Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). PIP 3 significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP 3 -binding protein (Grp1-PH), which sequestered intracellular PIP 3 . 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of ␥-epithelial sodium channel (␥-ENaC) were truncated by PCRbased mutagenesis (␥ T -ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing ␣-and ␤-ENaC only or with either ␥-ENaC or ␥ T -ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasma membrane. These data suggest that PIP 3 mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of ␥-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between ␥-ENaC and PIP 3 . ENaC2 is a member of the ENaC/Deg superfamily of ion channels responsible for sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney (reviewed in Ref. 1). Since 1994, when ENaC was initially cloned from rat colon (2), the biophysical properties and molecular structure of ENaC have been extensively studied. Several lines of evidence suggest that ENaC is composed of three subunits, ␣, ␤, and ␥, and that all three subunits are required to form a functional ␣␤␥-ENaC channel complex (2-9).Studying the mechanisms that regulate ENaC function is important because abnormal channel activity leads to several severe diseases. Constitutive activation of any component of ENaC subunits can cause Liddle's syndrome, an autosomal dominant inherited disease that causes excessive sodium retention and hypertension. Conversely, loss of function mutations in ␣-, ␤-, or ␥-ENaC causes pseudohypoaldosteronism type I, a hypotensive condition characterized by an inability to retain salt. These syndromes highlight the importance of normal ENaC activity in the kidney to maintain fluid and sodium homeostasis. The proper regulation of ENaC activity is also very important in the lung, because transgenic mice lacking functional channels die within 40 h of birth from fluid filled airways (10). Additionally, increases in intracellular Cl Ϫ concentrations that secondarily lead to changes in ENaC activity play an important role in the pathophysiology of cystic fibrosis (11).Anionic phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), are normally located in the inner leaflet of the plas...

Cyclosporine A (CsA) is an efficient immunosuppressant used for reducing allograft rejection but with a severe side effect of causing hypertension. We hypothesize that the renal epithelial sodium channel (ENaC) may participate in CsA-induced hypertension. In the present study, we used the patch-clamp cell-attached configuration to examine whether and how CsA stimulates ENaC in A6 distal nephron cells. The data showed that CsA significantly increased ENaC open probability. Since CsA is an inhibitor of the ATP-binding cassette A1 (ABCA1) transporter, we employed 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), another ABCA1 inhibitor, and found that DIDS mimicked the effects of CsA on ENaC basal and cholesterol-induced activity but without any additive effect if combined with CsA. CsA and DIDS also had an identical effect on reduced ENaC activity caused by cholesterol extraction. ABCA1 protein was detected in A6 cells by Western blot analysis. Confocal microscopy data showed that both CsA and DIDS facilitated A6 cells to uptake cholesterol. Since enhanced ENaC activity is known to cause hypertension, these data together suggest that CsA may cause hypertension by stimulating ENaC through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells.

To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-alpha (TNF-alpha), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-alpha (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-alpha was mimicked by exogenous sphingomyelinase (0.1 U/ml) and C(2)-ceramide (50 microM), but neither C(2)-dihydroceramide, a membrane-impermeable analog of C(2)-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. C(2)-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. C(2)-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GF109203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-alpha inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS.

The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca 2ϩ channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer 45 (15,16,27). Mice lacking Trpv5 resulted in a 6-to 10-fold increase in urinary Ca 2ϩ excretion, and ultimately in defects in bone mineralization (16).Urinary Ca 2ϩ excretion is an important factor for kidney stone formation and bone health. For instance, urine Ca 2ϩ excretion correlates with bone loss in calcium-stone-forming patients with idiopathic hypercalciuria (3). Interestingly, African Americans exhibit lower urinary Ca 2ϩ excretion than whites (8,25,29,34,35,40,45), and the risk of kidney stones in African American is lower than that in whites (32,36,38). Furthermore, African Americans have higher bone mass (5, 41) and lower incidence of osteoporosis-related fractures than whites (4, 6, 7). The mechanism underlying the lowered urinary Ca 2ϩ in African Americans is not well understood. A lower 25-hydroxyvitamin D concentration in African Americans could result in an increased parathyroid hormone level and in turn renal Ca 2ϩ conservation. However, Braun and colleagues (8) observed significantly lower urinary Ca 2ϩ excretion in adolescent African American girls than white girls in a wide range of controlled Ca 2ϩ intake, whereas the 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone values were not significantly different between the two groups. The difference in bone mass and urinary Ca 2ϩ excretion between blacks and whites is not restricted to Americans; a similar finding was reported in South Africa (10). Thus genetic rather than social and environmental factors may play a major role in the superior renal Ca 2ϩ conservation mechanism in African descendents.Because TRPV5 is a key protein that regulates Ca 2ϩ reabsorption, genetic variations of TRPV5 may influence urinary Ca 2ϩ excretion. Interestingly, TRPV5 gene is one of the four contiguous genes, including EPHB6, TRPV6, TRPV5, and KEL, in chromosome 7q34-35 with striking evidence of a recent selective sweep in European Americans based on the analysis on the single nucleotide polymorphisms (SNPs) of over 100 genes from 24 African Americans and 23 European Americans provided by SeattleSNPs program (1, 37). Akey and colleagues (2) further showed that the TRPV6 haplotype, defined by three nonsynonymous SNPs, is nearly fixed in populations outside Africa, suggesting that these variations may confer a selective advantage, e.g., efficiency in Ca 2ϩ absorption from dairy products or resistance to a pathogen, after early humans migrated out of Africa. The TRPV6 variant with the three nonsynonymous SNPs exhibited increased Ca 2ϩ transport ability and may play a role in absorptive hypercalciuria and ki...

Anionic phospholipids (APs) present a variety of lipids in the cytoplasmic leaflet of the plasma membrane, including phosphatidylinositol (PI), PI-4-phosphate (PI(4) P), phosphatidylserine (PS), PI-4,5-bisphosphate (PI(4,5) P 2 ), PI-3,4,5-trisphosphate (PI(3,4,5)P 3 ), and phosphatidic acid (PA). We previously showed that PI(4,5)P 2 and PI(3,4,5)P 3 upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5) P 2 and PI(3,4,5)P 3 respectively target β-and γ-ENaC subunit. To determine whether PI(4,5)P 2 and PI(3,4,5)P 3 selectively bind to β and γ subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P 2 , PI(3,4,5)P 3 , and PA, but not PI, non-selectively bind to not only β and γ but also α subunit. To determine how these APs regulate ENaC, we performed insideout patch-clamp experiments and found that PS, but not PI or PI(4)P, maintained ENaC activity, that PI(4,5)P 2 and PI(3,4,5)P 3 stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with α-, β-, and γ-ENaC. Further, the data from cell-attached patch-clamp and confocal microscopy experiments indicate that PA, a product of phospholipase D, may provide one of the pathways for inhibition of ENaC by endothelin receptors.