A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model.

Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca²⁺ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability ("leaky") or excessive activity ("hyperactive"). We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca²⁺ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca²⁺ uptake in response to elevated extracellular Ca²⁺ concentrations ([Ca²⁺]_ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca²⁺ uptake, achieving a 50% inhibitory concentration (EC₅₀) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations. In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody's applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs. Plain English summary: Connexins (Cxs) are proteins essential for communication between cells, forming gap junction channels (GJCs) that connect neighboring cells and hemichannels (HCs) that link the inside of cells to their surroundings. Mutations in the GJB1 gene, which makes Cx32, cause X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare nerve disorder that currently has no cure. Our research studied the effects of a specially designed antibody, abEC1.1-hIgG1, on both normal and mutated Cx32 HCs using a well-established cell model for studying these channels. We discovered that the antibody significantly reduced the movement of calcium ions (Ca²⁺), which are vital for nerve health, in both normal and mutated channels. Further analysis showed that the antibody's success might be due to how it binds to certain areas of the channels. Our findings suggest that abEC1.1-hIgG1 could be developed into a treatment for CMTX1 and similar conditions. Moreover, this research could help in creating even more effective antibodies for targeting faulty hemichannels. [ABSTRACT FROM AUTHOR]