| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
A cytoplasmic protein that activates L-type Ca^<2+> channel was investigated by using both electrophysiological and biochemical methods. First, we attempted to establish a purification procedure using lipid vesicles into which bovine cardiac Ca^<2+> channels were reconstituted. The Ca^<2+> channel activating protein (CCAP) was eluted from a gel-filtration column as a mass having an apparent molecular weight (M'_r) of 200-300 x 10^3. On DEAE-sepharose chromatography, CCAP was eluted at 100-150 mM KCl. The partially Purified CCAP had M'_r of about 100 x 10^3 on SDS-polyacrylamid gel electrophoresis. These biochemical properties were clearly different from the cAMP- dependent protein kinase or any subunit of the Ca^<2+> channel. Second, CCAP could restore the activity of Ca^<2+> channel in inside-out patches of cardiac myocytes. Neither number of channels nor single channel conductance was affected by CCAP, but open-state probability of the channel was dramatically increased by CCAP. Third, we investigated tissue distribution of CCAP. A supernatant fraction of the homogenate from brain, heart, skeletal muscle, liver or kidney was applied to cardiac Ca^<2+> channels which were previously led to 'run-down' in inside-out patches. The tissue extracts from brain, heart, skeletal muscle and liver, but not kidney, recovered the channels from the run-down.
These results suggest that the activity of the L-type Ca^<2+> channel is maintained by the cyto- plasmic protein CCAP. It would be important. in future studies to characterize this protein further and to clarify possible regulatory mechanisms in which CCAP is involved.
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