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J. Biol. Chem., Vol. 281, Issue 12, 7994-8009, March 24, 2006

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Mutation of a Conserved Threonine in the Third Transmembrane Helix of - and -Connexins Creates a Dominant-negative Closed Gap Junction Channel^*

Derek L. Beahm¹, Atsunori Oshima¹², Guido M. Gaietta, Galen M. Hand, Amy E. Smock, Shoshanna N. Zucker, Masoud M. Toloue³, Anjana Chandrasekhar³, Bruce J. Nicholson³, and Gina E. Sosinsky⁴

From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260 and National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0608

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the - and -connexin subgroups but not the -subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.

Received for publication, June 15, 2005 , and in revised form, December 15, 2005.

^* This work was supported by National Institutes of Health Grants GM065937 (to G. E. S.), GM072881 (to G. E. S.), GM048773 (to B. J. N.), and CA048049 (to B. J. N.) and National Science Foundation Grant MCB-0131425 (to G. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Biophysics, Kyoto University, Kyoto 606-8502, Japan.

³ Present address: Dept. of Biochemistry, University of Texas Health Sciences Center, San Antonio, TX 78229-3900.

⁴ To whom correspondence should be addressed: University of California, San Diego, 1070 Basic Science Bldg. MC 0608, 9500 Gilman Dr., La Jolla, CA 92093-0608. Tel.: 858-534-0128; Fax: 858-534-7497; E-mail: gsosinsky{at}ucsd.edu.

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