| Co-Investigator(Kenkyū-buntansha) |
MIZUNO Norikazu TOKYO INSTITUTE OF TECHNOLOGY, 生命理工学部, 寄附講座教員 (90212232)
KOZASA Tohru UNIVERSITY OF TEXAS WE MEDICAL CTR, 医学部, 助教授
NAKAJIMA Yasuko UNIVERSITY OF ILLINOIS AT CHICAGO, 医学部, 教授
KAZIRO Yoshito TOKYO INSTITUTE OF TECHNOLOGY
NAKAJIMA Shigehiro UNIVERSITY OF ILLINOIS AT CHICAGO
KAWANO Takeharu TOKYO INSTITUTE OF TECHNOLOGY
SATOH Takaya TOKYO INSTITUTE OF TECHNOLOGY
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| Research Abstract |
Heterotrimeric GTP-binding regulatory proteins (G proteins) mediate a variety of extracellular signals from receptors to effector molecules. G protein-coupled inward rectifier K+ channels (GIRKs) are known to be one of the effector molecules, and are involved in the inhibition of neuronal excitability and the development of central neurvous system. To investigate the regulation mechanism of GIRK by G proteins, we examined the heteromultimeric complex formation of different GIRK and the interaction of the GIRKs with G protein betagamma subunits using co-immunoprecipitation technique. We cloned cDNAs coding for three types of GIRKs, GIRK1, GIRK2, and GIRK4, from rat brain. The GIRK cDNAs were subcloned into mammalian expression vector, and expressed in human embryonal kidney 293 cells. Using specific antibody against each GIRK,the GIRK was immunoprecipitated. The immunoprecipitate was analyzed by immunoblotting. The results of co-immunoprecipitation of the two different GIRKs in three different combination indicated that GIRK1, GIRK2, and GIRK4 intreract each other. to from a heteromultimer complex. Interaction of GIRK with G protein betagamma subunit (Gbetagamma) was studied using various mutants of Gbeta and Ggamma. We found that the association of GIRK with Gbetagamma is mediated mainly through Gbeta, but not Ggamma, and Gbeta alone can interact with GIRK.Moreover, it was suggested that the membrane localization of Gbetagamma is required for association of GIRK.Futhermore, we succeeded in measuring the activity of GIRK which was transiently expressed together with Gbetagamma in 293 cells and in primary cultured neuron. Pilot experiments using the Gbeta mutants suggested that the association of GIRK with Gbeta alone fails to activate the GIRK and the Gbetagamma dimer functions as an activator of GIRK.Futher studies including electrophysical and molecular biological experiments should throw a new linght on the molecular mechanisms of regulation of GIRK.
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