Biophysical characterization of connexin hemichannels in model cells

Background. Connexin (Cx) hemichannels (HCs) mediate the exchange of solutes between cytoplasm and extracellular milieu. Dysregulation or aberrant functioning of Cx HCs has been implicated in a plethora of pathological conditions. Mutations in the GJB1 gene, encoding Cx32, cause X-linked Charcot Marie Tooth type 1 (CMTX1) disease, a rare hereditary demyelinating disease of the peripheral nervous system (PNS) for which there is no cure or treatment. Monoclonal antibodies (mAbs) targeting the extracellular domains of HCs are promising therapeutic tools and the development of computational approaches is becoming more and more important to adapt mAbs to a specific target. Aim and methods. This thesis project aimed at characterizing the biophysical properties of Cx HCs with measurements of Ca2+ and dye uptake in model cells overexpressing Cx32, the CMT1X mutant p.D178Y, or wild type (WT) Cx43, together with a genetically encoded biosensor (GCaMP6s). These assays were used to quantify the effects of a fully human inhibitory immunoglobulin G1 mAb, abEC1.1-hIgG1, on Cx32 and its mutant, as well as to characterize a mutated version of the mAb, abEC1.1-1R5, obtained through a computational approach and targeted to Cx43 HCs. Results. Ca2+ transients elicited by restoring a normal extracellular Ca2+ concentration ([Ca2+]ex, 2 mM) after bathing the cells in a “low” [Ca2+]ex (60-200 μM) medium were mainly due to HCs, since they were significantly suppressed (>90%) by flufenamic acid (FFA), a widely used HC inhibitor. Comparing this novel approach to an established dye uptake assay based on DAPI highlighted the greater sensitivity of Ca2+ uptake to discriminate between different conditions such as [Ca2+]ex and the presence of HCs inhibitors (FFA, abEC1.1-hIgG1). An atomistic computational model revealed that the p.D178Y mutation of Cx32 weakened the interaction of the HC with serine 56 (S56) of the mAb, but the rest of the epitope at the outer surface of the HC maintained the binding with the mAb, ensuring it could also inhibit the mutant HC at sub-micromolar concentrations. An unrelated set of preliminary experiments with the mAb mutant abEC1.1-1R5 failed to show a significant effect on cytosolic Ca2+ load and kinetics of Ca2+ uptake in HaCaT cells expressing native Cx43 HCs. Conclusion. These results suggest abEC1.1-hIgG1 has potential for therapeutic intervention to combat CMTX1, thus broadening the scope of the mAb for targeting diseases associated with connexin HCs. The study also paves the way for the intelligent design of new mAbs with improved characteristics and higher affinity for mutant HCs and for other Cxs.