Membrane topology and quaternary structure of cardiac gap junction ion channels.
Membrane topology and quaternary structure of cardiac gap junction ion channels.
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DOI：
10.1016/0022-2836(92)90253-g
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发表时间：
1992-02
影响因子：
5.6
通讯作者：
M. Yeager;N. Gilula
中科院分区：
文献类型：
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作者：
M. Yeager;N. Gilula
用针对α1连接蛋白（Cx43）的7个不同位点的抗肽抗体、心脏组织中内源性蛋白酶的切割和离体心脏缝隙连接的天然和蛋白酶切割的二维膜晶体的电子显微镜图像分析，研究了含有α1连接蛋白（Cx43）的大鼠心脏缝隙连接离子通道的膜拓扑结构和四级结构。使用斑点免疫印迹、Western免疫印迹、免疫荧光和免疫电镜确定肽抗体的特异性。基于亲水性分析预测的折叠，五种抗体针对胞质结构域中的位点，两种抗体针对两个细胞外环结构域。分离的缝隙连接不能标记的两个细胞外环抗体使用薄切片免疫金电子显微镜。这与细胞外间隙区域的已知狭窄性一致，该细胞外间隙区域可能阻止抗体探针的渗透。然而，冷冻切片使得细胞外结构域可用于免疫标记。至少Mr = 5100（残基（101至142））的胞质“环”结构域容易接近肽抗体标记。天然Mr = 43，000的蛋白质可以在膜的胞质侧被蛋白酶切割，产生Mr = 30，000的膜结合片段。Western免疫印迹显示蛋白酶切割发生在蛋白质的羧基尾部，并且切割位点位于氨基酸残基252-271之间。免疫电子显微镜显示，蛋白酶切割后释放出分子量为13，000的羧基末端肽，并且通过非共价相互作用不再与分子量为30，000的膜结合片段结合。心脏缝隙连接的二维膜晶体的电子显微镜图像分析显示，离子通道是由蛋白质亚基的六边形排列形成的。蛋白酶切割α 1多肽不会显著改变这种四级排列。因此，Mr 13，000羧基末端结构域不参与形成跨膜离子通道。心脏和肝脏间隙连接的相似六聚体结构表明α或β连接蛋白形成的差距连接通道的分子设计保守。
The membrane topology and quaternary structure of rat cardiac gap junction ion channels containingα1, connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic “loop” domain of at leastMr= 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The nativeMr= 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in anMr≈ 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252–271. Immunoelectron microscopy demonstrated that theMr≈ 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to theMr≈ 30,000 membrane-bound fragmentvianon-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of theα1polypeptide. Therefore, theMr≈ 13,000 carboxy-terminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexons indicates conservation in the molecular design of the gap junction channels formed by α or β connexins.