| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
It has been predicted from our previous studies that N-type Ca^<2+> channels in neurons are negatively regulated by G proteins via direct interactions of G protein α- (Gα) and βγ-subunits (Gβγ) with the C-terminus and the intracellular loop between repeat I and II (I-II loop) of the channel α1B subunit, respectively. To confirm these interactions in living cells, fluorescence resonance energy transfer (FRET) analysis was employed in the present study. Go1α and Gβ1 were tagged on their N-termini with ECFP, a variant of the green fluorescence protein (GFP), or DsRed, a new red fluorescent protein. Also, α1B was tagged on the I-II loop (α1B(L)) or the C-terminus (α1B(C)) with another variant of GFP, EYFP, or DsRed. When ECFP/DsRed-Go1α or ECFP/DsRed-Gβ1 was expressed in BHK cells in which both α2 and β1a subunits of Ca^<2+> channels had been stably expressed (BHK6 cells), fluorescences of ECFP/DsRed-Go1α and ECFP/DsRed-Gβ1 were found in plasma membranes and throughout the cytoplasm but not detectable in cell nuclei. By contrast, EYFP/DsRed- α1B showed that α1B was localized to plasma membranes and intra-cytoplasmic vesicles in a patch-like pattern, without depending on whether EYFP/DsRed was tagged at the I-II loop or the C-terminus. When δ-opioid receptor, EYFP-α1B and ECFP-G protein (ECFP-Go1α or EGFP-Gβ1) were co-expressed in BHK6 cells, cellular distribution patterns of α1B and G protein were superimposed. The ratio between the fluorescence intensities of acceptor (EYFP) and donor (ECFP) was changed by receptor stimulation in BHK6 cells expressing in a combination of EYFP-α1B(C) and ECFP-Go1α. These results indicate that Gα actually interacts with N-type Ca^<2+> channels through the C-terminus of α-1B in living cells.
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