Control mechanism of gap junctional permeability by phosphorylation was examined in a model system in which gap junctional hemichannels were reconstituted in lipid vesicles. Connexin 43 (Cx43) and Connexin 32 (Cx32) were immunoaffinity-purified from rat brain and liver respectively and were reconstituted into unilamellar phospholipid liposomes. The activity of reconstituted channels was measured by monitoring the permeability of liposomes containing connexins. The liposomes containing the connexin proteins were fractionated on the basis of permeability to urea/sucrose in an iso-osmolar density gradient and the sucrose-permeable and impermeable liposomes were separated in the gradient. The liposomes permeable to sucrose were also permeable to a communicating dye molecule, Lucifer Yellow. It was found that the channel activities of the reconstituted connexin hemichannels were pH-sensitive and directly dependent on the phosphorylation states of connexin proteins. The permeability of liposomes containing Cx43 channels was increased by treating the liposomes with calf intestinal phosphatase (CIP). Moreover liposomes formed with Cx43 which was dephosphorylated by CIP treatment revealed increased permeability to sucrose. The permeability of liposomes containing Cx32 channels was decreased by treating the connexins contained in liposomes with protein kinase C and protein kinase A, and the changes of permeability were reversed by dephosphorylating connexin by CIP treatment. The permeability of liposomes containing Cx43 was also decreased by phosphorylating the connexin by MAP kinase treatment. Data provide evidence that the gap junctional permeability is directly dependent on the phosphorylation of gap junction proteins.
인산화에 의한 간극연접의 여닫이 조절기작을 연구하기 위해 단일막 리포좀에 컨넥신 32와 컨넥신 43 간극연접통로를 재구성하고 이를 이용해 여닫이 조절기작을 조사하였다. 컨넥신 32 와 컨넥신 43은 각각 쥐의 뇌와 간에서 면역 친화 크로마토그래피에 의해 순수 분리되었고 단일막 리포좀에 재구성되었다. 간극연접 단백질을 포함한 리포좀은 원심분리시 슈크로즈 농도 구배 상에서 슈크로즈에 대한 투과도의 차이에 따라 침전속도의 차이를 보였다. 슈크로오즈에 대한 투과도가 높은 리포좀은 잘 알려진 간극연접 투과물질인 Lucifer Yellow에 대해서도 높은 투과도를 보였고 이는 재구성된 간극연접통로가 실제 세포에 존재하는 간극연접통로의 크기와 유사하다는 것을 의미한다. 이와 같은 시스템을 이용해 간극연접의 여닫이 조절에 관한 여러 실험을 수행한 결과 간극연접통로의 여닫이는 pH 변화와 컨넥신 단백질의 인산화 정도에 따라 조절된다는 것을 밝혔다. 특히 MAP 인산화 효소가 컨넥신 43 단백질의 직접적인 인산화를 통해 재구성된 간극연접의 여닫이를 조절할 수 있음을 증명하였다. 이러한 결과는 간극연접 단백질의 인산화는 간극연접 통로의 직접적인 구조 변화를 통해 간극연접의 여닫이를 조절할 수 있다는 것과 MAP 인산화 효소가 세포내에서 간극연접의 여닫이를 조절하는데 있어 중요한 역할을 하는 효소라는 것을 의미한다.
