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

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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.