Elucdidation of physiological and pathophysiological roles of Na^+/Ca^<2+> exchanger using drug discovery and genetic engineering

利用药物发现和基因工程阐明Na^/Ca^<2>交换器的生理和病理生理作用

• 批准号: 12670102
• 负责人: IWAMOTO Takahiro
• 金额: $ 2.24万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670102/
• 关键词: Na^+; Ca^<2+> exchanger; mutagenesis; knockout mice; transgenic mice; pathophysiological model; inhibitor; 虚血再灌流障害

The Na^+/Ca^<2+> exchanger (NCX) is a membrane protein that exchanges 3 to 4 Na^+ ions for each Ca^<2+> ion. The mammalian NCX forms a multigene family of homologous proteins comprising three isoforms, NCX1, NCX2, and NCX3. NCX1 is highly expressed in the heart, brain, and kidney and expressed at much lower levels in many other tissues, whereas the expression of NCX2 and NCX3 is limited mainly to the brain. In this study, we tried to elucidate physiological and pathophysiological roles of NCX using NCX inhibitors and NCX-engineered mice. From characterizing NCX inhibitors, we found that KB-R7943 and SN-6 preferentially block NCX3 and NCX1, respectively, and both specifically inhibit the reverse mode of Na^+/Ca^<2+> exchange. The following analyses of NCX1/NCX3 chimeras revealed that these two inhibitors might interact with different sites on NCX molecule. More recent data have shown that the potency of NCX inhibitors is related to the extent of NCX inactivation caused by high Na^+i_1 concentration. We have reported before that NCX inhibitors can improve the ischemia/reperfusion-induced renal injury. In this study, we analyzed the ischemia/reperfusion-induced renal injury in NCX1-knockout mice (heterozygous). Expectedly, the ischemia/reperfusion-induced renal dysfunction in the heterozygous mice was markedly attenuated compared with cases in the wild-type mice. These results suggest that Ca^<2+> overload via the reverse mode of NCX1 seems to play an important role in the pathogenesis of the ischemia/reperfusion-induced renal failure.

Na^+/ca^<2+>交换器（NCX）是一种膜蛋白，可为每个Ca^<2+>离子交换3至4 Na^+离子。哺乳动物NCX形成了一个多基因的同源蛋白家族，其中包括三种同工型NCX1，NCX2和NCX3。 NCX1在心脏，大脑和肾脏中高度表达，在许多其他组织中表达得多，而NCX2和NCX3的表达主要限制在大脑中。在这项研究中，我们试图使用NCX抑制剂和NCX工程小鼠阐明NCX的生理和病理生理作用。通过表征NCX抑制剂，我们发现KB-R7943和SN-6优先阻断NCX3和NCX1，并且都特别抑制了Na^+/Ca^<2+>交换的反向模式。 NCX1/NCX3嵌合体的以下分析表明，这两个抑制剂可能与NCX分子上的不同位点相互作用。最近的数据表明，NCX抑制剂的效力与高Na^+I_1浓度引起的NCX失活的程度有关。我们之前已经报道说，NCX抑制剂可以改善缺血/再灌注引起的肾脏损伤。在这项研究中，我们分析了NCX1-KNOCKOUT小鼠（杂合）中缺血/再灌注诱导的肾损伤。与野生型小鼠相比，杂合小鼠的缺血/再灌注诱导的肾功能障碍显着衰减。这些结果表明，通过NCX1的反向模式的Ca^<2+>过载似乎在缺血/再灌注诱导的肾衰竭的发病机理中起重要作用。

项目成果

脇本公嗣: "Targeted disruption of Na^+/Ca^<2+> exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat."J.Biol.Chem.. 275・47. 36991-36998 (2000)

Kimitsugu Wakimoto：“Na^+/Ca^<2+> 交换基因的靶向破坏导致心肌细胞凋亡和心跳缺陷。” J.Biol.Chem.. 275・47 (2000)。

Takahiro Iwamoto: "Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Na^+/Ca^<2+> exchanger"Mol.Pharmacol.. 59・3. 524-531 (2001)

Takahiro Iwamoto：“影响心脏 Na^+/Ca^<2+> 交换器对异硫脲衍生物抑制剂 KB-R7943 敏感性的结构域”Mol.Pharmacol.. 59・3 (2001)。

Iwamoto, T., Uehara, A., Imanaga, I., and Shigekawa, M.: "The Na^+/Ca^<2+> exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca^<2+> affinity"J. Biol. Chem.. 275(49). 385

Iwamoto, T.、Uehara, A.、Imanaga, I. 和 Shigekawa, M.：“Na^ /Ca^<2> 交换器 NCX1 具有相反方向的可重入环结构域，其中包含保守的天冬氨酸，其突变改变了其表观 Ca2+

Iwamoto, T.: "Structure, function and pathophysiology of Na^+/Ca^<2+> exchanger"Jap. J. Circ. Res.. 24(3). 101-113 (2001)

Iwamoto, T.：“Na^/Ca^<2>交换器的结构、功能和病理生理学”Jap。

Shigekawa, M., and Iwamoto, T.: "Cardiac Na^+-Ca^<2+> exchanger. Molecular and pharmacological aspects"Circ. Res.. 88. 864-876 (2001)

Shigekawa，M.和Iwamoto，T.：“心脏Na ^ -Ca ^ 2 交换器。分子和药理学方面”Circ。