Sequence specific antibodies to connexins block intercellular calcium signaling through gap junctions
Sequence specific antibodies to connexins block intercellular calcium signaling through gap junctions
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DOI：
10.1016/s0143-4160(98)90069-0
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发表时间：
1998-01-01
期刊：
影响因子：
4
通讯作者：
Evans, WH
中科院分区：
文献类型：
--
作者：
Boitano, S;Dirksen, ER;Evans, WH
在原代气道上皮细胞培养中，单个细胞的机械刺激诱导细胞间Ca2+波，这被认为是通过间隙连接介导的。为了直接研究间隙连接在这种多细胞反应中的作用，我们评估了细胞内负载的序列特异性连接蛋白（间隙连接蛋白）抗体对细胞间Ca2+波传播的影响。对胞质环（Des 1，产生于氨基酸102-112 + 116-124；Des 5，产生于氨基酸108-119）或连接蛋白32的羧基尾部（Gap 9，氨基酸264-283）的抗体电穿孔抑制细胞间Ca2+波的传播。Des 1抗体的抑制作用被从类似细胞质环序列中获得的肽（Des 5肽）共同负载竞争性地逆转。相反，与Des 5肽共载不改变Gap 9抗体对细胞间Ca2+波传播的抑制作用。在连接蛋白32的胞外环（Gap 11，氨基酸151-187）或细胞质上的氨基端（Gap 10，氨基酸1-21）的肽序列上培养抗体，不会抑制机械诱导的细胞间通讯。对连接蛋白43 （Gap 15，氨基酸131-142）或连接蛋白26 （Des 3，氨基酸106-119）胞质环肽序列产生的抗体也对干扰细胞间通讯无效。这些数据表明，气道细胞培养中机械诱导的Ca2+波是通过由连接蛋白32组成的间隙连接传播的。
Mechanical stimulation of a single cell in primary airway epithelial cell cultures induces an intercellular Ca2+ wave that has been proposed to be mediated via gap junctions. To investigate directly the role of gap junctions in this multicellular response, the effects of intracellularly-loaded sequence-specific connexin (gap junction) antibodies on the propagation of intercellular Ca2+ waves were evaluated. Electroporation of antibodies to the cytosolic loop (Des 1, generated to amino acids 102-112 + 116-124; and Des 5, amino acids 108-119), or to the carboxyl tail (Gap 9, amino acids 264-283) of connexin 32 inhibited the propagation of intercellular Ca2+ waves. The inhibitory effect of Des 1 antibody was competitively reversed by the co-loading of a peptide derived from a similar cytosolic loop sequence (Des 5 peptide). Conversely, the inhibitory effects on intercellular Ca2+ wave propagation of Gap 9 antibody was not altered by co-loading with the Des 5 peptide. Antibodies raised to peptide sequences within the extracellular loop (Gap 11, amino acids 151-187), or the cytoplasmically located amino terminus (Gap 10, amino acids 1-21) of connexin 32 did not inhibit mechanically-induced intercellular communication. Also ineffective in perturbing intercellular communication were antibodies raised to peptide sequences of the cytosolic loops of connexin 43 (Gap 15, amino acids 131-142) or connexin 26 (Des 3, amino acids 106-119). These data suggest that mechanically-induced Ca2+ waves in airway cell cultures are propagated through gap junctions made up of connexin 32 proteins.