Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels".

Hemichannels (HCs) are large pore hexameric structures composed of connexin (Cx) subunits that allow the exchange of ions, metabolites, and a variety of other molecules between cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluo-rometric measurements of Ca2+ uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate its usefulness for the characteriza-tion of new monoclonal antibodies (mAb) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same method-ology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.