Development of a ligand for purification of the ATP-sensitive K^+ channe protein

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Using the patch-clamp technique applied to guinea-pig ventricular myocytes, we studied the activating mechanism of the ATP-sensitive K^+ channels (I_<K.ATP>) by the K^+ channel openers, nicorandil and pinacidil, and the inhibitory action of the channel by glibenclamide. Furthermore, we examined the opening actions of I_<K.ATP> by the synthetic compounds for photoaffinity labeling or photoaffinity chromatography based on the structures from cromakalim, nicorandil or glibenclamide. Among these more than 10 compounds, nicorandil analogues were shown to be no effects on the channel openings. Although the cromakalim analogues were effective open I_<K.ATP>, their actions were not stable because of low water solubility. We prepared a photoaffinity column from another synthetic chromalalim analogue. Although this compound showed a binding affinity to the K^+ channel proteins of 150 kDa from the rat brain, it had a low affinity to the heart preparations, suggesting that the K^+ channel proteins … More of the two tissue might not have identical properties. Then, we prepared two glibenclamide analogues for photoaffinity labeling. They show a high affinity to bind the membrane fraction of the heart. We conducted photoaffinity labeling using these compounds and obtained a single band protein on the column chromatography. This purified protein was shown to have no homology to the known channel proteins but had similarity to another soluble protein. This purified protein had ATP binding capacity and was present on the cardiac plasma membrane fraction as revealed by the antibody using immunohistochemical technique. From these results, we suspect that the target protein or associated proteins composing I_<K.ATP> might be identified. To further confirm the nature of this purified protein, the experiment using reconstituted technique of the lipid bilayer was currently undertaken. At the same time, we carried out the molecular biology study to find out cDNA clone for I_<K.ATP> from cDNA library and synthesized oligonucleotides in the heart preparations. We also succeeded the expression experiments of cloned small also succeeded the expression experiments of cloned small K^+ channel from kidney. Less