Molecular Analysis of Connexin 43 and Connexin 30.3 Variants Associated with Skin Disease
Abstract
The connexin family of gap junction-forming proteins are expressed in most cells of the human body. The critical roles of connexins in physiology and lifelong health are highlighted by the link between ~1000 mutations in 11 connexin-encoding genes and at least 30 developmental and early life disorders, including deafness, cataracts, and skin diseases. Collectively, these disorders are common and affect approximately 1 in 1500 live births. Among these, 33 gene mutations in the genes encoding Cx30.3, Cx31, and Cx43 are clinically associated with erythrokeratodermia variabilis et progressiva (EKVP), a rare incurable skin disorder characterized by painful erythematous and hyperkeratotic lesions that worsen with age. EKVP treatment involves symptom management that generally fails to achieve long-term clearance of disease. As such, there is a pressing need to understand the breadth of mechanisms that underpin this debilitating skin condition to guide future research into effective targeted therapeutics for EKVP. Here, we utilized rat epidermal keratinocytes (REKs) to investigate the consequences of connexin variant expression on connexin and keratinocyte physiology. In the first study, we discovered that the Cx43-P283L variant exhibited a modest increase in its lifespan, possibly due to impaired phosphorylation on nearby residues, while the Cx43-T290N variant appeared benign. In our second study, we discovered a trio of variants, Cx30.3-G12D, Cx30.3-T85P, and Cx30.3-F189Y, failed to form functional gap junctions due to a trafficking defect resulting in their entrapment within the secretory pathway. However, these Cx30.3 variants formed gap junctions when intermixed with co-expressed Cx30.3, Cx26, Cx30, or Cx43. Dye uptake studies also revealed these variants assembled into leaky hemichannels (HCs) and/or otherwise impaired cell membrane integrity. Finally, in our third study we showed the Cx30.3-R22H, Cx30.3-S26Y, Cx30.3-P61R, Cx30.3-C86S, Cx30.3-E99K, Cx30.3-T130M, and Cx30.3-M190L variants each altered one or more aspects of the connexin lifecycle, including protein stability and channel function. Additionally, the expression of Cx30.3-P61R and Cx30.3-S26Y impaired keratinocyte viability. Collectively, the range of molecular changes identified are complex and may all be sufficient to cause EKVP. Overall, this project enhanced our understanding of 12 EKVP-associated connexin variants and laid the foundation for the development of targeted treatments for this debilitating disorder.