A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis.

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Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost-effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modified Escherichia coli cells deficient in potassium uptake (LB2003 cells). These cells cannot grow in low-potassium medium, and hemichannel function is assayed by the reversion of the no-growth phenotype. Since functional hemichannels are permeable to potassium, they allow for its uptake and cell growth. The simple reading of bacterial growth in low-potassium medium distinguishes functional hemichannels (growth) from those inhibited (no growth). This assay is simple, robust, inexpensive, and reliable, and is easily scaled to high-throughput multiwell platforms. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of competent LB2003 cells resistant to kanamycin Basic Protocol 2: Growth complementation assay Support Protocol: Evaluation of cytotoxic effects of potential connexin hemichannel inhibitors.