Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

Abstract

We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K + [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCD c14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterolrich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCD c14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5) P 2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I g [PI(4)P5K I g], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I g diffusion into planar regions and elevated PI(4,5)P 2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P 2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

“…which are Cho-rich microdomains are mainly located in microvilli [13], these data suggest that CCD cells can synthesize Cho and that Cho accumulates in microvilli with PIP 2 to form a unique membrane microdomain to regulate ENaC activity.…”

“…Another exciting discovery is that extraction of Cho out of the outer leaflet of the apical membrane with MβCD induces Madin-Darby canine kidney cells to lose their microvilli [12]. We have recently shown that MβCD can also excise the microvilli of CCD principal cells to release vesicles from the apical membrane [13]. These studies together suggest a hypothesis that depletion of the outer leaflet Cho may reduce ENaC density by destruction of the microvilli.…”

“…We have recently shown that the ENaC activator PIP 2 is located in microvilli and that its levels are regulated by lovastatin [13]. To determine whether depletion of membrane Cho Fig.…”

“…In contrast, the determination of PIP 2 distribution between microvilli and planar region is feasible. We have already shown that PIP 2 is predominantly localized in microvilli [13]. However, it is unclear whether ENaC and Cho are also localized in microvilli and, if so, whether Cho affects the microvillar localization of PIP 2 and ENaC.…”

“…We have shown that anionic phospholipids including PIP 2 can bind to all three ENaC subunits and directly stimulate ENaC in distal nephron A6 cells [18,19]. Recently, we have also shown that PIP 2 is predominantly located in microvilli and that PIP 2 distribution between microvilli and non-microvillar planar region depends on Cho synthesis in the CCD cells [13]. In the outer leaflet, Cho interacts with sphingolipids via hydrogen bonds to form membrane microdomains, also referred as lipid rafts [20][21][22].…”