Is 240kDa protein, a substrate of G-kinase, IP_3 receptor of smooth muscle?

我们正在研究硝酸甘油对血管平滑肌的松弛机制，发现从猪主动脉平滑肌中提纯的240 kDa蛋白被环GMP依赖的蛋白激酶(G-Kinase)磷酸化。240 kDa蛋白的胰蛋白酶多肽的氨基酸序列与小脑IP_3受体非常相似，240 kDa蛋白与IP_3高度结合。这些结果有力地表明240 kDa蛋白是动脉平滑肌的IP_3受体。如果该蛋白是IP_3受体，则它应该具有钙通道特性。本项目的最终目的是确定240 kDa蛋白是否具有钙通道活性，并检测240 kDa蛋白与G-激酶之间的相互作用。为此，我们选择了平面双层法，以天冬氨酸和胆固醇为成膜剂，建立了高阻平面膜(约500GOMEGA)。阿拉美辛作为通道形成剂，产生电压依赖性的通道活性。使用甲氧西林可以获得电生理技术的技能。由于人工脂质体与平面双层的融合是非常困难的，我们首先尝试将骨骼肌和平滑肌SR作为天然脂质体进行通道活性的测定。利用骨骼肌SR，我们在10%的实验中观察到了一些通道活动，而在实验中只观察到了3%的单通道活动。最后，我们尝试将240 kDa蛋白的脂质体融合到平面膜中，但没有观察到任何通道活性，这一结果表明我们需要更多的平面膜实验经验。我们将继续实验，增加相对容易建立的方法的骨骼肌SR通道活性的概率，然后转移到测定平滑肌SR和240 kDa蛋白的Ca~(2+)通道活性。

We are investigating relaxation mechanisms of nitroglycerin of vascular smooth muscle and found a 240kDa protein purified from porcine aortic smooth muscle was phosphorylated by cyclic GMP-dependent protein kinase (G-kinase). Amino acid sequences of tryptic peptides of 240kDa protein are very similar to cerebellar IP_3 receptor and 240kDa protein binds IP_3 with high affinity. These results strongly suggest 240kDa protein is an IP_3 receptor of arterial smooth muscle. If the protein is IP_3 receptor, it should have Ca^<2+> channel characteristics. The final aim of this project is to determine whether 240kDa protein has Ca^<2+> channel activity or not and examine the interaction between 240kDa protein and G-kinase. For this object, we chose planar bilayr method.Using asolectin and cholesterol as membrane forming lipids, high resistance planar membrane (around 500GOMEGA) was established. Alamethicin used as a channel forming agent produced voltage dependent channel activity. Using alamethicin we could obtained the skill of electrophysiological technique. As fusion of artificial liposome into planar bilayrs is very difficult, at the first step we tried to determine channel activity of skeletal and smooth muscle SR as a natural liposome. Using skeletal muscle SR,we observed some channel activity with 10% of experiment but single channel activity was observed only 3% of experiment. We failed to get any channel activity of smooth muscle SR.Finally we tried to fuse liposomes of 240kDa protein into planar membrane, but no channel activity was observed.These results indicate that we need more experience of planar membrane experiment. We will continue the experiment to increase probability of channel activity of skeletal muscle SR which is relatively easy to establish the methods, then move to measure Ca^<2+> channel activity of smooth muscle SR and 240kDa protein.