Connexin32 gap junction channels in stably transfected cells: unitary conductance.
Connexin32 gap junction channels in stably transfected cells: unitary conductance.
复制标题
稳定转染细胞中的 Connexin32 间隙连接通道：单一电导。
DOI：
10.1016/s0006-3495(91)82159-2
复制
发表时间：
1991
影响因子：
3.4
通讯作者：
Spray,DC
中科院分区：
文献类型：
--
作者：
Moreno,AP;Eghbali,B;Spray,DC
对SKHep 1细胞，这是来自一个高转移性的人肝癌，研究了使用全细胞电压钳技术与贴片型电极含有氯化铯作为主要离子物种。在81个细胞对中的12个中，可检测到通过连接膜的电流;在其余69个细胞对中，连接电导小于我们的记录装置的噪声限值（最差情况：10 pS）。宏观交界电导（gj）在小百分比的对，它是可检测的范围从100到600 pS。在最低电导对中或在短时间暴露于解偶联剂氟烷降低电导后测定的单一结电导（γ j）为25-35 pS。为了研究由连接蛋白32形成的间隙连接通道的特性，用含有编码大鼠肝细胞的主要间隙连接蛋白连接蛋白32的cDNA的质粒稳定转染亲本SKHep 1细胞系。在98对电压钳位连接蛋白32转染的SKHep 1细胞中，有85对的宏观gj大于1 nS;解离后gj随时间增加（从接种后2 h的1.8 +/- 0.6 [平均值+/- SE; n=7] nS至24 h的9.3 +/- 2.2 [n = 9] nS（最大值））。在低电导对和通过暴露于氟烷或庚醇降低gj后，测量转染的SKHep 1细胞对之间的间隙连接通道的单位电导。在可测量超过100个转变的10个实验中，转染细胞中γ j值的直方图显示两个峰; 120-130 pS和25-35 pS。较小的尺寸对应于偶尔在亲本细胞中检测到的通道。因此，我们得出结论，连接蛋白32形成120-130 pS大小级别的间隙连接通道。
Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25–35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n=7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120–130 pS and 25–35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120–130 pS size class.