Mutations in Cx30 that are linked to skin disease and non-syndromic hearing loss exhibit several distinct cellular pathologies

Abstract

Connexin 30 (Cx30), a member of the large gap junction protein family, plays a role in the homeostasis of the epidermis and inner ear through gap junctional intercellular communication (GJIC). Here, we investigated the underlying mechanisms of four autosomal dominant Cx30 gene mutations linked to hearing loss and/or various skin diseases. First, the T5M mutant linked to non-syndromic hearing loss formed functional gap junction channels and hemichannels, similar to wild type Cx30. The loss-of-function V37E mutant associated with Clouston syndrome or keratitis-ichthyosis-deafness syndrome was retained in the endoplasmic reticulum and significantly induced apoptosis. The G59R mutant linked to Vohwinkel and Bart-Pumphrey syndromes was retained primarily in the Golgi apparatus and exhibited loss of gap junction channel and hemichannel function, but did not cause cell death. Lastly, the A88V mutant related to Clouston syndrome also significantly induced apoptosis, although through an endoplasmic reticulum-independent mechanism. Collectively, we discovered that four unique Cx30 mutants may cause disease through different mechanisms that also likely include their selective transdominant effects on co-expressed connexins, highlighting the overall complexity of connexin-linked diseases and the importance of GJIC in disease prevention.

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

“…Whether these clinical presentations are associated with the disruption of the normal Cx30 life cycle or function, or whether they are due to any gainof-or loss-of-function mutant effect on other co-expressed connexins, remains poorly understood. Surprisingly, we have previously shown that some Cx30 mutants acquire new properties and can affect the localization of Cx43 (Berger et al, 2014), even though here we show that wild-type Cx30 remains in distinct subplaque domains from Cx43. One explanation for connexin segregation could be due to the wealth of Cx43 binding partners while the interactome for Cx30 is limited due to the small C-terminal domain (Laird, 2010).…”

“…We have previously shown that the presence of GFP does not affect the ability for Cx30 to traffic to the cell surface and form functional gap junction channels and hemichannels (Berger et al, 2014). While a few Cx30 plaques were dynamic, splitting up and Fig.…”

“…We and others have shown that loss-of-function Cx30 gene mutations can lead to severe deafness and devastating skin diseases or both (Grifa et al, 1999;Essenfelder et al, 2004;Schutz et al, 2010;Wang et al, 2011;Berger et al, 2014;Bosen et al, 2014). Yet the cellular life cycle of this connexin remains poorly studied.…”

“…All other experiments used the following DNA constructs: mouse Cx30 encoded within the pBluescript vector was kindly provided by Dr C. Naus (University of British Colombia, BC). To create a GFP-tagged construct, Cx30 cDNA was subcloned into a pEGFP-N1 (Clontech) expression vector, as described previously (Berger et al, 2014). Sar1 and Sar1 H79G cDNA were cloned into pDsRed2-N1 (Clontech) vectors as described previously (Thomas et al, 2005).…”

Endoplasmic Reticulum Stress in Hearing Loss

“…Several deafnessassociated Cx26, Cx29, Cx30, and Cx31 mutants have been shown to be trapped in the ER and are unable to transport to the plasma membrane in transfected cells [98][99][100][101][102]. Moreover, Bip/GRP78 is upregulated in Cx31R180X-or Cx31E183K-overexpressed cells, suggesting that ER stress is elevated by these Cx31 mutations [101].…”