Background

Connexins (Cx) are integral transmembrane proteins that oligomerise into connexons at the cell surface. Connexons dock with similar hexameric protein complexes on adjacent cells to form a bidirectional conduit for gap junction intercellular communication (GJIC; Bosco et al., 2011). Gap junctions play an important role in synchronizing cellular activity and allow for the direct exchange of small ions, metabolites and secondary messengers, crucial for cellular function, survival and tissue homeostasis (Spray and Hanani, 2019). Under normal conditions, undocked connexons, known as hemichannels, remain mostly closed; however, in a pathophysiological context hemichannel-mediated intercellular communication can predominate, permitting the release of 1-2 kDa molecules, including adenosine triphosphate (ATP) into the extracellular milieu (Solini et al., 2015; Hills et al., 2018; Taruno, 2018; Siamantouras et al., 2019). Altered hemichannel activity has previously been linked to inflammation, fibrosis and the progression of various disease states (Roy et al., 2017; Hills et al., 2018; Cea et al., 2020; Sáez et al., 2020). Consequently, quantitative characterisation of hemichannel activity can inform on the onset and progression of disease, e.g., chronic kidney disease (CKD), along with highlighting potential targets for therapeutic intervention (Price et al., 2020).

Depending on the constituent connexin isoform, several factors are known to open hemichannels, including temperature, pH, enteric pathogenic infections and low extracellular calcium ([Ca²⁺]_e) (Trexler et al., 1999; Ceelen et al., 2011; Lopez et al., 2016; Pinto et al., 2017). Whilst various methods can determine expression and localisation of connexin hemichannel subunits at the cell surface, e.g., biotinylation combined with immunoblot analysis and immunocytochemistry respectively, these do not provide a measure for functional changes in connexin-mediated cell communication. In recent years, a range of different methods have been used to evaluate the functional implications of altered hemichannel activity and/or function (Johnson et al., 2016). One of these techniques involves electrophysiological characterisation using whole-cell patch clamp recordings. Although dual whole-cell voltage recordings offer a highly attractive method to attain vast amounts of information on hemichannel permeability selectivity, opening/closing kinetics, open probability latency and presence of subconductance states, it is not without its drawbacks (Brokamp et al., 2012). Not only is this technique highly complex, the membrane potential needed to gate hemichannels is cell-type specific, a difficult hurdle to overcome (Veenstra, 2001). Quantification of hemichannel-permeable molecules into the local extracellular environment (e.g., ATP release) via luminescence based assays is much simpler, yet provides minimal, indirect information with regards to hemichannel activity and would benefit from the use of potentially costly general or local connexin inhibitors (Musil and Goodenough, 1991; Brokamp et al., 2012; Sáez and Green, 2018; Price et al., 2020). Although these techniques provide excellent methodological approaches for identifying cell-surface connexin expression and hemichannel open probability, it is the use of simpler hemichannel-permeable, membrane impermeant dye uptake studies that are routinely used (Johnson et al., 2016). Dye uptake studies make use of controlled parameters and utilise altered [Ca²⁺]_e to gate hemichannels, providing a simple, efficient and reliable procedure. Several fluorescent tracers, including Lucifer Yellow, ethidium bromide, 4’,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and 5(6)-carboxyfluorescein can be used to quantify hemichannel activity, with the extent of dye uptake proportional to the number of active cell-surface hemichannels (Lopez et al., 2016). Unfortunately, the majority of these tracers are hazardous and carcinogenic and require careful handling; however, this step-by-step protocol utilises the non-hazardous, 376 Da polyanionic fluorescent probe, 5(6)-carboxyfluorescein, to quantitively measure hemichannel activity in proximal tubular epithelial cells in vitro.