|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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.