The Cx family of integral membrane proteins are

The Cx family of integral membrane proteins [13] are key components of the Gap Junction channels that provide GJIC, which is indispensable in the preservation of tissue integrity and homeostasis in multicellular organisms. Gap Junction channels form a conduit for exchange of ions (Ca2+), second messengers [adenosine 3′,5′-cyclic phosphate (cAMP), inositol 1,4,5 triphosphate (IP3)], small metabolites (sugars, aminoacidsand nucleotides), etc., between the cytoplasm of the adjacent ER 27319 maleate [14,15]. Cx proteins typically are constituted of four α helical transmembrane domains, intracellular N and C termini, a cytoplasmic loop and two extracellular loops (E1 & E2), each of which contain three conserved Cys residues vital for its function. [[16], [17], [18], [19]]. The function of connexin to alter cellular physiology due to Arsenic exposure is not very well understood, except that the cellular level changes of expression occur in response to Arsenic toxicity. A study with liver epithelial cells (WBF344) exposed to Arsenic trioxide (6.25–50 μM) for up to 8 h showed a dose and time dependent decrease in Cx43 [20]. Human Aortic Endothelial cells also show significant decrease in Cx43 composed gap junctions when treated with 1, 10, 100 and 1000 ng/ml of As2O3 [21]. Studies documented in literature reveal that the decrease of Cx43 levels can be inhibited by protease inhibitors. This points to the degradation of Cx43 as a key cause for the decrease in the occurrence of gap junctional channels due to deleterious binding of Arsenic [22]. In prior studies, however, mechanistic aspects were not emphasized to show how GJIC is affected by Arsenic. A myriad of pathophysiological and epidemiological studies on the toxic effects of Arsenic shows it as a potent carcinogen. In our study, we investigate mechanisms of Arsenic interaction with Cx43 and its effect on GJIC, transportation, localization, oligomerization and folding. Our combined in silico and in vitro cellular and biochemical studies indicate that Arsenic can interact with Cx43, thereby inhibiting the oligomerization of Cx43 into hemichannels. The end-result of Arsenic binding is loss of functional gap junctions on the cell surface.
Results
Discussion In this current study, combined in silico structural modelling and in vitro molecular, biochemical and functional data revealed that Arsenic binding to Cx43 may result in conformational change in the protein resulting in altered trafficking of Cx43 to the cell surface, decreasing Cx43 plaque formation, thus causing disruption of cell-cell communication. Traditional molecular biology Cys mutation studies confirming Arsenic binding are problematic to perform here, as Cys disulfide bonds are critical to Cx43 structural stability and their mutation renders the molecule dysfunctional. Nonetheless, the Hard-Soft Acid-Base principle [24] clearly explains why Cys-thiol in Cx43 must be the only chemical group reactive to the Arsenic compounds as has been noted in many Arsenic biotransformation studies [25]. The presence of Arsenic in situ, when Cx43 is being synthesized, folded and transported has potentially different implications. For example, in the initial stages while the protein is being synthesized, if As+3 is bound to free sulfhydryl Cys then it can directly lead to a dysfunctional protein due to misfolded regions, which otherwise require to be tethered together by disulphide bonds for proper folding. As+3 binding consequences arise due to either non-formation or arbitrary formation (i.e., scrambled) of disulfide bonds, especially in the extracellular domain where all six Cysthiol groups are proximal (within 7.0 Å) to each other. Lack of disulfide bonds may cause improper packing of the protein resulting in its dysfunction. Such proteins may be oligomerization deficient and also poorly transportable to the cell surface. Interestingly, replacement of disulphide (SS) bonds by As bridge bonds (SAsS) is not stereochemically equivalent, since the dimensions of the disulfide bond (~2.05 Å) are always shorter than the SAsS linkage (~2.8 Å). The interchange may possibly loosen the packing of the transmembrane helices causing more fluctuation and flexibility, leading to loss of stereochemical integrity required for the gap junction channel function. More importantly, breakage of the Cys54 disulfide which is most proximal to E43, D46, E47 perturbs the Ca+2 receptor site leading to total impairment of Ca+2 binding, which is integral to gap junction gating mechanisms. This fact is already experimentally demonstrated by Toyofuku et al. [26], who mutated Cys 54 to Ser to show that disruption of the Cys 54 – Cys 198 dislufide bond leads to total loss of intercellular Ca2+signaling. They also showed that it is important to hold the sheet comprised of strand-turn-strand residues 183–200 that forms the docking surface for the hemichannel in place. This sheet also includes a small strand comprising residues 53–55, which contains the crucial Cys 54 that holds the upstream Ca+2 binding loop in place. In absence of disulfide bonds stabilizing the whole sheet, the protein has been found to be Ca2+ signaling deficient. The observations together strongly suggest that As+3 affects Cx43 at all stages after its synthesis, starting from its nascent state, to organelle transport, membrane translocation and gap junction channel formation.