Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells
Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells
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DOI：
10.1016/s0006-291x(03)00868-4
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发表时间：
2003-06-13
影响因子：
3.1
通讯作者：
D'Andrea, P
中科院分区：
文献类型：
--
作者：
Beltramello, M;Bicego, M;D'Andrea, P
人连接蛋白26和30通过双顺反子pIRES-EGFP表达载体或EYFP标记的嵌合体表达。HCx26-PIRES瞬时转染HeLa细胞后，对622 Da的染料具有较强的穿透性，但对759 Da分子的穿透性明显降低。在相同条件下，hCx26-EYFP融合产物的通透性与hCx26-PIRES相当，但在759Da处扩散显著增加，这可能是由于选择了大的荧光连接斑块的结果。在hCx30-EYFP中，染料转移被限制在457Da。HCx26-EYFP和hCx30-EYFP形成的荧光斑块在共聚焦连续切片上重建时，呈不规则状，常有长突起或深凹陷。在hCx26-pIRES和稳定转染鼠Cx26的HeLa细胞进行免疫染色后，观察到相似的斑块。稳定转染组的组织电导(Tg(I))(28.8+/-1.8 ns)显著低于瞬时转染组(43.5+/-3.3 ns)。这些差异反映在归一化结电导(G(J))与跨结电压(V-j)的函数依赖关系上。在mCx26和hCx26中，G(J)的半激活电压分别接近+/-95 mV和+/-58 mV。HCx30转染体的相应参数为Tg(J)=45.2+/-3.5 nS，V-0=+/-34 mV。这些结果突出了mCx26和hCx26在该表达系统中的意外差异，强化了通道通透性可能与Cx水平表达有关的概念，并表明hCx30与GFP颜色突变体的融合产生了适合通透性和门控研究的通道。(C)2003年埃尔塞维尔科学公司(美国)。版权所有。
Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26-EYFP fusion products was comparable to that of hCx26-pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30-EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26-EYFP and hCx30-EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26-pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg(i)) displayed significantly smaller values (28.8 +/- 1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5 +/- 3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (G(j)) on transjunctional voltage (V-j). The half-activation voltage for G(j) was close to +/-95 and +/-58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg(j) = 45.2 +/- 3.5 nS and V-0 = +/-34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies. (C) 2003 Elsevier Science (USA). All rights reserved.