Assembly of the cochlear gap junction macromolecular complex requires connexin 26
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
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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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 6

Discontinuous gap junctions with excessive endocytosis in CX26-mutant mice.

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Discontinuous gap junctions with excessive endocytosis in CX26-mutant mi...
(A–K) Ultrastructure of gap junctions and caveolae in 5-week-old CX26R75W+ (B) and Cx26f/f P0-Cre (D–K) mice compared with their respective littermate controls (A and C). Horizontal ultrathin sections of ISCs revealed that most of the gap junctions (GJs, brackets) were remarkably shorter or discontinuous (B, D, and K) compared with those of the control ISCs (A and C). (B) A caveolar vesicle (asterisk) with dissociated plasma membrane (arrows) around a short gap junction in a CX26R75W+ mouse. In both mutant mice, numerous caveolae and vesicles (B and K, asterisks; E and F, arrowheads; G–J) were observed. (E and F) Abnormally condensed intermembrane layers (asterisks with brackets) at the cell borders were observed in Cx26f/f P0-Cre mice. (G–J) In Cx26f/f P0-Cre mice, the plasma membrane showed a small cave-like shape (G), an invagination with a neck (H, arrows), and the formation of a vesicle-like structure (I); the vesicle was separated from the plasma membrane (J). (K) A caveolar vesicle (asterisk) is shown at the edge of discontinuous short gap junctions (inset) with clear internal layers. (L) Number of caveolae or caveolar vesicles that were counted around each cell border (mean ± SEM with dot plots): n = 23 and 39 cells for the littermate control and CX26R75W+ mice, respectively (***P = 3.2 × 10–5); n = 42 and 97 for the littermate control and Cx26f/f P0-Cre mice, respectively (***P = 8.4 × 10–10). Scale bars: 100 nm and 20 nm (insets).