Journal of Cell Science RESEARCH ARTICLE Mutations in Cx30 that are linked to skin disease and non- syndromic hearing loss exhibit several distinct cellular pathologies Amy C. Berger 1, *, John J. Kelly 2, *, Patrick Lajoie 2 , Qing Shao 2 and Dale W. Laird 1,2,` 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 investigate the underlying mechanisms of four autosomal dominant Cx30 gene mutations that are 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 the 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, which is linked to the development of Clouston syndrome, also significantly induced apoptosis but through an endoplasmic- reticulum-independent mechanism. Collectively, we discovered that four unique Cx30 mutants might cause disease through different mechanisms that also likely include their selective trans- dominant effects on coexpressed connexins, highlighting the overall complexity of connexin-linked diseases and the importance of GJIC in disease prevention. KEY WORDS: Connexin, Gap junction, Hemichannel, Hearing loss, Skin disease, Mutation, Vohwinkel syndrome, Bart-Pumphrey syndrome, Clouston syndrome, Keratitis-ichthyosis-deafness syndrome INTRODUCTION Gap junctions are clusters of specialized intercellular channels that regulate the direct exchange of ions and various hydrophilic cellular metabolites that are smaller than 1000 Da, a process known as gap junctional intercellular communication (GJIC) (Alexander and Goldberg, 2003). Two inter-docked connexons (hemichannels), one from each of two apposing cells, form a functional gap junction channel. Each connexon is composed of six oligomerized connexin (Cx) subunits, and, to date, the connexin family consists of 21 members in human (So ¨hl and Willecke, 2003; So ¨hl and Willecke, 2004). Interestingly, the primary function of gap junction channels is to facilitate intercellular communication; however, hemichannels have also been reported to exist and function at the cell surface in an undocked state, permitting the transfer of molecules between extracellular and intracellular environments (Anselmi et al., 2008; Burra and Jiang, 2011; Tong et al., 2007). Hemichannels that are formed from single or multiple types of connexins are termed homomeric and heteromeric, respectively, and gap junction channels are characterized as homotypic or heterotypic according to whether their channels are composed of the same or different connexions, respectively (Burra and Jiang, 2011). The composition of the channel depends on the connexin expression profile of each cell and tissue type, as well as the natural compatibility of the connexins in order for them to intermix (Beyer et al., 2013; Burra and Jiang, 2011; Laird, 2006). Connexins are highly expressed in virtually all tissues in the human body, and GJIC plays an essential role in the regulation of cellular and physiological processes including proliferation, differentiation, apoptosis, growth and development (Alexander and Goldberg, 2003; Choudhry et al., 1997; Decrock et al., 2009; Kumar and Gilula, 1996; McLachlan et al., 2007). In the inner ear and the skin, proper connexin expression and function directly relate to the maintenance of cochlear homeostasis and epidermal differentiation, respectively (Langlois et al., 2007; Wangemann, 2006; Zhao et al., 2006). In the cochlea, Cx26, Cx29, Cx30, Cx31 and Cx43, found in the epithelial and connective tissue gap junction networks, play a crucial role in sound transduction. Cx26, and possibly other connexins, are thought to be involved in the recycling of K + through the supporting cells back to the endolymphatic space, for potential re-entry into sensory cells when activated by an acoustic stimulus (Kikuchi et al., 2000; Nickel and Forge, 2008). Interestingly, at least seven connexins, including Cx26, Cx30 and Cx43, are temporally and spatially expressed at the protein level in the human epidermis with overlapping distribution in the various non-cornified epidermal strata (Di et al., 2001; Kretz et al., 2004). GJIC plays an important role in epidermal differentiation: however, it is also critical to the wound healing process (Churko and Laird, 2013; Langlois et al., 2007). Here, we focus on Cx30, which, in human, is predominantly expressed in the inner ear and epidermis (Di et al., 2001; Nickel and Forge, 2008). Connexin mutations have been linked to a number of different diseases ranging from developmental disorders to congenital cataracts (Laird, 2006). Mutations in the genes encoding Cx26, Cx30, Cx30.3 and Cx31, in particular, have been linked predominantly to hearing loss and various skin diseases (Di et al., 2001). Importantly, mutations in Cx30 and Cx26, the most predominant connexins in the inner ear (Hoang Dinh et al., 2009), are the leading cause of nearly half of the cases of inherited prelingual non-syndromic hearing loss (Bitner-Glindzicz, 2002; Chang et al., 2009; Schutz et al., 2010; Wang et al., 2011). In particular, seven distinct single amino acid substitutions in 1 Department of Physiology and Pharmacology, University of Western Ontario, London, ON N6A 5C1, Canada. 2 Department of Anatomy and Cell Biology, University of Western Ontario, London, ON N6A 5C1, Canada. *These authors contributed equally to this work ` Author for correspondence (Dale.Laird@schulich.uwo.ca) Received 9 July 2013; Accepted 22 January 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1751–1764 doi:10.1242/jcs.138230 1751