Molecular mechanism of PIP2 regulation of ROMK channel

描述（由申请人提供）：ROMK K+通道在皮质集合管（CCD）中的K+分泌和亨利氏袢粗升支（TAL）中的K+再循环中发挥重要作用。ROMK基因突变是Bartter病的遗传病因之一。ROMK通道的活性受多种信号通路调节，如蛋白激酶A（PKA）通路、蛋白激酶C（PKC）通路和细胞内pH（pHi）。近年来发现，膜磷脂，磷脂酰肌醇-4，5-二磷酸（PIP 2），也调节ROMK通道的活性。然而，PIP 2调节ROMK的机制仍不清楚。假设PIP 2通过直接的PIP 2-通道相互作用将通道的C-末端胞质结构域锚定到质膜来调节ROMK通道开放。为了检验该假设，将使用共聚焦荧光成像系统检查活细胞中ROMK 1的C-末端胞质结构域的绿色荧光蛋白融合蛋白与质膜PIP 2的结合。将使用棕榈酰化作为替代的膜锚来检查ROMK 1的近端C-末端锚定到质膜对爪蟾卵母细胞中的通道活性的影响。这项拟议的研究培训将为申请人长期研究肾脏生理学和疾病中的离子通道做好准备。

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.