Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability

Cinzia Ambrosi, Daniela Boassa, Jennifer Pranskevich, Amy Smock, Atsunori Oshima, Ji Xu, Bruce J. Nicholson, Gina E. Sosinsky

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Abstract

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), C×26T135A in M3 produces a closed channel; 2), C×26M34A in M1 severely decreases channel activity; 3), C×26P87L in M2 has been implicated in defective channel gating; and 4), C×26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP3 transfer. These mutations do not affect C×26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, C×26M34A and C×26V84L are stable as hexamers or dodecamers, but C×26T135A and C×26P87L oligomers are not. This instability is also found in C×26T135A and C×26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type C×26 and C×26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of C×26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.

Original languageEnglish (US)
Pages (from-to)1809-1819
Number of pages11
JournalBiophysical Journal
Volume98
Issue number9
DOIs
StatePublished - May 5 2010

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ASJC Scopus subject areas

  • Biophysics

Cite this

Ambrosi, C., Boassa, D., Pranskevich, J., Smock, A., Oshima, A., Xu, J., Nicholson, B. J., & Sosinsky, G. E. (2010). Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability. Biophysical Journal, 98(9), 1809-1819. https://doi.org/10.1016/j.bpj.2010.01.019