| Budget Amount *help |
¥11,500,000 (Direct Cost : ¥11,500,000)
Fiscal Year 2003 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Fiscal Year 2002 : ¥7,200,000 (Direct Cost : ¥7,200,000)
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| Research Abstract |
Two mechanisms that link presynaptic defects to postsynaptic aspects of neuronal differentiation and maturation have been revealed. Firstly, in N-type, Ca^<2+> channel-deficient mice we created, positive, regulation by automic nervous system was ablated. This is reasonable, considering that noradrenalin release from presynaptic terminal is predominantly controlled by N-type channels in wild-type mice. However, interestingly, cardiac contraction, vascular constriction, and sensitivity of receptors to their agonists were upregulated in the mutant mice. Secondly, we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca^<2+> channel subtypes among wild-type control, tottering (tg) and rolling Nagoya (tg^<rol>) that carry mutations in the P/Q-type Ca^<2+> channel α_<1A> (Cav2.l) subunit gene. The EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg^<rol>, which was associated
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with altered properties of the postsynaptic glutamate receptors. The climbing fiber mediated EPSC was more dependent on other Ca^<2+> channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. Thus, presynaptic Ca^<2+> channels control postsynaptic excitability and function and thereby regulate differentiation and maturation synapses. We showed that G protein-coupled, metabotropic glutamate receptor subtype 1 (mGluR1) and P/Q-type Cav2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. mGluR1 inhibited Cav2.1-mediated [Ca^<2+>]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary alpha2/delta and beta1 subunits, respectively, in a ligand-independent manner and was enhanced by pre activation of mGluR1 in a ligand-dependent manner. Furthermore, in dihdropyridine (DHP)-sensitive L-type Ca^<2+> channel Cav1.2 (α1c), Ser1142 was the recognition site for both the DHP agonist BAYk8644 and Phosphatase inhibited by okadaic acid. This suggests that upregulation of L-type channels by DHP agonist and that by Ser phophorylation are mediated by a common mechanism involving Ser1142. Finally, we carried out two-hybrid screening using the β4 subunit as a bate to isolate a clone encoding the protein regulating fusion of synaptic vesicles. Functional and molecular characterization of the protein is under way. Less
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