| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
1.Modulation of neuronal Ca^<2+> by cyclic AMP-dependent protein kinase (PKA)
To understand the functional and structural basis for the modulation of neuronal Ca^<2+> by PKA, P/Q-type α_<1A> and N-type α_<1B> Ca^<2+> channels were expressed in Xenopus oocytes and in cultured cells. PKA inhibitors decreased α_<A1> channel activities, and this effect was abolished by protein phosphatase inhibitors. On the other hand, PKA catalytic subunit applied intracellularly increased the channel activities. In cell-attached patches, PKA inhibitors significantly decreased single-channel activities of α_<1A> without affecting unitary conductance. By contrast, α_<1B> Ca^<2+> channels were not markedly affected by PKA inhibitors. Chimeric channels between α_<1A> and α_<1B>, and mutant α_<1A> channels revealed that one threonine residue in the C-terminus of α_<1A> is critical for the PKA-dependent regulation of P/Q-type Ca^<2+> channels. The evidence for PKA-dependent phosphorylation of this threonine res
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idue was confirmed by immunoblot analysis using a phospho-site-selective antiserum. Based on these results, the threonine is identified as a potential site of channel regulation by PKA-dependent phosphorylation.
2.Interaction of neuronal Ca^<2+> channels with dihydropyridine (DHP) derivatives
Amlodipine, a DHP derivative, which inhibits N-type α_<1B> Ca^<2+> channels as well as L-type α_<1C> channels as demonstrated in our previous study (J.Pharmacol.Exp.Ther. 291,464-473,1999), failed to influence R-type α_<1E> channels. Therefore, mutant α_1 subunits with amino acid substitutions between α_<1B> and α_<1E> were constructed to identify the interaction site(s) on α_<1B> for amlodipine by electrophysiological and radioligand binding analyses. One leucine residue on the segment 6 of repeat IV of the α<1B> subunit was identified as critical for the formation of a high affinity binding site for amlodipine in α_<1B> channels, and also for the inhibition of α_<1B> channels by amlodipine. These results suggest that the presence of the high affinity binding site for amlodipine is required for the amlodipine-induced inhibition of α_<1B> channels. Less
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