Abstract
Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.
Highlights
Starting from the equilibrated configuration of two abEC1.1 antibodies simultaneously docked to a hCx26 hemichannel (Xu et al, 2017) (Figure 1), we analyzed the last 10 ns of the molecular dynamics (MD) simulation and searched for antibody-connexin residue pairs that interacted stably
We concluded that abEC1.1 binds Asn54, Thr55, Leu56, Gln57, and Pro58 in EC1, as well as Pro175, Asn176 and Thr177 in EC2
Each antibody appears to interact directly with four different protomers (P) of the connexon: the first antibody interacts with P1, P2, P3, and P4; the second with P4, P5, P6, and P1, the pair of diametrically opposed protomers, P1 and P4, is able to interact with both binding antibodies at the same time
Summary
Connexins are a family of integral transmembrane proteins (indicated by Cx followed by their molecular weight in kDa, e.g., hCx26 for human connexin 26, encoded by the GJB2 gene) which form hexameric plasma membrane structures known as “connexons.” A connexon may function as a regular plasma membrane channel, termed “hemichannel,” or dock head-to head with another connexon from an opposing cell and self-assemble into a gap junction intercellular channel (Mammano, 2018).Partial high-resolution crystal structures have been determined only for hCx26 (Maeda et al, 2009) and sheep Cx46/50 (Myers et al, 2018). Due to the relatively high sequence similarity across the family, all connexin proteins are thought to share a topology similar to that of hCx26 or Cx46/50, which comprise 4 transmembrane helices (TM1-4) connected by 2 extracellular loops (EC1, EC2) and 1 intracellular loop (ICL). The ICL, connecting TM2–TM3, and the cytoplasmic C-terminal domain (CTD) were not resolved (Maeda et al, 2009; Myers et al, 2018). The fairly conserved sequences of EC1 and EC2 suggest the extracellular vestibule of all hemichannels has a relatively rigid three-dimensional (3D) structure. In MD simulations lasting ∼100 ns, it appears to be the stiffest part of the hemichannel (Zonta et al, 2012) due to the presence of six conserved cysteine residues, three in each loop, forming intramolecular disulfide bonds between EC1 and EC2 (Maeda et al, 2009; Myers et al, 2018)
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