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Cholesterol inhibits ROMK channels by holding PI(4,5)P 2 in microvilli (1181.17)
Author(s) -
Liu BingChen,
Yang LiLi,
Lu XiaoYu,
Song Xiang,
Li XueChen,
Chen Guangping,
Eaton Douglas,
Shen BaoZhong,
Ma HePing
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1181.17
Subject(s) - chemistry , apical membrane , cholesterol , lipid raft , ion channel , filipin , patch clamp , potassium channel , microbiology and biotechnology , biophysics , biochemistry , biology , membrane , receptor
The kidney plays an important role in maintaining ion balance in the blood. K + balance is tightly regulated by a K + channel called ROMK1. We used cultured mouse cortical collecting duct principal cells (mpkCCD c14 line) to determine how ROMK1 is regulated by cholesterol. Previous studies suggest that cholesterol inhibits ROMK channels via a direct mechanism. Surprisingly, our data demonstrate that ROMK1 channels are not located in cholesterol‐rich lipid rafts in cultured CCD cells. Patch‐clamp data demonstrated that a small‐conductance K + (ROMK1) channel in mpkCCD c14 cells was inhibited by cholesterol and activated by extraction of cholesterol with methyl‐beta‐cyclodextrin (MbetaCD). However, confocal microscopy data showed that ROMK1 was not in the microvilli where cholesterol‐rich lipid rafts are located, but in the planar region of the apical membrane of mpkCCD c14 cells, and that phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P 2 ), a well‐known activator of ROMK channels, was found only in the microvilli under resting conditions. Interestingly, after cholesterol synthesis was inhibited by lovastatin, PI(4,5)P 2 diffused into planar region with a consequence of activation of ROMK channels. Activation of ROMK1 channels by MbetaCD was reversed by addition of exogenous cholesterol, but the activation did not occur when PI(4,5)P 2 was sequestered with its antibody. These results suggest that cholesterol inhibits ROMK1 channels, at least in part, by limiting PI(4,5)P 2 diffusion from microvilli to planar region of the renal CCD cell apical membrane. Since we have reported that cyclosporine A (CsA), an immunosuppressant drug which causes hyperkalemia, can alter the levels of cholesterol in renal epithelial cells, this study provides a basis for researchers to determine the mechanism of CsA‐induced hyperkalemia. Grant Funding Source : supported by NIH 5R01‐DK067110 to HPM and by NSF of China 81130028 to B‐ZS