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

内向整流钾通道广泛存在于心肌细胞、神经细胞、骨骼肌、血细胞、破骨细胞和内皮细胞等多种细胞中，在维持静息膜电位、调节动作电位时程和控制细胞兴奋性等方面发挥重要作用。1.在小鼠大脑粗膜组分中检测到IRK 1和GIRK 1的表观分子量分别为-83 kDa和-65 kDa，并且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位移到~ 58 kDa，而IRK 1没有变化，这表明IRK 1可能具有O-连接的寡糖链。2.用去污剂处理粗膜组分使GIRK 1溶解约40%的总含量，但几乎不提取IRK 1，几乎所有的IRK 1都保留在不溶性组分中。这些结果表明，这些通道蛋白以不同的方式存在于细胞器中。在神经细胞中，IRK 1可能与PSD-95或PSD-95样蛋白相互作用，PSD-95或PSD-95样蛋白是一种细胞骨架蛋白，对去污剂提取具有抗性。有趣的是，约70 - 80%的两种通道蛋白通过去污剂处理从细胞中提取。由于培养的神经元细胞不能形成成熟的突触连接，这两种通道蛋白不能正确地与细胞骨架蛋白相互作用，再次表明在成熟的突触连接中IRKI与PSD-95家族蛋白相互作用。

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.