Mechanisms of Regulation of Calcium Channels

用膜片钳和生化方法研究了心肌细胞和平滑肌细胞钙通道的调节机制。将环磷酸腺苷依赖性蛋白激酶(PKA)应用于豚鼠心室肌细胞由内向外的膜片钙通道，可增加该通道的开放概率。然而，蛋白激酶C(PKC)并不影响通道的活动。用Fura-2光荧光法检测重组为脂质体的钙通道也得到了类似的结果。PKA而不是PKC磷酸化了200kD的膜蛋白，膜蛋白被光亲和性钙通道配体叠氮多宾特异性标记，表明该磷蛋白是该通道的α-1亚基。未见GTP结合蛋白直接激活钙通道的证据。我们观察了PKA和PKC对豚鼠结肠带肌细胞钙通道的影响。福司可林和佛波酯(TPA)对细胞贴附斑块的通道活动均无显著影响。外加PKA或冈田酸不改变钙通道的活动或时程，支持PKA直接磷酸化和调节心肌钙通道的假说。然而，不支持PKC对该通道的直接磷酸化。平滑肌细胞中的钙通道似乎不受通道蛋白与PKA或PKC的磷酸化的调节。

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.