Assessing Hemichannel and Gap Junctional Channel Activities in HaCaT Keratinocyte Cells

Gap junctions (GJ), formed by connexins (Cx), are mediators of intercellular communication between adjacent cells by allowing the transfer of small molecules. Connexins assemble to form hemichannels, which are then transported to the plasma membrane. There, they can either function independently or dock with adjacent hemichannels to form gap junctions. The epidermis is an avascular tissue and depends on connexin-mediated communication to maintain epidermal cell homeostasis and dysregulation of connexins can lead to human disorders. Keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the GJB2 gene, encoding Cx26 protein, and the severity of the phenotypes varies among individuals carrying distinct mutations. The effects of mutations on channel functions can be assessed by various techniques. Here, we describe approaches to assess hemichannel and gap junctional activity in HaCaT cells that constitutively express Cx26 KID syndrome mutations. Ethidium bromide (EtBr) can be used in scrape loading assay to investigate GJ channel activity, and neurobiotin in dye uptake assay can be used to determine hemichannel functionality using fluorescence microscopy.