired. In contrast, 3243H4 was unable to form functional channels with either itself or the other parent, connexin32. Together, these results suggest that these connexins are not composed of functionally exchangeable regions and that multiple domains, namely the middle cytoplasmic portion and the second extracellular domain, can influence the interactions between connexins present in adjacent cells. Furthermore, they indicate that voltage gating is not strictly intrinsic behavior for a given connexin, but can be modulated by the partner connexins to which they are paired. Finally, the finding that 3243H4 is functional only in heterotypic configurations, and cannot form homotypic channels, suggests the existence of a novel form of selectivity: self-discrimination. The latter property may represent another mechanism that operates to control the extent of communication between cells.

REFERENCES

Barrio
L. C.
,
Suchyna
T.
,
Bargiello
T.
,
Xu
L. X.
,
Roginski
R. S.
,
Bennett
M. V. L.
,
Nicholson
B. J.
(
1991
).
Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.
Proc. Nat. Acad. Sci. USA
88
,
8410
8414
Barrio
L. C.
,
Handler
A.
,
Bennett
M. V. L.
(
1993
).
Inside-outside and transjunctional voltage dependence of rat connexin43 channels expressed in pairs of Xenopus oocytes.
Biophys. J
64
,
191
–.
Bennett
M. V. L.
,
Barrio
L. C.
,
Bargiello
T. A.
,
Spray
D. C.
,
Hertzberg
E.
,
Sáez
J. C.
(
1991
).
Gap junctions: new tools, new answers, new questions.
Neuron
6
,
305
320
Bennett
M. V. L.
,
Verselis
V. K.
(
1992
).
Biophysics of gap junctions.
Semin. Cell Biol
3
,
29
47
Beyer
E. C.
,
Paul
D. L.
,
Goodenough
D. A.
(
1987
).
Connexin43: a protein from rat heart homologous to a gap junction protein from liver.
J. Cell Biol
105
,
2621
2629
Beyer
E. C.
,
Kistler
J.
,
Paul
D. L.
,
Goodenough
D. A.
(
1989
).
Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues.
J. Cell Biol
108
,
595
605
Beyer
E. C.
,
Paul
D. L.
,
Goodenough
D. A.
(
1990
).
Connexin family of gap junction proteins.
J. Membr. Biol
116
,
187
194
Bruzzone
R.
,
Haefliger
J.-A.
,
Gimlich
R. L.
,
Paul
D. L.
(
1993
).
Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol. Biol.
Cell
4
,
7
20
Dahl
G.
,
Levine
E.
,
Rabadan-Diehl
C.
,
Werner
R.
(
1991
).
Cell/cell channel formation involves disulfide exchange.
Eur. J. Biochem
197
,
141
144
Dahl
G.
,
Werner
R.
,
Levine.
E.
,
Rabadan-Diehl
C.
(
1992
).
Mutational analysis of gap junction formation.
Biophys. J
62
,
172
182
Ebihara
L.
,
Beyer
E. C.
,
Swenson
K. I.
,
Paul
D. L.
,
Goodenough
D. A.
(
1989
).
Cloning and expression of a Xenopus embryonic gap junction protein.
Science
243
,
1194
1195
Eghbali
B.
,
Kessler
J. A.
,
Spray
D. C.
(
1990
).
Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin32.
Proc. Nat. Acad. Sci. USA
87
,
1328
1331
Eghtedarzadeh
M. K.
,
Henikoff
S.
(
1986
).
Use of oligonucleotides to generate large deletions.
Nucl. Acids. Res
14
,
5115
–.
Fishman
G. I.
,
Spray
D. C.
,
Leinwand
L. A.
(
1990
).
Molecular characterization and functional expression of the human cardiac gap junction channel.
J. Cell Biol
111
,
589
598
Fishman
G. I.
,
Moreno
A. P.
,
Spray
D. C.
,
Leinwand
L. A.
(
1991
).
Functional analysis of human cardiac gap junction channel mutants.
Proc. Nat. Acad. Sci. USA
88
,
3525
3529
Goodenough
D. A.
,
Paul
D. L.
,
Jesaitis
L.
(
1988
).
Topologicaldistribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions.
J. Cell Biol
107
,
1817
1824
Haefliger
J.-A.
,
Bruzzone
R.
,
Jenkins
N. A.
,
Gilbert
D. J.
,
Copeland
N. J.
,
Paul
D. L.
(
1992
).
Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping.
J. Biol. Chem
267
,
2057
2064
Hennemann
H.
,
Dahl
E.
,
White
J. B.
,
Schwarz
H.-J.
,
Lalley
P. A.
,
Chang
S.
,
Nicholson
B. J.
,
Willecke
K.
(
1992
).
Two gap junction genes, connexin 31.1 and 30.3, are closely linked on mouse chromosome 4 and preferentially expressed in skin.
J. Biol. Chem
267
,
17225
17233
Hennemann
H.
,
Schwarz
H.-J.
,
Willecke
K.
) (
1992
).
Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and-45 cDNAs.
Eur. J. Cell Biol
57
,
51
58
Hennemann
H.
,
Suchyna
T.
,
Lichtenberg-Frate
H.
,
Jungbluth
S.
,
Dahl
E.
,
Schwarz
J.
,
Nicholson
B. J.
,
Willecke
K.
(
1992
).
Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung.
J. Cell Biol
117
,
1299
1310
Hertzberg
E. L.
,
Disher
R. M.
,
Tiller
A. A.
,
Zhou
Y.
,
Cook
R.
(
1988
).
Topology of the M r 27,000 liver gap junction protein. Cytoplasmic localization of amino-and carboxyl termini and a hydrophilic domain which is protease-hypersensitive.
J. Biol. Chem
263
,
19105
19111
Hoh
J. H.
,
John
S. A.
,
Revel
J.-P.
(
1991
).
Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.
J. Biol. Chem
266
,
6524
6531
Horton
R. M.
,
Cai
Z.
,
Ho
S. N.
,
Pease
L. R.
(
1990
).
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.
BioTechniques
8
,
528
535
Krieg
P. A.
,
Melton
D. A.
(
1984
).
Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
Nucl. Acids Res
12
,
7057
7070
Levine
E.
,
Werner
R.
,
Neuhaus
I.
,
Dahl
G.
(
1993
).
Asymmetry of gap junction formation along the animal-vegetal axis of Xenopus oocytes.
Dev. Biol
156
,
490
499
Makowski
L.
,
Caspar
D. L. D.
,
Phillips
W. C.
,
Goodenough
D. A.
(
1977
).
Gap junction structures. II. Analysis of the X-ray diffraction data.
J. Cell Biol
74
,
629
645
Methfessel
C.
,
Witzemann
V.
,
Takahashi
T.
,
Mishina
M.
,
Numa
S.
,
Sakmann
B.
(
1986
).
Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.
Pflugers Arch. Ges. Physiol
407
,
577
588
Milks
L. C.
,
Kumar
N. M.
,
Houghten
R.
,
Unwin
N.
,
Gilula
N. B.
(
1988
).
Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.
EMBO J
7
,
2967
2975
Moreno
A. P.
,
Fishman
G. I.
,
Spray
D. C.
(
1992
).
Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human gap junction channels.
Biophys. J
62
,
51
53
Noda
M.
,
Ikeda
T.
,
Kayano
T.
,
Suzuki
H.
,
Takeshima
H.
,
Kurasaki
M.
,
Takahashi
H.
,
Numa
S.
(
1986
).
Existence of distinct sodium channel messenger RNAs in rat brain.
Nature
320
,
188
192
Paul
D. L.
(
1986
).
Molecular cloning of cDNA for rat liver gap junction protein.
J. Cell Biol
103
,
123
134
Paul
D. L.
,
Ebihara
L.
,
Takemoto
L. J.
,
Swenson
K. I.
,
Goodenough
D. A.
(
1991
).
Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes.
J. Cell Biol
115
,
1077
1089
Reed
K. E.
,
Westphale
E. M.
,
Larson
D. M.
,
Wang
H.-Z.
,
Veenstra
R. D.
,
Beyer
E. C.
(
1993
).
Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.
J. Clin. Invest
91
,
997
1004
Risek
B.
,
Klier
F. G.
,
Gilula
N. B.
(
1992
).
Multiple gap junction genes are utilized during rat skin and hair development.
Development
116
,
639
651
Risley
M. S.
,
Tan
I. P.
,
Roy
C.
,
Sáez
J. C.
(
1992
).
Cell-, age-and stage-dependent distribution of connexin43 gap junctions in testes.
J. Cell Sci
103
,
81
96
Rubin
J. B.
,
Verselis
V. K.
,
Bennett
M. V. L.
,
Bargiello
T. A.
(
1992
).
A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32.
Proc. Nat. Acad. Sci. USA
89
,
3820
3824
Rubin
J. B.
,
Verselis
V. K.
,
Bennett
M. V. L.
,
Bargiello
T. A.
(
1992
).
Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.
Biophys. J
62
,
183
195
Rup
D. M.
,
Veenstra
R. D.
,
Wang
H.-Z.
,
Brink
P. R.
,
Beyer
E. C.
(
1993
).
Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression.
J. Biol. Chem
268
,
706
712
Spray
D. C.
,
Harris
A. L.
,
Bennett
M. V. L.
(
1981
).
Equilibrium properties of a voltage-dependent junctional conductance.
J. Gen. Physiol
77
,
77
93
Spray
D. C.
,
Moreno
A. P.
,
Eghbali
B.
,
Chanson
M.
,
Fishman
G. I.
(
1992
).
Gating of gap junction channels as revealed in cells stably transfected with wild type and mutant connexin cDNAs.
Biophys. J
62
,
48
50
Stevenson
B. R.
,
Siliciano
J. D.
,
Mooseker
M. S.
,
Goodenough
D. A.
(
1986
).
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.
J. Cell Biol
103
,
755
766
Stuhmer
W.
,
Conti
F.
,
Suzuki
H.
,
Wang
X.
,
Noda
M.
,
Yahagi
N.
,
Kubo
H.
,
Numa
S.
(
1989
).
Structural parts involved in activation and inactivation of the sodium channel.
Nature
339
,
597
603
Suchyna
T.
,
Xu
L. X.
,
Gao
F.
,
Fourtner
C. R.
,
Nicholson
B. J.
(
1993
).
Identification of a proline residue as a transduction element involved in the voltage gating of gap junctions.
Nature
365
,
847
849
Swenson
K. I.
,
Jordan
J. R.
,
Beyer
E. C.
,
Paul
D. L.
(
1989
).
Formation of gap junctions by expression of connexins in Xenopus oocyte pairs.
Cell
57
,
145
155
Swenson
K. I.
,
Piwnica-Worms
H.
,
McNamee
H.
,
Paul
D. L.
(
1990
).
Tyrosine phosphorylation of the gap junction protein connexin43 is requiredfor the pp60v- src -induced inhibition of communication.
Cell Regul
1
,
989
1002
Werner
R.
,
Levine
E.
,
Rabadan-Diehl
C.
,
Dahl
G.
(
1989
).
Formation of hybrid cell-cell channels.
Proc. Nat. Acad. Sci. USA
86
,
5380
5384
Werner
R.
,
Levine
E.
,
Rabadan-Diehl
C.
,
Dahl
G.
(
1991
).
Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes.
Proc. R. Soc. Lond. B Biol. Sci
243
,
5
11
White
T. W.
,
Bruzzone
R.
,
Goodenough
D. A.
,
Paul
D. L.
(
1992
).
Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol. Biol.
Cell
3
,
711
720
White
T. W.
,
Bruzzone
R.
,
Wolfram
S.
,
Goodenough
D. A.
,
Paul
D. L.
(
1993
).
The selective formation of heterotypic intercellular channels by different connexins may involve the second extracellular domain. Mol. Biol.
Cell
4
,
328
–.
Wilders
R.
,
Jongsma
H. J.
(
1992
).
Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.
Biophys. J
63
,
942
953
Willecke
K.
,
Heynkes
R.
,
Dahl
E.
,
Stutenkemper
R.
,
Hennemann
H.
,
Jungbluth
S.
,
Suchyna
T.
,
Nicholson
B. J.
(
1991
).
Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung.
J. Cell Biol
114
,
1049
1057
Yancey
S. B.
,
John
S. A.
,
Ratneshwar
L.
,
Austin
B. J.
,
Revel
J.-P.
(
1989
).
The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains.
J. Cell Biol
108
,
2241
2254
Yeager
M.
,
Gilula
N. B.
(
1992
).
Membrane topology and quaternary structure of cardiac gap junction ion channels.
J. Mol. Biol
223
,
929
948
Zhang
J.-T.
,
Nicholson
B. J.
(
1989
).
Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.
J. Cell Biol
109
,
3391
3401
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