| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1991: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1990: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
Mechanisms of regulation of Ca channels in cardiac and smooth muscle cells were investigated with patch-clamp and biochemical methods. Cyclic AMP-dependent protein kinase (PKA), applied to the Ca channels in inside-out patches from guinea-pig ventricular cells, increased the open probability of the channels. Protein kinase C (PKC), however, did not affect the activity of the channel. Similar results were obtained in the Ca channles reconstituted into liposomes, examined with photofluorometry using fura-2. PKA but not PKC phosphorylated a 200KD membrane protein, which was specifically labelled with a photoaffinity Ca-channel ligand azidopine, suggesting that the phosphoprotein was the alpha-1-subunit of the channel. Evidence for a direct activation of the Ca channel by GTP-binding protein was not obtained.
Effects of PKA and PKC were examined in the Ca channel of smooth muscle cells from guinea pig taenia coli. Neither forskolin nor a phorbol ester (TPA) significantly affected the activity of the channels in cell-attached patches. PKA or okadaic acid, applied to the inside-out patches, did not change the activity or time course of rundown of the Ca channels.
The present results support the hypothsis that PKA directly phosphorylates and regulates the cardiac Ca channel. However, direct phosphorylation by PKC of the channel is not supported. The Ca channel in the smooth muscle cells does not seem to be regulated by phosphorylation of the channel protein with PKA or PKC.
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