내분비계장애물질이 커넥신 채널에 미치는 영향

Abstract

Direct transmission of ions, second messengers, and cellular metabolites between adjacent cells is mediated by gap junction channels. Connexins (Cx) are membrane proteins and monomers for forming gap junction channels. Reproductive organ is the site where the most connexin types are expressed. One major target of endocrine disruptors is reproductive organ. It is quite reasonable to assume that endocrine disruptor may affect gap junction channels in some way. The work of present this thesis was performed to find out gap junction channel blockers using endocrine disruptors and to examine their effect on the plaque formation of gap junction channel. To examine the effect of endocrine disruptors on gap junction channels as channel blockers, Cx46 hemichannels expressed in Xenopus oocyte were used. It is shown that the outward currents of Cx46 hemichannel elicited by depolarizing potentials are decreased by the extracellular application of endocrine disruptors. Bisphenol A (10 nM) reduced the peak current of Cx46 hemichannel up to average 18.01%, and its effect was reversible. Octylphenol (1 uM) reduced the peak current of Cx46 hemichannel up to average 28.21%, and its effect was reversible. Bisphenol A and octylphenol are likely to block Cx46 hemichannel gating and thus are potential candidates for gap junction channel blockers. It is likely that endocrine disruptors can occupy the pore of Cx46 hemichannel and thus obstruct the ionic flux. In fact, octylphenol and bisphenol A have the smallest molecular weights among endocrine disruptors used in this study. To further examine the effect of endocrine disruptors on gap junction channels, the transient expression of Cx43-EGFP fusion constructs in HeLa cells was used. The potency of endocrine disruptors was monitored by the appearance of gap junction plaque formation. The fluorescent pattern of Cx43-EGFP positive HeLa cells was characterized by variable fluorescent structures including long gap junction plaques and internalized vesicles. Gap junction plaques were variable in size and shape and internalized within hours. Internalized vesicles were derived from gap junction plaques and localized to either nuclear peripheries or membrane boundaries. Octylphenol (1 uM) was appeared to disturb the plaque formation of Cx43-EGFP. Octylphenol has the smallest molecular weights among endocrine disruptors used in this study. If octylphenol has a property to be incorporated into the cell membrane rather than diffused into the cytoplasm, it is assumed that inserted octylphenol prevent gap junction channels from aggregating to form the gap junction plaques. The work presented in this thesis is part of our on-going effort to gain insight into the relationship between endocrine disruptors and reproductive system. Single-channel studies using conductive hemichannels promise to provide more detailed mechanistic picture of the effects of endocrine disruptors on gap junction channels.