Molecular mechanism of PIP2 regulation of ROMK channel

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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.