Molecular mechanism of PIP2 regulation of ROMK channel

描述（由申请人提供）：ROMK K+通道在皮质集管（CCDs）的K+分泌和Henle’s袢厚升肢（TALs）的K+循环中起重要作用。ROMK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控，如蛋白激酶A （PKA）通路、蛋白激酶C （PKC）通路和细胞内pH （pHi）。最近发现膜磷脂，磷脂酰肌醇- 4,5 -二磷酸（PIP2）也调节ROMK通道的活性。然而，PIP2调控ROMK的机制尚不清楚。据推测，PIP2通过直接的PIP2通道相互作用，将通道的c端胞质结构域锚定在质膜上，从而调节ROMK通道的打开。为了验证这一假设，我们将使用共聚焦荧光成像系统检测活细胞中ROMK1 c端胞质结构域的绿色荧光蛋白融合蛋白与质膜PIP2的结合。将romk1近端c端锚定在质膜上对爪蟾卵母细胞通道活性的影响将使用棕榈酰化作为替代的膜锚点进行研究。这项研究培训将为申请人长期研究肾脏生理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.