The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton
The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton
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DOI：
10.1096/fj.01-0855com
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发表时间：
2002-08-01
期刊：
影响因子：
4.8
通讯作者：
Haase, H
中科院分区：
文献类型：
--
作者：
Hohaus, A;Person, V;Haase, H
Ahnak是一种广泛表达的5643个氨基酸的巨型蛋白质，参与细胞分化和信号转导。在最近的一项研究中，我们证明了ahnak与心脏L型Ca 2+通道的调节β 2亚基的关联。在这里，我们确定了最羧基末端的ahnak区（aa 5262-5643）与重组β 2a以及与β 2和β 1a同种型天然肌肉钙离子通道使用面板的GST融合蛋白。平衡沉降分析显示，β 2a亚基羧基末端（aa 195- 606）和氨基末端（aa 1-200）截短物的Kd值分别为55 +/- 11 nM和328 +/- 24 nM。同一羧基端ahnak区（aa 5262-5643）与G-肌动蛋白结合并与F-肌动蛋白共沉淀。人左心室组织的共聚焦显微镜定位羧基末端ahnak部分的肌膜，包括T-管系统和心肌细胞的闰盘。这些结果表明，ahnak提供了一个结构基础的肌膜下的细胞结构，并赋予肌动蛋白为基础的细胞骨架的L型钙通道的调节作用。
Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed K-d values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl- terminal (aa 195- 606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.