Molecular mechanism of PIP2 regulation of ROMK channel

PIP2调控ROMK通道的分子机制

• 批准号: 6724528
• 负责人: JASON Z LEE
• 金额: $ 0.82万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724528
• 关键词: Bartter's syndrome; Xenopus oocyte; binding sites; chimeric proteins; confocal scanning microscopy; fatty acylation; green fluorescent proteins; intermolecular interaction; kidney; membrane activity; palmitates; phosphatidylinositols; postdoctoral investigator; potassium channel; protein binding

DESCRIPTION (provided by applicant): ROMK K+ channels play essential roles in K+ secretion in cortical collecting ducts (CCDs) and K+ recycling in the thick ascending limbs (TALs) of Henle's loop. Mutation of ROMK is one of the genetic causes for Bartter's disease. The activity of ROMK channels is regulated by multiple signaling pathways, such as the protein kinase A (PKA) pathway, the protein kinase C (PKC) pathway and intracellular pH (pHi). It has recently been found that membrane phospholipid, phosphatidylinositol-4, 5-bisphosphate (PIP2), also regulates the activity of ROMK channel. However, the mechanism of PIP2 regulation of ROMK remains unknown. It is hypothesized that PIP2 regulates ROMK channel opening by anchoring the C-terminal cytoplasmic domain of the channels to the plasma membrane via a direct PIP2-channel interaction. To test this hypothesis, binding of green fluorescent protein fusion proteins of the C-terminal cytoplasmic domain of ROMK1 to plasma membrane PIP2 in living cells will be examined using confocal fluorescent imaging system. Effects of anchoring of the proximal C-terminus of ROMK 1 to the plasma membrane on channel activity in Xenopus oocytes will be examined using palmitoylation as an alternative membrane anchor. This proposed research training will prepare the applicant for his long-term research interest in studying ion channels in renal physiology and diseases.

描述（由申请人提供）：ROMK K+通道在Henle循环的厚升节（TALS）中在皮质收集管道（CCD）和K+回收中起着重要作用。罗克突变是巴特氏病的遗传原因之一。 ROMK通道的活性受多种信号通路的调节，例如蛋白激酶A（PKA）途径，蛋白激酶C（PKC）途径和细胞内pH（PHI）。最近发现，膜磷脂，磷脂酰肌醇-4、5-双磷酸盐（PIP2）也调节了ROMK通道的活性。但是，ROMK的PIP2调节机制仍然未知。假设PIP2通过将通道的C末端细胞质结构域锚定在质膜上通过直接的PIP2通道相互作用来调节ROMK通道的打开。为了检验这一假设，将使用共聚焦荧光成像系统检查ROMK1的C末端细胞质结构域与质膜PIP2的绿色荧光蛋白融合蛋白与质膜PIP2的结合。将使用棕榈酰化作为替代性膜锚进行检查，将检查ROMK 1近端C末端对质膜的C末端对异武卵母细胞中通道活性的影响。该拟议的研究培训将使申请人为他在研究肾脏生理和疾病中的离子渠道的长期研究兴趣做好准备。