Connexin43 mutations linked to skin disease have augmented hemichannel activity

Miduturu Srinivas,Leping Li,Anthony G Cocozzelli,Nefeli Slavi,Thomas W White,Thomas F Jannace,Caterina Sellitto

doi:10.1038/s41598-018-37221-2

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.

Highlights

Connexins (Cx) are a family of proteins that make gap junction channels, and allow the direct passage of small molecules between adjacent cells[1,2]
Even in the case of Cx43, the initial characterization of the Cx43-G8V mutation linked to PPKCA1 in transfected cells showed increased whole cell membrane current, Ca2+ influx, and cell death when compared to wild-type Cx43, which could have been mediated by an increase in the activity of hemichannels[22]
We further observed that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional hemichannels with greatly increased membrane current flow, a feature not shared by wild-type Cx43

Summary

Introduction

Connexins (Cx) are a family of proteins that make gap junction channels, and allow the direct passage of small molecules between adjacent cells[1,2]. Cx31 mutations causing EKVP were shown to result in increased hemichannel activity, ATP leakage, and necrotic cell death when expressed in transfected cells[38]. Even in the case of Cx43, the initial characterization of the Cx43-G8V mutation linked to PPKCA1 in transfected cells showed increased whole cell membrane current, Ca2+ influx, and cell death when compared to wild-type Cx43, which could have been mediated by an increase in the activity of hemichannels[22]. We further observed that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional hemichannels with greatly increased membrane current flow, a feature not shared by wild-type Cx43 These results suggest that the augmentation of hemichannel function shared by all three mutations may play a role in the pathophysiology of human Cx43 mutations linked to skin disease

Methods

Results

Conclusion