LOCALIZATION OF THE RECEPTOR-SITE FOR ALPHA-SCORPION TOXINS BY ANTIBODY MAPPING - IMPLICATIONS FOR SODIUM-CHANNEL TOPOLOGY
LOCALIZATION OF THE RECEPTOR-SITE FOR ALPHA-SCORPION TOXINS BY ANTIBODY MAPPING - IMPLICATIONS FOR SODIUM-CHANNEL TOPOLOGY
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DOI：
10.1073/pnas.86.24.10161
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
CATTERALL, WA
中科院分区：
文献类型：
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作者：
THOMSEN, WJ;CATTERALL, WA
位点定向和单克隆抗体识别RII钠通道的不同细胞外区域。亚基已被用来确定组成。α受体的序列。通过评价抗体在电压依赖性结合中对重组大鼠脑钠通道和大鼠脑突触体的影响。在测试的6种抗体中，位于结构域I跨膜段S5和S6之间的胞外环上的两个识别氨基酸残基355-371和382-400，以及位于结构域IV类似环上的一个识别氨基酸残基1686-1703的抗体抑制了30-55%的结合。抑制浓度-(EC50 = 0.4-2 .mu)。M)和时间（t1/2 = 40-80 min）相关。五种不同的单克隆抗体在I结构域识别相同的细胞外环，完全抑制结合，其EC50值与位点定向抗体相似。抗体效应的动力学研究与毒素受体位点的缓慢可逆竞争是一致的。我们的研究结果表明，结构域I和IV的S5和S6节段之间的胞外环至少包含部分α。支持通道蛋白三级结构域I和IV相邻的膜拓扑模型，并且四个同源结构域各包含六个跨膜序列。
Site-directed and monoclonal antibodies recognizing different extracellular regions of the RII sodium channel .alpha. subunit have been used to determine the sequences that comprise the receptor for .alpha.-scorpion toxins by evaluating the effect of antibody in voltage-dependent binding of radiolabeled toxin isolated from Leiurus quinquestriatus to both reconstituted rat brain sodium channel and rat brain synaptosomes. Of six antibodies tested, two recognizing amino acid residues 355-371 and 382-400 located on an extracellular loop between transmembrane segments S5 and S6 of domain I and one recognizing residues 1686-1703 of a similar loop of domain IV inhibit binding by 30-55%. Inhibition is concentration-(EC50 = 0.4-2 .mu.M) and time- (t1/2 = 40-80 min) dependent. Five different monoclonal antibodies recognizing the same extracellular loop in domain I inhibit binding completely with similar EC50 values as observed for site-directed antibodies. Kinetic studies of the antibody effect are consistent with a slowly reversible competition for the toxin receptor site. Our results suggest that the extracellular loops between segments S5 and S6 of domains I and IV comprise at least part of the .alpha.-scorpion toxin receptor site and support the membrane topology models in which domains I and IV are adjacent in the tertiary structure of the channel protein and six transmembrane sequences are contained in each of the four homologous domains.