A Novel E. Coli-Based Assay for Rapid Screening of Hemichannel Function

Abstract

Connexin hemichannels (HCs) are involved in the pathophysiology of several disorders including deafness, stroke and cardiac infarct. This makes HCs an attractive therapeutic target. Most available pharmacological inhibitors are either non-selective/non-isoform specific, which hampers their translational application. This is in part due to the absence of simple screening assays for the discovery of inhibitors. Here we report a simple bacterial growth-complementation assay to assess connexin HC function. We used the E. coli strain LB2003 as host-system. LB2003 cells cannot grow in low-[K+] medium due to the deletion of K+ import transporters, but growth can be complemented by supplementing the medium with [K+]O, or transforming the cells for expression of K+-selective channels or connexin HCs. We found growth-complementation by expression of functional human connexin 26 (Cx26) or 43 (Cx43). Connexin HCs function in the bacteria was validated using known inhibitors of HCs (Ca2+ and Mg2+). We found that aminoglycosides (kanamycin A & B) inhibit Cx26 and Cx43 HC function in a dose-dependent manner with IC50s in the low micro molar range. Our results present the first HC functional assay amenable for high-throughput scaling, and identified kanamycin as a connexin HC inhibitor with immediate translational potential since it is FDA approved.This work was supported by the National Institutes of Health grants R01GM79629 and 3R01GM079629- 03S1, the American Heart Association Texas Affiliate Inc. grant 14GRNT18750014, Welch Grant- BI-1757 and a Texas Tech University Health Sciences Center Preliminary Data Program grant.