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  • Protease Inhibitor Cocktail (EDTA-Free, 200X in DMSO) The st

    2022-01-15

    The structural modelling studies gave a view of arsenic mediated covalent modifications possible in Cx43. In order to look for the biochemical effects of Arsenic on Cx43 and Cx43 mediated GJIC formation, and in order to further decipher the mechanism involved in Arsenic induced decreases in expression of Cx43, we used HeLa cells, which does not express Cx43, along with stably Cx43 transfected HeLa-43 Protease Inhibitor Cocktail (EDTA-Free, 200X in DMSO) which exhibit profuse expression of Cx43. The transfected cell line was an effective model to track the changes in the expression of Cx43 post NaAsO2 treatment. This in vitro model was especially efficient for experiments like ICP-MS analysis of immune-precipitated Cx43. The experiments were also conducted in the Cx43 endogenously expressing mouse fibroblast cell line NIH3T3. Immunofluorescence microscopy results showed that Cx43 accumulates inside HeLa-43 cells and the characteristic gap junctional plaques on the surface were absent, demonstrating that Cx43 might be retained in an intracellular compartment. The mouse embryonic fibroblast cell line NIH3T3 cells also showed a profound reduction in Cx43 staining post NaAsO2 treatment. The surface biotinylation experiment revealed significant decrease of surface level Cx43 in Arsenic treated cells. The Lucifer yellow dye transfer in NaAsO2 treated cells was found to be reduced, indicating reduced number of functional gap junctions post NaAsO2 treatment and Immunoblot analysis also revealed decreased levels of total Cx43. Few prior studies have examined mechanistic aspects of disruption of functional gap junctions on the cell surface by Arsenic; thus, the current results provide important and new insights. Musil and Goodenough [23] experiments revealed that the fraction of Cx43 which is assembled into gap junction plaques on the cell surface does not solubilize in the detergent at 4 °C. In the present study, detergent solubilization experiments revealed a significant increase in solubility of Cx43 in TritonX-100 at 4 °C which corresponds to the pool of Cx43 present in the intracellular compartment. There was also a decrease in the insoluble fraction of Cx43 which corresponds to the membrane bound pool of Cx43. Hence, it was evident from the findings thatCx43 was unable to assemble into gap junction plaques on the cell surface. Cx43 monomers oligomerized to form hexamers called hemichannels in order to get docked onto the cell membrane. Oligomerization of Cx43, unlike other connexins, occurs after exit from ER, most likely in the trans-Golgi network [27]. To find out the oligomerization state of Cx43 on Hela-43 cells and NIH3T3 cells after treatment with NaAsO2, we carried out sucrose gradient velocity sedimentation studies. The findings revealed that there was a significant reduction of hexamers, suggesting that Cx43 oligomerization was inhibited by Arsenic. Colocalization of Cx43 with Calnexin (ER marker) revealed that Cx43 was unable to get transported beyond ER. Gap junction proteins are highly dynamic structures, as the Cx43 pool is continuously replaced on the cell surface [19,28,29]. Connexins are known to have fast turnover rate (half-life is equal to 1.5–5 h), in almost all the tissues. The regulation of connexin turnover rate is considered to be an indispensable mechanism of cells, to control functional gap junction levels on the cell surface [30]. The finding of this current study reveals that Arsenic decreases the pool of Cx43 on the cell surface. This study is the first of its kind in deciphering the mechanism of Arsenic induced disruption of GJIC. Our future aim is to develop an in vivo (mouse) model to rationalize the in vitro and in silico models to find the effect of GJIC in different tissues. The results may reveal the pathways which are altered by Arsenic exposure in humans. GJIC has an indispensable function in regulating tissue homeostasis and is thus a decisive factor in the life and death balance of cells. A better understanding of the mechanism(s) of action of Arsenic induced alteration of GJIC and alteration of gap junction proteins (Cxs) will help in deciphering the mechanism that explains the risks associated with exposure to Arsenic.