Kwak B R, Sáez J C, Wilders R, Chanson M, Fishman G I, Hertzberg E L, Spray D C, Jongsma H J
Department of Physiology, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Pflugers Arch. 1995 Sep;430(5):770-8. doi: 10.1007/BF00386175.
The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.
研究了蛋白激酶G（PKG）的膜渗透性激活剂8-溴鸟苷3':5'-环一磷酸（8Br-cGMP）对大鼠和人连接蛋白43（Cx43）的作用，Cx43是哺乳动物心脏中最丰富的间隙连接蛋白，在SKHep1细胞中进行了外源表达。在双细胞全电压钳制条件下，8Br-cGMP使大鼠Cx43转染细胞中的间隙连接电导（gj）降低了24.0±3.7%（平均值±标准误，n = 5），而相同处理对人Cx43转染细胞中的gj没有影响。在大鼠Cx43转染细胞中观察到的跨连接电压阶跃引起的gj松弛最适合用三个指数来拟合。在8Br-cGMP存在下，衰减相的时间常数和幅度发生了变化。在氟烷存在下解析出了单个大鼠和人Cx43间隙连接通道。在对照条件下，检测到三种单通道电导状态（γj），分别约为20、40 - 45和70 pS，中等大小的事件最常被观察到。在8Br-cGMP存在下，大鼠Cx43转染细胞中的γj分布向较小尺寸偏移，而人Cx43转染细胞中未出现这种情况。对大鼠Cx43或人Cx43转染细胞的亚汇合培养物中的Cx43进行免疫印迹分析表明，8Br-cGMP在两种细胞中均未引起Cx43电泳迁移率的变化。然而，8Br-cGMP显著改变了[32P]掺入大鼠Cx43的基础水平，而[32P]掺入人Cx43的情况不受影响。我们得出结论，8Br-cGMP调节SKHep1细胞中大鼠Cx43的磷酸化，但不调节人Cx43的磷酸化。大鼠Cx43的这种cGMP依赖性磷酸化与gj降低有关，这是由于最低电导状态的相对频率增加以及这些通道动力学的变化共同导致的。
