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 5

Changes in the endocytosis proteins CAV1 and CAV2 in CX26-mutant mouse cochlea.

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Changes in the endocytosis proteins CAV1 and CAV2 in CX26-mutant mouse c...
The CAV1 isoform preference shifted from α to β in CX26R75W+ cochlea (A). The CAV1β and CAV2 increased in both mutant mice (A and C) at 8 weeks of age, as shown by Western blotting. Protein levels of CAV1β (B) and CAV2 (D) are expressed relative to the amount present in each littermate control (mean ± SEM, n = 5). P = 0.009 and 0.03 for CAV1β and CAV2, respectively, in CX26R75W+ cochlea. P = 0.007 and 0.01 for CAV1β and CAV2, respectively, in Cx26f/f P0-Cre cochlea. (E–K) Accumulation of CAV2 and CAV1 in 3-week-old non-Tg littermate controls (E and I) and CX26R75W+ mice (F–H, J, and K) with S-GJPs (brackets in F) was occasionally observed at the inter-GJP space (G, arrow) and on the surface of the GJPs (H, arrowheads), differing from that seen in littermate controls (I and E). The boxed regions in F and J are magnified in G, H, and K, respectively. (L) Numbers of cells with accumulated CAV1 or CAV2 were counted in five animals from each group (mean ± SEM, n = 10; P = 0.0003, 6.1 × 10–5, 0.001 and 0.002, from left to right). (M–N) In adult mice, vesicles positive for CAV1 (arrowheads) were frequently detected on GJPs in Cx26f/f P0-Cre mice (K), but not in their littermate controls (J). Boxed regions are magnified in the bottom right corner. Scale bars: 10 μm and 5 μm (insets). *P < 0.05; **P < 0.01; ***P < 0.001.