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Characterization of the distribution and localization of IRK channel families in the developing neuronal cells.

发育中神经元细胞中 IRK 通道家族的分布和定位特征。

基本信息

批准号：
09670060
负责人：
OMORI Koichiro
金额：
$ 1.73万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The inwardly rectifying K^+ channels have been found in a variety of cell types including cardiac myocytes, neuronal cells, skeletal muscle, blood cells, osteoclasts and endothelial cells, and play important roles in maintenance of the resting membrane potential, regulation of the action potential duration and thereby controlling the excitability of the cells.We examined the localization and distribution of IRK1, one of the classical inwardly rectifying K^+ channel, and GIRK1 that is gated by betagamma subunit of G protein, by using specific antibodies in neuronal cells.1.The apparent molecular weights of-83 kDa and -65 kDa for the IRK1 and GIRK1, respectively, were detected in mouse cerebral crude membrane fraction, and were larger than the respective expected molecular weight. When these channel proteins were digested with N-glycosidase F, GIRKI shifted to -58 kDa, while IRK1 showed no change, suggesting that IRK1 may have 0-linked oligosaceharide chains.2.The treatment of the crude membrane fraction with detergents solubilized GIRK1 by -40% of the total contents but hardly extracted IRK1, almost all of which retained in the insoluble fraction. The findings indicate that these channel proteins exist in the cellular organelle in different manner. In the neuronal cells, IRKl may interacts with PSD-95 or PSD-95 like protein which is one of thecytoskeletal protein and resistant to the detergent extraction.3.We performed the same experiment as described above in cultured rat fetal neurons after cultivation for 4 weeks. Interestingly, -70-80% of both channel proteins were extracted from the cells by the detergent treatment. As neuronal cells in culture can not form mature synaptic junction, both channel proteins could not interact with cytoskeletal proteins correctly, suggesting again the interaction with PSD-95 family protein for IRKI in mature synaptic junction.

内向整流K^+通道已在多种细胞类型中发现，包括心肌细胞、神经元细胞、骨骼肌、血细胞、破骨细胞和内皮细胞，并在维持静息膜电位、调节动作电位持续时间从而控制细胞的兴奋性方面发挥重要作用。我们检查了IRK1的定位和分布，IRK1是经典的K^+通道之一。利用神经细胞特异性抗体检测内向整流K^+通道和G蛋白β亚基门控的GIRK1。1.在小鼠脑粗膜组分中检测到IRK1和GIRK1的表观分子量分别为-83 kDa和-65 kDa，均大于各自的预期分子量。当这些通道蛋白用N-糖苷酶F消化时，GIRKI移动到-58 kDa，而IRK1没有变化，表明IRK1可能具有0连接的寡糖链。 2.用去污剂处理粗膜组分，使GIRK1溶解了-40%的总含量，但几乎没有提取IRK1，几乎全部保留在不溶部分。研究结果表明这些通道蛋白以不同的方式存在于细胞器中。在神经元细胞中，IRK1可以与PSD-95或PSD-95样蛋白相互作用，PSD-95或PSD-95样蛋白是细胞骨架蛋白之一并且对去污剂提取具有抗性。 3.培养4周后，我们在培养的大鼠胎儿神经元中进行了与上述相同的实验。有趣的是，通过去污剂处理，从细胞中提取了-70-80%的两种通道蛋白。由于培养的神经元细胞不能形成成熟的突触连接，两种通道蛋白都不能与细胞骨架蛋白正确相互作用，这再次表明成熟突触连接中IRKI与PSD-95家族蛋白的相互作用。