Connexin43 mutations linked to skin disease have augmented hemichannel activity

Abstract

Mutations in the gene (GJA1) encoding connexin43 (Cx43) are responsible for several rare genetic disorders, including non-syndromic skin-limited diseases. Here we used two different functional expression systems to characterize three Cx43 mutations linked to palmoplantar keratoderma and congenital alopecia-1, erythrokeratodermia variabilis et progressiva, or inflammatory linear verrucous epidermal nevus. In HeLa cells and Xenopus oocytes, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional gap junction channels with the same efficiency as wild-type Cx43, with normal voltage gating and a unitary conductance of ~110 pS. In HeLa cells, all three mutations also localized to regions of cell-cell contact and displayed a punctate staining pattern. In addition, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D significantly increase membrane current flow through formation of active hemichannels, a novel activity that was not displayed by wild-type Cx43. The increased membrane current was inhibited by either 2 mM calcium, or 5 µM gadolinium, mediated by hemichannels with a unitary conductance of ~250 pS, and was not due to elevated mutant protein expression. The three Cx43 mutations all showed the same gain of function activity, suggesting that augmented hemichannel activity could play a role in skin-limited diseases caused by human Cx43 mutations.

Connexin 43 Mutations Lead to Increased Hemichannel Functionality in Skin Disease

“…Results from immunofluorescent staining revealed proper membrane trafficking to areas of cell contact, as well as the inability of untransfected cells to form gap junctions. In contrast, the unitary conductance of transfected HeLa cells was similar to wild-type Cx43, displaying the capability to form gap junctional channels [69]. These results demonstrate that these mutations efficiently form functioning gap junctions without differences in electrophysiology, and suggest that increasing hemichannel function, evident by the mutations studied, might influence skin pathologies associated with Cx43 [69].…”

“…This, in turn, can intensify Cx43 hemichannel activity; therefore, resulting in ATP leakage and Ca 2+ overload [79,80]. In a previous study, we demonstrated that three Cx43 mutations linked to diseases of the epidermis, Cx43-G8V, Cx43-E227D, and Cx43-A44V, formed functioning hemichannels that mediated increased membrane current flow, suggesting a potential common feature of genetic skin disease related to this protein [69]. Currently, ten of the identified human connexin genes are linked to twenty-eight genetic diseases.…”

“…Currently, there are one syndromic and three non-syndromic skin conditions specifically associated with Cx43 (Table 1). Typically, mutations in GJA1 encoding Cx43 result in a syndromic, autosomal-dominant disorder known as ODDD, which is characterized by craniofacial structure, syndactyly of fingers or toes, and in rare circumstances skin disease [68,69]. Hearing loss is also observed; however, unlike other connexin-related diseases, ODDD hearing loss is conductive rather than sensorineural [47,68].…”

Pannexin 1 activation and inhibition is permeant‐selective

“…; Srinivas et al . ). It should be noted that comparison of uptake of different solutes may be challenged by distinct sensitivity of the methods of detection.…”

“…Ethidium permeation was observed in Panx1-expressing oocytes and HEK293T cells at the resting membrane potential, but Panx1-mediated ion conductance occurred exclusively at positive membrane potentials, in agreement with previous reports (Locovei et al 2006;Silverman et al 2008;Chekeni et al 2010;Zhan et al 2012;Hansen et al 2014b). This ability of a given channel to offer ethidium permeability while excluding atomic ions has previously been demonstrated in aquaporins, some of which readily allow permeation of larger solutes, such as urea and glycerol, while not supporting membrane currents (Tsukaguchi et al 1998;Stroud et al 2003), and in Cx43 hemichannels, which permit ethidium uptake at negative potentials, but membrane conductance only at highly positive membrane potentials, if at all (Contreras et al 2003;Wang et al 2012;Valiunas, 2013;Hansen et al 2014a,b;Nielsen et al 2017;Nielsen et al 2019;Srinivas et al 2019). It should be noted that comparison of uptake of different solutes may be challenged by distinct sensitivity of the methods of detection.…”