Molecular Cloning and Functional Expression of the Mouse Gap Junction Gene Connexin-57 in Human HeLa Cells*
- 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
Abstract
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 α 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.
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 GenBank™/EMBL Data Bank with accession number(s) AJ010741.
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↵** 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.
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↵2 D. Manthey, F. Bukauskas, C. G. Lee, C. A. Kozak, and K. Willecke, unpublished observations.
- Abbreviations:
- 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
-
- Received November 9, 1998.
- Revision received February 5, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
