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

开发用于纯化 ATP 敏感 K^ 通道蛋白的配体

• 批准号: 02557039
• 负责人: HIRAOKA Masayasu
• 金额: $ 9.54万
• 依托单位: Tokyo Medical and Dental University, Medical Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557039/
• 关键词: ATP-sensitive K^+ channel protein; Cromakalim; Glibenclamide; photoaffinity labeling method; ATP binding capacity; Nicorandil; ATP感受性K^+チャネル; 単一チャネル電流; グリベンクロマイド; K^+チャネル蛋白

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

应用豚鼠心室肌细胞膜片钳技术，研究了<K.ATP>钾通道开放剂尼可地尔和吡那地尔对ATP敏感性钾通道（I_）的激活机制，以及格列本脲对该通道的抑制作用。此外，我们还研究了<K.ATP>基于色满卡林、尼可地尔和格列本脲结构的光亲和标记或光亲和层析化合物对I_2的开放作用。在这10多个化合物中，尼可地尔类似物显示对通道开放没有影响。色满卡林类似物虽然是有效的开环化合物，<K.ATP>但由于其水溶性较低，作用不稳定。我们从另一种合成的chromalalim类似物制备了光亲和柱。尽管该化合物显示出与大鼠大脑中150 kDa的K^+通道蛋白的结合亲和力，但它与心脏制备物的亲和力较低，这表明K^+通道蛋白 ...更多信息 这两种组织的性质可能不同。然后，我们制备了两个格列本脲类似物的光亲和标记。它们显示出与心脏的膜部分结合的高亲和力。我们使用这些化合物进行了光亲和标记，并在柱层析上获得了单一条带的蛋白质。这种纯化的蛋白质被证明与已知的通道蛋白没有同源性，但与另一种可溶性蛋白质具有相似性。这种纯化的蛋白具有ATP结合能力，并存在于心脏质膜组分上，如使用免疫组织化学技术通过抗体所揭示的。根据这些结果，我们推测可能鉴定出组成I_的靶蛋白或相关蛋白<K.ATP>。为了进一步证实这种纯化蛋白的性质，目前正在进行使用脂质双层重构技术的实验。同时，我们进行了分子生物学研究，从cDNA文库中寻找I_2的cDNA克隆<K.ATP>，并在心脏标本中合成了寡核苷酸。成功地进行了小钾离子通道的表达实验，成功地进行了小钾离子通道的表达实验。少

项目成果

FURUKAWA T.,Fan Z.,SAWANOBORI T.,HIRAOKA M.: "Modification of the adenosine 5'-triphosphate-sensitive K^+ channel by trypsin in guinea-pig ventricular myocytes." J.Physiol.(1993)

FURUKAWA T.、Fan Z.、SAWANOBORI T.、HIRAOKA M.：“豚鼠心室肌​​细胞中胰蛋白酶对腺苷 5-三磷酸敏感 K^ 通道的修饰。”

NAKAYAMA H.,HATANAKA Y.,YOSHIDA E.,OKA K.et al.: "Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electro-plax sodium channel." Biochem.Biophys.Res.Commun.184. 900-907 (1992)

NAKAYAMA H.、HATANAKA Y.、YOSHIDA E.、OKA K.等人：“电信号钠通道的结构域 III 和 IV 中带有河豚毒素衍生物的光标记位点。”

Nakayama H., Hatanaka Y., Taki M., Kanaoka Y.: "Chemical and immunological characterization of binding sites for tetrodotoxin and dihydropyridine that are close proximity to the external mouth of ion pore in sodium and calcium channels." J. Protein Chem.1

Nakayama H.、Hatanaka Y.、Taki M.、Kanaoka Y.：“河豚毒素和二氢吡啶结合位点的化学和免疫学特征，这些结合位点靠近钠和钙通道离子孔的外口。”

Fan Z.,Nakayamm K.,Sawanobori T.,Hiraoka M.: "Aromtic aldehydes and aromatic detones open ATP-sensitive K^+ channels en guinea-pig ventricular myocytes." Pflugers Arch.421. 409-415 (1992)

Fan Z.、Nakayamam K.、Sawanobori T.、Hiraoka M.：“芳香醛和芳香爆能打开豚鼠心室肌​​细胞的 ATP 敏感 K^ 通道。”

Nakayama H.,Hatanaka Y.,Taki M.kanaoka Y.: "chemical and immunological characterization of binding sites for tetrodotoxin and dihydro-pyridine that are close proximity to the extermal mouth of ion pore in the sodium and calcium channels." J.protein Chem.1

Nakayama H.、Hatanaka Y.、Taki M.kanaoka Y.：“河豚毒素和二氢吡啶结合位点的化学和免疫学特征，这些结合位点靠近钠和钙通道中离子孔的外口。”