Retamal Mauricio A, Schalper Kurt A, Shoji Kenji F, Bennett Michael V L, Sáez Juan C
Núcleo Milenio Inmunología e Inmunoterapia, Departamento de Ciencias Fisiológicas, Pontificia Universidad Católica de Chile, Santiago 4860, Chile.
Proc Natl Acad Sci U S A. 2007 May 15;104(20):8322-7. doi: 10.1073/pnas.0702456104. Epub 2007 May 9.
Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca(2+) and enhanced by low Ca(2+), positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La(3+), by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.
许多细胞中的非连接膜含有连接蛋白间隙连接半通道（或连接子），这些半通道可以打开，允许小分子通透。在正常细胞外Ca(2+) 浓度下，Cx43半通道很少打开，而在低Ca(2+) 浓度、正膜电位以及关键残基去磷酸化的情况下，其打开频率会增加。在此，我们报告在其他条件正常的情况下，降低细胞内氧化还原电位会增加Cx43半通道的开放概率。我们研究了转染野生型Cx43、C末端连接增强型绿色荧光蛋白（Cx43-EGFP）的Cx43以及N末端连接增强型绿色荧光蛋白（EGFP-Cx43）的HeLa细胞中的染料摄取和单通道活性。膜渗透性还原剂二硫苏糖醇[（DTT）10 mM]增加了表达Cx43和Cx43-EGFP细胞的染料摄取速率，但未增加亲本细胞或表达EGFP-Cx43细胞的染料摄取速率。诱导的染料摄取被La(3+)、一种肽间隙连接和半通道阻滞剂（间隙26）以及氟芬那酸阻断。DTT在正电压下增加了Cx43-EGFP半通道的开放。浴槽中加入膜非渗透性还原剂还原型谷胱甘肽不会增加染料摄取，但记录微管中的谷胱甘肽在正电压下增加了半通道的开放，这表明其作用于细胞内。DTT对表面Cx43或Cx43-EGFP的水平或细胞内pH几乎没有影响。这些发现表明，降低细胞内氧化还原电位可能通过作用于连接蛋白C末端的细胞质半胱氨酸残基来增加Cx43和Cx43-EGFP半通道的开放。
