Renal defects in KCNE1 knockout mice are mimicked by chromanol 293B in vivo: identification of a KCNE1-regulated K+ conductance in the proximal tubule.

KCNE1 is a protein of low molecular mass that is known to regulate the chromanol 293B and clofilium-sensitive K+ channel, KCNQ1, in a number of tissues. Previous work on the kidney of KCNE1 and KCNQ1 knockout mice has revealed that these animals have different renal phenotypes, suggesting that KCNE1 may not regulate KCNQ1 in the renal system. In the current study, in vivo clearance approaches and whole cell voltage-clamp recordings from isolated renal proximal tubules were used to examine the physiological role of KCNE1. Data from wild-type mice were compared to those from KCNE1 knockout mice. In clearance studies the KCNE1 knockout mice had an increased fractional excretion of Na+, Cl−, HCO3(−) and water. This profile was mimicked in wild-type mice by infusion of chromanol 293B, while chromanol was without effect in KCNE1 knockout animals. Clofilium also increased the fractional excretion of Na+, Cl− and water, but this was observed in both wild-type and knockout mice, suggesting that KCNE1 was regulating a chromanol-sensitive but clofilium-insensitive pathway. In whole cell voltage clamp recordings from proximal tubules, a chromanol-sensitive, K+-selective conductance was identified that was absent in tubules from knockout animals. The properties of this conductance were not consistent with its being mediated by KCNQ1, suggesting that KCNE1 regulates another K+ channel in the renal proximal tubule. Taken together these data suggest that KCNE1 regulates a K+-selective conductance in the renal proximal tubule that plays a relatively minor role in driving the transport of Na+, Cl− and HCO3(−).

A Kir2.3-like K+ conductance in mouse cortical collecting duct principal cells.

K(+) channels play an important role in renal collecting duct cell function. The current study examined barium (Ba(2+))-sensitive whole-cell K(+) currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba(2+) in a dose- and voltage-dependent fashion, with the K (d) decreasing with hyperpolarization. The electrical distance of block by Ba(2+) was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba(2+) entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba(2+) ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K(+) channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K(+) conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K(+) handling in the principal cells of the cortical collecting duct.

Adaptive downregulation of a quinidine-sensitive cation conductance in renal principal cells of TWIK-1 knockout mice.

TWIK-1, a member of the two-pore domain K(+) channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K(+) conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (G (Quin)). G (Quin) demonstrated outward rectification and had a relatively low K(+) to Na(+) selectivity ratio. Loss of G (Quin) would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K(+) conductance and Na(+) uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that G (Quin) contributes to the resting membrane potential in the cortical collecting duct and that a fall in G (Quin) could be an adaptive response in TWIK-1 knockout ducts.

Molecular mechanisms of cerebrospinal fluid production.

The epithelial cells of the choroid plexuses secrete cerebrospinal fluid (CSF), by a process which involves the transport of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. The unidirectional transport of ions is achieved due to the polarity of the epithelium, i.e. the ion transport proteins in the blood-facing (basolateral) membrane are different to those in the ventricular (apical) membrane. The movement of ions creates an osmotic gradient which drives the secretion of H(2)O. A variety of methods (e.g. isotope flux studies, electrophysiological, RT-PCR, in situ hybridization and immunocytochemistry) have been used to determine the expression of ion transporters and channels in the choroid plexus epithelium. Most of these transporters have now been localized to specific membranes. For example, Na(+)-K(+)ATPase, K(+) channels and Na(+)-2Cl(-)-K(+) cotransporters are expressed in the apical membrane. By contrast the basolateral membrane contains Cl(-)- HCO(3) exchangers, a variety of Na(+) coupled HCO(3)(-) transporters and K(+)-Cl(-) cotransporters. Aquaporin 1 mediates water transport at the apical membrane, but the route across the basolateral membrane is unknown. A model of CSF secretion by the mammalian choroid plexus is proposed which accommodates these proteins. The model also explains the mechanisms by which K(+) is transported from the CSF to the blood.

Volume regulation is defective in renal proximal tubule cells isolated from KCNE1 knockout mice.

The membrane protein KCNE1 has been implicated in cell volume regulation. Using a knockout mouse model, this study examined the role of KCNE1 in regulatory volume decrease (RVD) in freshly isolated renal proximal tubule cells. Cell diameter was measured using an optical technique in response to hypotonic shock and stimulation of Na(+)-alanine cotransport in cells isolated from wild-type and KCNE1 knockout mice. In HEPES buffered solutions 64% of wild-type and 56% of knockout cells demonstrated RVD. In HCO3- buffered solutions 100% of the wild-type cells showed RVD, while in the knockout cells the proportion of cells displaying RVD remained unchanged. RVD in the knockout cells was rescued by valinomycin, a K+ ionophore. In wild-type HCO3- dependent cells the K+ channel inhibitors barium and clofilium inhibited RVD. These data suggest that mouse renal proximal tubule is comprised of two cell populations. One cell population is capable of RVD in the absence of HCO3-, whereas RVD in the other cell population has an absolute requirement for HCO3-. The HCO3- dependent RVD requires the normal expression of KCNE1.

A hypertonicity-activated nonselective conductance in single proximal tubule cells isolated from mouse kidney.

The whole-cell patch-clamp technique was used to examine nonselective conductances in single proximal tubule cells isolated from mouse kidney. Single cells were isolated in either the presence or absence of a cocktail designed to stimulate cAMP. Patches were obtained with Na+ Ringer in the bath and Cs+ Ringer in the pipette. On initially achieving the whole-cell configuration, whole-cell currents were small. In cAMP-stimulated cells, with 5 mM ATP in the pipette solution, whole-cell currents increased with time. The activated current was linear, slightly cation-selective, did not discriminate between Na+ and K+ and was inhibited by 100 microM gadolinium. These properties are consistent with the activation of a nonselective conductance, designated G(NS). Activation of G(NS) was abolished with pipette AMP-PNP, ATP plus alkaline phosphatase or in the absence of ATP. In unstimulated cells G(NS) was activated by pipette ATP together with PKA. These data support the hypothesis that G(NS) is activated by a PKA-mediated phosphorylation event. G(NS) was also activated by a hypertonic shock. However, G(NS) does not appear to be involved in regulatory volume increase (RVI), as RVI was unaffected in the presence of the G(NS) blocker gadolinium. Instead, the ATP sensitivity of G(NS) suggests that it may be regulated by the metabolic state of the renal proximal tubule cell.

Na+-alanine uptake activates a Cl- conductance in frog renal proximal tubule cells via nonconventional PKC.

Hyposmotically induced swelling of frog renal proximal tubule cells activates a DIDS-sensitive, outwardly rectifying Cl- conductance via a conventional protein kinase C (PKC). This study examines whether Na+-alanine cotransport similarly activates a DIDS-sensitive Cl- conductance in frog renal proximal tubule cells. On stimulation of Na+-alanine cotransport, the DIDS-sensitive current (I(DIDS-Ala)) increased markedly over time. I(DIDS-Ala) exhibited outward rectification, a Na+/Cl- selectivity ratio of 0.19 +/- 0.03, and an anion selectivity sequence Br- = Cl- > I- > gluconate-. Activation of I(DIDS-Ala) was dependent on ATP hydrolysis and PKC-mediated phosphorylation and was inhibited by hyperosmotic conditions. Activation could be not ascribed to a conventional PKC isoform, as I(DIDS-Ala) was not affected by removing Ca2+ or by phorbol ester treatment, suggesting a role for a nonconventional PKC isoform, either novel or atypical. We conclude that Na+-alanine cotransport activates a DIDS-sensitive Cl- conductance via a nonconventional PKC isoform. This contrasts with the hyposmotically activated Cl- conductance, which requires conventional PKC activation.

Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.

1. The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henle's loop. Mutations in Kir1.1 account for some types of Bartter's syndrome. 2. Here we report that stable transfection of Kir1.1b (ROMK2) in Madin-Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26 C. However, whole-cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26C. 3. To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26 C. The increased level of Kir1.1b at the plasma membrane at 26 C was due to an increase in protein stability. 4. Confocal microscopic investigation of EGFP-Kir1. 1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5. These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.

The regulation of Na(+)-dependent anionic amino acid transport by the rat mammary gland.

The regulation of anionic amino acid transport, using radiolabelled D-aspartate as a tracer, by rat mammary tissue explants has been examined. Na(+)-dependent D-aspartate uptake by mammary tissue increased between late pregnancy and early lactation and again at peak lactation but thereafter declined during late lactation. In contrast, the Na(+)-independent component of D-aspartate uptake by mammary explants did not change significantly with the physiological state of the donor animals. Premature weaning of rats during peak lactation markedly decreased Na(+)-dependent D-aspartate uptake by mammary tissue. In addition, premature weaning also reduced the effect of reversing the trans-membrane Na(+)-gradient on the fractional loss of D-aspartate from mammary tissue explants. Unilateral weaning of rats during peak lactation revealed that milk accumulation per se reduced the Na(+)-dependent moiety of D-aspartate uptake by mammary tissue suggesting that the transport of anionic amino acids is regulated to match supply with demand. Treating lactating rats with bromocryptine reduced D-aspartate uptake by mammary tissue explants suggesting that the transport of anionic amino acids by the rat mammary gland is regulated by prolactin.

Substrate specificity of the mammary tissue anionic amino acid carrier operating in the cotransport and exchange modes.

The substrate specificity of the rat mammary tissue high affinity, Na+-dependent anionic amino acid transport system has been investigated using explants and the perfused mammary gland. D-Aspartate appears to be transported via the high affinity, Na+-dependent L-glutamate carrier. Thus, D-aspartate transport by rat mammary tissue was Na+-dependent and saturable with respect to extracellular D-aspartate with a Km and Vmax of 32.4 microM and 49.0 nmol/2 min per g of cells respectively. The uptake of D-aspartate by mammary explants was cis-inhibited by L-glutamate and L-aspartate, but not by D-glutamate. L-glutamate uptake by mammary tissue explants was cis-inhibited by beta-glutamate, L-cysteate, L-cysteine sulfinate and dihydrokainate but not by DL-alpha-aminoadipate. In addition, dihydrokainate, but not DL-alpha-aminoadipate inhibited D-aspartate and L-glutamate uptake by the perfused gland. D-Aspartate efflux from mammary tissue explants was trans-accelerated by external L-glutamate in a dose-dependent fashion (50-500 microM). The effect of L-glutamate on D-aspartate efflux was dependent on the presence of extracellular Na+. D-Aspartate, L-aspartate and L-cysteine sulfinate (at 500 microM) also markedly trans-stimulated D-aspartate efflux from mammary tissue explants. In contrast, L-cysteine. D-glutamate, L-leucine, dihydrokainate and DL-alpha-aminoadipate were either weak stimulators of D-aspartate efflux or were without effect. D-Aspartate efflux from the perfused mammary gland was trans-stimulated by L-glutamate but not by D-glutamate and only weakly by L-cysteine (all at 500 microM). It appears that the mammary tissue high affinity anionic amino acid carrier can operate in the exchange mode with a similar substrate specificity to that of the co-transport mode.

Mammary protein synthesis is acutely regulated by the cellular hydration state.

The effect of cell-volume pertubations on mammary tissue protein synthesis has been examined. Cell-swelling, induced by a hyposmotic shock, increased the rate of incorporation of radiolabelled leucine and methionine into trichloroacetic acid precipitable material. The incorporation of radiolabel under both isosmotic and hyposmotic conditions was inhibited by cycloheximide. The increases in mammary protein synthesis as a result of cell-swelling may be attributable to an increase in casein synthesis. Conversely, cell-shrinking, as a consequence of a hyperosmotic challenge, almost abolished mammary protein (casein) synthesis. The finding that cell-volume pertubations had no significant effect on steady-state casein mRNA levels suggests that the regulation, within the time course of the experiments, is at the level of translation. The results strongly suggest that mammary cell volume may be an important cellular signal in the control of mammary protein synthesis in general and casein synthesis in particular.

The mechanism of L-glutamate transport by lactating rat mammary tissue.

The transport of L-glutamate by lactating rat mammary gland has been examined using both tissue explants and a perfused mammary preparation. L-Glutamate uptake by mammary tissue explants was predominantly via a Na(+)-dependent pathway: Li+, choline+ and NMDG+ could not substitute for Na+. L-Glutamate efflux from preloaded explants was also influenced by the transmembrane Na(+)-gradient. These results are consistent with (Na(+)-glutamate) cotransport. The Na(+)-dependent system for L-glutamate transport in tissue explants was saturable (Km = 112.5 +/- 19.7 microM; Vmax = 71.3 +/- 10.4 nmol/min per g cells) and selective for anionic amino acids. Thus, D- and L-aspartate were high affinity inhibitors of L-glutamate uptake whereas neutral amino acids were relatively ineffective. D-Aspartate inhibited L-glutamate uptake in a competitive fashion. L-Glutamate uptake by the perfused mammary gland was (a) Na(+)-dependent (b) saturable (Km = 18.1 +/- 4.9 microM; Vmax = 40.3 +/- 3.7 nmol/min per g tissue) and (c) selective for anionic amino acids. The results suggest that the (Na(+)-glutamate) cotransporter is situated in the blood-facing aspect of the mammary epithelium.