Assembly of the cochlear gap junction macromolecular complex requires connexin 26
Kazusaku Kamiya, … , Osamu Minowa, Katsuhisa Ikeda
Kazusaku Kamiya, … , Osamu Minowa, Katsuhisa Ikeda
Published March 3, 2014
Citation Information: J Clin Invest. 2014;124(4):1598-1607. https://doi.org/10.1172/JCI67621.
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Research Article Cell biology

Assembly of the cochlear gap junction macromolecular complex requires connexin 26

Abstract

Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

Authors

Kazusaku Kamiya, Sabrina W. Yum, Nagomi Kurebayashi, Miho Muraki, Kana Ogawa, Keiko Karasawa, Asuka Miwa, Xueshui Guo, Satoru Gotoh, Yoshinobu Sugitani, Hitomi Yamanaka, Shioko Ito-Kawashima, Takashi Iizuka, Takashi Sakurai, Tetsuo Noda, Osamu Minowa, Katsuhisa Ikeda

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Figure 4

Disruption of cochlear GJPs is reproduced by human cDNA clones for CX30 and CX26, with or without mutations in HeLa cells, and leads to functional differences in dye transfer depending on the resultant GJP sizes.

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Disruption of cochlear GJPs is reproduced by human cDNA clones for CX30 ...
(A–E) Clear differences in GJP formation were observed in HeLa cells that expressed the indicated connexin(s), which made homomeric or heteromeric channels. Cells were colabeled with anti-CX26 (green) and anti-CX30 (red) antibodies and were counterstained with DAPI (blue). L-GJPs were observed only when normal CX26 was expressed alone (A) or was coexpressed with CX30 (C). The other combinations (B, D, and E) formed S-GJPs. (F–Q) In HeLa cells that expressed CX30 and CX26, cells with smaller GJPs demonstrated decreased NB transfer. (F, G, L, and M) Shown are HeLa cells that expressed WT CX30 alone (CX30) or coexpressed WT CX30 and WT CX26 (CX30/CX26), or the indicated CX26 mutants (CX30/Cx26R75Q or CX30/CX26R75W). CX26 and CX30 were colocalized, and the GJP size differed across cell lines. (H and N) Quantitative analysis of the GJP length (mean ± SEM). ***P = 3.3 × 10–17 (H) or P = 7.7 × 10–12 (N). (I, J, O, and P) Digital fluorescence images of HeLa cells expressing the indicated connexin(s) after NB scrape-loading. (K and Q) Quantitative analysis of intercellular NB transfer after scrape-loading. Columns represent the mean distance (± SEM) of NB transfer from the scrape line. ***P = 9.6 × 10–9 (K) or P = 3.2 × 10–11 (Q). Scale bars: 10 μm and 5 μm (insets).