Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.
Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.
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DOI：
10.1083/jcb.134.4.1019
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发表时间：
1996-08
期刊：
影响因子：
--
通讯作者：
Johnson RG
中科院分区：
文献类型：
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作者：
Li H;Liu TF;Lazrak A;Peracchia C;Goldberg GS;Lampe PD;Johnson RG
在缝隙连接的组装过程中，一个细胞质膜中的半通道被认为与相对的膜中的另一个细胞对齐并对接，形成细胞到细胞的通道。我们在这里报告了非连接性、质膜连接蛋白43(Cx43)半通道的存在和性质。当细胞外钙水平降低时，细胞从培养液中摄取5(6)-羧基荧光素，证明了半管的开放。染料吸收表现出与缝隙结通道相似的特性。例如，使用不同的染料，摄取水平与分子大小相关：5(6)-羧基荧光素(约32%)、7-羟基香豆素-3-羧酸(约24%)、Fura-2(约11%)和荧光素-葡聚糖(约0.4%)。辛醇和庚醇也减少了大约50%的染料摄取率。对一个转染Cx43反义表达载体的Novikoff细胞克隆的详细分析表明，根据摄取实验，染料摄取水平降低，显微注射实验中细胞间染料转移速率相应降低。此外，在反义转染体的电生理学研究中，与对照细胞相比，在细胞外钙减少时，膜电阻的下降更为有限。HeLa细胞对染料摄取的研究也显示，Cx43基因的转染率大大增加。综上所述，这些观察表明，Cx43负责这些培养细胞的半通道功能。表达Cx43的正常大鼠肾(NRK)细胞也获得了类似的染料摄取结果。在这些细胞系统中，染料摄取可以被12-O-十四烷基佛波醇-13-乙酸酯激活的蛋白激酶C以及在LA25-NRK细胞中被温度敏感的酪氨酸蛋白激酶pp60v-src显著抑制。我们认为，Cx43半通道存在于细胞膜上，在质膜上受多条信号通路调控，可能是缝隙连接组装的重要阶段。
During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7- hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.