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

Derek L Beahm,Atsunori Oshima,Guido M Gaietta,Galen M Hand,Amy E Smock,Shoshanna N Zucker,Masoud M Toloue,Anjana Chandrasekhar,Bruce J Nicholson,Gina E Sosinsky

doi:10.1074/jbc.m506533200

Abstract

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 alpha- and beta-connexin subgroups but not the gamma-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.

Highlights

Intercellular communication is a fundamental feature of all multicellular organisms
The genetic characterizations of connexin diseases have led to studies focused on understanding how single amino acid changes in connexin sequences result in macroscopic symptoms seen in patients and whether these mutations arise from defects in synthesis, trafficking, docking, or channel function
There has been great interest in finding connexin mutants that form gap junction structures but fail to establish open channels, because such mutants would allow us to distinguish functions of connexins associated with the protein or the structures per se, and those associated with their function in intercellular communication

Summary

Introduction

Intercellular communication is a fundamental feature of all multicellular organisms. Gap junctions are one means by which cells communicate with each other and arise as tissue cells grow and abut each other. Gap junction structures are found throughout vertebrates and invertebrates, the primary sequences of constituent proteins are different from each other even though electron micrographs and physiological assays indicate similar quaternary structure and functionality. As we show in dye transfer and electrophysiological assays, the channels carrying this mutation are nonfunctional and may mimic a “closed pore” state This residue is strictly conserved among the ␣- and ␤-connexins. We show electron microscopic data indicating small differences in the mutant from wild-type structure. We have mutated this residue at the equivalent positions in three connexin isoforms, and we found that function is eliminated in all three cases in intercellular channels in a dominant-negative fashion. In addition to being interesting from a structure/function relationship point of view, this mutation is potentially very useful as a tool for knocking out connexin functionality without having to delete connexin genes or for examining potential heteromeric interactions between different connexins

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Conclusion