Fukatsu Kazumi, Bannai Hiroko, Zhang Songbai, Nakamura Hideki, Inoue Takafumi, Mikoshiba Katsuhiko
Division of Molecular Neurobiology and Division of Neural Signal Information Nippon Telegraph and Telephone Company-The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
J Biol Chem. 2004 Nov 19;279(47):48976-82. doi: 10.1074/jbc.M408364200. Epub 2004 Sep 10.
Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) plays an important role in neuronal functions; however, the lateral diffusion of IP3R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP3R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP3R1 showed lateral diffusion with an effective diffusion constant of approximately 0.3 microm2/s. Depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP3R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP3R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP3R1 diffusion constant. The diffusion of IP3R type 3 (IP3R3), one of the isoforms of IP3Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP3R1 and actin filaments. This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling.
1,4,5-三磷酸肌醇受体1型（IP3R1）在神经元功能中发挥着重要作用；然而，IP3R1在内质网膜上的侧向扩散及其在活神经元中的调节机制仍不清楚。我们在培养的大鼠海马神经元中表达了绿色荧光蛋白标记的IP3R1，并通过光漂白后的荧光恢复技术观察了其侧向扩散。IP3R1表现出侧向扩散，有效扩散常数约为0.3平方微米/秒。肌动蛋白丝的耗尽增加了IP3R1的扩散常数，表明IP3R1的扩散通过肌动蛋白丝受到负调控。我们还发现，与IP3R1结合并含有肌动蛋白-血影蛋白结合区域的蛋白4.1N负责这种对IP3R1扩散常数的肌动蛋白调节。显性负性4.1N的过表达和4.1N与IP3R1结合的阻断增加了IP3R1的扩散常数。IP3R3是IP3Rs的亚型之一，缺乏与4.1N的结合能力，其扩散不依赖于肌动蛋白丝，但在添加4.1N结合序列后变得依赖于肌动蛋白丝。这些数据表明，4.1N作为IP3R1和肌动蛋白丝之间的连接蛋白。这种对IP3R1扩散的肌动蛋白丝依赖性调节可能对细胞内Ca2+信号的时空调节很重要。
