The aquaporins are a family of water channels expressed in several water-transporting tissues, including the kidney. We have used peptide-derived, affinity-purified polyclonal antibody to aquaporin-3 (AQP-3) investigate its localization and regulation Immunoblotting experiments showed expression both renal cortex medulla, with greatest base inner medulla. Subcellular fractionation membranes, using progressively higher centrifugation speeds, revealed that AQP-3 is present predominantly 4,000...

Currents through individual Na channels in the apical membrane of rat cortical collecting tubule were resolved by using patch-clamp technique. In cell-attached patches, had a conductance 5 pS with 140 mM NaCl pipet. The was saturable function external Na, maximal value about 8 and half saturation at 75 Na. excised inside-out selectivity for over K estimated from reversal potentials to be least 10:1. underwent spontaneous transitions between open closed states. Both states mean lifetimes 3-4...

The terminal part of the inner medullary collecting duct exhibits a high degree water permeability that is independent increased intracellular cAMP and not accounted for by activity known renal epithelial channels CHIP28 (28-kDa channel-forming integral protein) WCH-CD (collecting channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed degenerate primers assuming putative would be member major intrinsic protein (MIP) family proteins. A cDNA fragment...

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation animals' mineralocorticoid status vivo using a low-Na diet or diuretic furosemide. Tubules were isolated and split open to expose luminal surface. Induction channel cell-attached patches tubules. No observed with control animals on normal diet. Channel could be induced by putting for at least 48 h. mean number per patch (NPo) maximal after 1 wk low Na. Channels also within 3 h...

Aldosterone controls the final sodium reabsorption and potassium secretion in kidney by regulating activity of epithelial channel (ENaC) aldosterone-sensitive distal nephron (ASDN). ASDN consists last portion convoluted tubule (late DCT), connecting (CNT), collecting duct (CD) (i.e., cortical CD [CCD] medullary [MCD]). It has been proposed that control transport CCD is essential for achieving balance. We have tested this hypothesis inactivating α subunit ENaC but leaving expression late DCT...

Low-conductance, K-selective channels were identified in the apical membrane of rat cortical collecting tubule (CCT) by use patch-clamp technique. Isolated, split tubules bathed K gluconate medium to depolarize cell while keeping intracellular concentration high. With pipette containing predominantly either Na+ or Li+ but no K, outward currents observed through that had a single-channel conductance (g) 9 pS and probability being open (Po) greater than 0.9, independent voltage (+/- 40 mV)...

The patch-clamp technique was used to identify individual Na channels in the apical membrane of rat cortical collecting tubule and evaluate effects cytoplasmic Ca2+ pH on channel activity. In excised, inside-out patches, probability a channels's being open (P0) increased with alkalinization solution bathing side patch. Estimates P0 were 0.05 at 6.4, 0.19 6.9, 0.41 7.4. Varying free concentration patch had no measurable effect P0. cell-attached addition ionophore ionomycin tubules final...

The patch-clamp technique was used to study the properties and density of conducting K Na channels in apical membrane rat cortical collecting tubule. predominant channel observed cell-attached patches (SK channels) had an outward single-channel conductance (with LiCl pipette) 10 pS. inward KCl 42 a high open probability that increased with depolarization. Kinetic analysis indicated presence single state two closed states. Increasing intake by maintaining animals on diet for 12-16 d number SK...

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in cortical collecting duct to investigate processing proteins rat kidney changes or K intake. When animals maintained on a low-Na diet for 7-9 days, abundance two forms alpha-subunit, apparent masses 85 and 30 kDa, increased. Salt restriction also increased beta-subunit produced an endoglycosidase H (Endo H)-resistant pool this subunit. The 90-kDa form...

High-conductance (maxi) K channels in the apical membrane of rat and rabbit cortical collecting tubules (CCT) were studied using patch-clamp technique. Principal cells (PC) intercalated (IC) distinguished with Hoffman modulation optics split-open tubules. IC further identified by staining fluorescent mitochondrial dye, rhodamine 123. Maxi-K their high conductance (greater than 80 pS) voltage-dependent kinetics. In CCT rats on a low-Na diet, maxi observed 11% cell-attached patches PC 79% IC....

Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells renal collecting tubule as well other epithelia. To examine selectivity AQP3, permeability to (Pf), urea (Pur), and glycerol (Pgly) Xenopus oocytes injected with cRNA encoding AQP3 was measured. Oocytes either human or rat aquaporin-1 (AQP1) were used controls. Although both aquaporins permit flow across membrane, only permeable (Pgly > Pur). The uptake into expressing linear up 165 mM. For...

High conductance, Ca-activated K channels were studied in the apical membrane of rat cortical collecting tubule (CCT) using patch-clamp technique. In cell-attached patches found mainly closed state at spontaneous potential. They spent progressively more time open as pipette potential was made negative relative to bath. excised these had a high selectivity for over Na and activated by micromolar concentrations Ca2+ on cytoplasmic side voltage-dependent manner. low conductance Rb blocked Ba...

Amiloride-sensitive Na(+) channel activity was examined in the cortical collecting ducts of a mouse line (SGK1(-/-)) deficient serum- and glucocorticoid-dependent protein kinase SGK1. This correlated with changes renal Na handling maturation epithelial (ENaC) protein. Neither SGK1(-/-) mice nor paired SGK1(+/+) animals expressed detectable activity, measured as amiloride-sensitive whole-cell current (I(Na)), under control conditions standard chow. Administration aldosterone (0.5 microg/h via...

Changes in apical surface expression of ion channels and transporters the superficial rat renal cortex were assessed using biotinylation immunoblotting during alterations dietary K intake. A high-K diet increased, a low-K decreased, both overall abundance β- γ-subunits epithelial Na channel (ENaC). In case γ-ENaC, effect was specific for 65-kDa cleaved form protein. The amount α-ENAC also increased with increasing total secretory K(+) (ROMK) decreased diet. ROMK high intake but not...

Expression of epithelial Na channel (ENaC) protein in the apical membrane rat kidney tubules was assessed by biotinylation extracellular surfaces renal cells and fractionation. Rat kidneys were perfused situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes solubilized labeled proteins isolated using neutravidin beads, surface β γENaC subunits assayed immunoblot. Surface αENaC Most at smaller...

The abundance of Na transport proteins in the luminal membrane rat kidney was assessed using situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, amounts epithelial channel (ENaC) beta-subunit (beta-ENaC) gamma-subunit (gamma-ENaC) Na-Cl cotransporter (NCC) protein surface fraction increased relative to controls by 1.9-, 3.5-, 1.5-fold, respectively. Na/H exchanger (NHE3) Na-K-2Cl (NKCC2) did not change significantly. increases ENaC subunits mimicked...

The epithelial Na+ channel (ENaC) is essential for homeostasis, and dysregulation of this underlies many forms hypertension. Recent studies suggest that mTOR regulates phosphorylation activation serum/glucocorticoid regulated kinase 1 (SGK1), which known to inhibit ENaC internalization degradation; however, it not clear whether contributes the regulation renal tubule ion transport. Here, we evaluated effect selective inhibitors on kidney K+ transport in WT Sgk1–/– mice, as well isolated...

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed cell-attached and inside-out excised patches from split-open tubules using patch-clamp technique. In containing a single channel open probability (Po) was variable, ranging 0.05 to 0.9. average Po 0.5. However, individual values not distributed normally, but mainly < or = 0.25 > 0.75. Mean times mean closed correlated directly inversely, respectively, with Po. where sufficient...

Renal potassium secretion occurs in the distal segments of nephron through apically located secretory (SK) channels. SK may correspond to ROMK channels cloned from rat kidney. In this study, localization at cellular level kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide ROMK. The specificity demonstrated by immunoblots membranes Xenopus oocytes expressing ROMK2. Immunoblots homogenates renal outer medulla and cortex revealed predominant...

The behavior of individual Na channels in the apical membrane rat cortical collecting tubule (CCT) was studied at different concentrations permeant ions and Li. Tubules were opened to expose their luminal surfaces bathed K-gluconate medium minimize tubule-to-tubule variation cell potential intracellular concentration. patch-clamp technique used resolve currents through channels. pipette filled with solutions containing variable either NaCl or LiCl. In one series experiments, changed without...

Epithelial Na channels were investigated using patch-clamp techniques in connecting tubule (CNT) segments isolated from rat kidney. Cell-attached patches with Li+ the patch pipette contained conductances for inward currents of 13-16 pS and slow opening closing kinetics, similar to properties cortical collecting (CCT). Macroscopic amiloride-sensitive (INa) also observed under whole cell clamp conditions. These undetectable cells control rats but large when animals infused aldosterone...

Na channels in the apical membrane of rat renal cortical collecting tubule were studied using patch-clamp technique. Channel activity was monitored cell-attached patches on tubules that split open to expose luminal surface. number (N), probability (Po), and single-channel currents (i) measured at 37 degrees C during continuous superfusion tubule. Addition amiloride (10 microM) or benzamil (0.5 superfusate resulted a twofold increase mean (NPo) after 2 min. The effect closely paralleled an i,...

Renal Na + and K excretion was measured in rats with varying dietary intake. The requirement for channel-mediated distal nephron reabsorption assessed by infusing the animals -sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, concentration urine 38 μM, corresponding to concentrations 9–23 μM tubular fluid, sufficient block >98% transport through apical channels (ENaC). With a control intake (0.6% KCl), reduced (U V) from 0.85 ± 0.15 0.05 0.01 μmol/min...

The epithelial Na channel (ENaC) forms a pathway for Na+ reabsorption in the distal nephron, and regulation of these channels is essential salt homeostasis. In rat kidney, ENaC subunits reached plasma membrane both immature fully processed forms, latter defined by either endoglycosidase H–insensitive glycosylation or proteolytic cleavage. Animals adapted to low-salt diet have increased surface expression that specific mature subunit proteins similar (three- fourfold) α, β, γENaC. Kidney...