Bao Xiaoyong, Lee Sung Chang, Reuss Luis, Altenberg Guillermo A
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-0437, USA.
Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):4919-24. doi: 10.1073/pnas.0603154104. Epub 2007 Mar 8.
Gap-junctional channels, permeable to large hydrophilic solutes of up to M(r) approximately 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits. The subunit composition was determined by luminescence resonance energy transfer. We show that all Cx43 subunits in the hemichannel hexamer have to be phosphorylated to abolish sucrose (M(r) 342) permeability. We also show that the hemichannel pores with all subunits phosphorylated by PKC have a sizable diameter, allowing for permeation of the small hydrophilic solute ethyleneglycol (M(r) 62). These results indicate that phosphorylation of Cx43 by PKC alters the hemichannel size selectivity and explain why PKC activity affects dye transfer between cells without consistent effects on electrical communication.
间隙连接通道对分子量高达约1000的大型亲水性溶质具有通透性，负责细胞间通讯。蛋白激酶C（PKC）对连接蛋白43（Cx43）的磷酸化作用会消除间隙连接通道和半通道对大型亲水性溶质的通透性，但不会影响对小型无机离子的通透性。在此，我们报告一种制备具有可控亚基组成的纯化半通道的方法，并将其应用于生成具有不同数量PKC磷酸化亚基的半通道。亚基组成通过发光共振能量转移来确定。我们发现，半通道六聚体中的所有Cx43亚基都必须被磷酸化才能消除蔗糖（分子量342）的通透性。我们还表明，所有亚基都被PKC磷酸化的半通道孔具有相当大的直径，允许小型亲水性溶质乙二醇（分子量62）通过。这些结果表明，PKC对Cx43的磷酸化改变了半通道的大小选择性，并解释了为什么PKC活性会影响细胞间的染料转移，而对电通讯没有一致的影响。
