Peracchia C, Wang X, Li L, Peracchia L L
Department of Physiology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642-8642, USA.
Pflugers Arch. 1996 Jan;431(3):379-87. doi: 10.1007/BF02207275.
The relationship among intracellular pH (pHi), -log10 intracellular Ca2+ concentration (pCai) and gap junctional conductance, the participation of Ca2+ stores, and the role of calmodulin in channel regulation have been studied in Xenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO2. The time courses of pHi [measured with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorscein (BCECF)], pCai (measured with the membrane-associated fura-C18) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with pCai than with pHi. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on CO2-induced uncoupling, coupled to negative results with Ca2+-free external solutions, point to a low-pHi -induced Ca2+ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO2 within 72 h. The effect of CO2 on junctional conductance was reduced by approximately 60% in 24 h, by approximately 76% in 48 h and by approximately 93% in 72 h. Oocytes that had lost gating sensitivity to CO2 partially recovered gating competency following calmodulin injection. The data suggest that lowered pHi uncouples gap junctions by a Ca2+- calmodulin-mediated mechanism.
在表达天然连接蛋白（Cx38）并暴露于用100%二氧化碳鼓泡的外部溶液中的非洲爪蟾卵母细胞中，研究了细胞内pH（pHi）、-log10细胞内Ca2+浓度（pCai）与缝隙连接电导之间的关系、Ca2+储存的参与情况以及钙调蛋白在通道调节中的作用。比较了用2',7'-双（2-羧乙基）-5,6-羧基荧光素（BCECF）测量的pHi、用膜相关的fura-C18测量的pCai以及用双电压钳制方案测量的连接电导的时间进程。获得的数据证实了先前的证据，即连接电导与pCai的关系比与pHi的关系更密切。细胞内注射钌红或1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）对二氧化碳诱导的解偶联有抑制作用，再加上无钙外部溶液的阴性结果，表明低pHi诱导内部储存（可能主要是线粒体）释放Ca2+。通过研究细胞内注射针对卵母细胞中表达的两种钙调蛋白mRNA的反义寡核苷酸对钙调蛋白表达的抑制作用，检验了钙调蛋白参与通道门控的假说。钙调蛋白mRNA在5小时内被永久消除。注射反义核苷酸的卵母细胞在72小时内逐渐失去与二氧化碳解偶联的能力。二氧化碳对连接电导的影响在24小时内降低约60%，在48小时内降低约76%，在72小时内降低约93%。对二氧化碳失去门控敏感性的卵母细胞在注射钙调蛋白后部分恢复了门控能力。数据表明，降低的pHi通过Ca2+-钙调蛋白介导的机制使缝隙连接解偶联。
