Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal AnkyrinG -: In vitro mapping of the interacting domains and association in synaptosomes
Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal AnkyrinG -: In vitro mapping of the interacting domains and association in synaptosomes
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DOI：
10.1074/jbc.m201760200
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发表时间：
2002-08-09
影响因子：
4.8
通讯作者：
Alcaraz, G
中科院分区：
文献类型：
--
作者：
Bouzidi, M;Tricaud, N;Alcaraz, G
轴突起始段和郎维尔结处的电压依赖性钠通道与锚蛋白（G）的结同种型（Ank（G）结）共定位。使用来自Nav1.2a亚基的细胞内区域和Ank（G）节点的Ank重复结构域的融合蛋白，我们在分离α亚基结构域I-II的细胞内环中定位了主要的相互作用位点。该57个氨基酸的区域以69 nM的K-D值结合Ank重复区。我们在胞内环III-IV中确定了另一个位点，并将锚蛋白重复结构域上的两个Nav1.2a结合位点映射到包含重复序列12-22的区域。锚蛋白重复结构域不结合β（1）和β（2）亚基胞质区域。我们发现，在培养的胚胎运动神经元中，β 2亚基的表达对于Ank（G）结与功能性钠通道在轴突起始段的共定位不是必需的。针对β（1）亚基胞内区、α亚基环III-IV和Ank（G）结的抗体不能从大鼠脑突触体的Triton X-100溶解物中共免疫沉淀Ank（G）结和钠通道。当使用最大化膜蛋白提取的增溶条件时，获得钠通道α亚基和270-和480-kDa Ank（G）结同种型的免疫共沉淀。然而，我们找不到锚蛋白与钠通道β（1）亚基发生免疫共沉淀的条件。
Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K-D value of 69 nM. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa Ank(G) node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.