I. D. Millar

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.

Biallelic Mutation of BEST1 Causes a Distinct Retinopathy in Humans

We describe a distinct retinal disorder, autosomal-recessive bestrophinopathy (ARB), that is consequent upon biallelic mutation in BEST1 and is associated with central visual loss, a characteristic retinopathy, an absent electro-oculogram light rise, and a reduced electroretinogram. Heterozygous mutations in BEST1 have previously been found to cause the two dominantly inherited disorders, Best macular dystrophy and autosomal-dominant vitreoretinochoroidopathy. The transmembrane protein bestrophin-1, encoded by BEST1, is located at the basolateral membrane of the retinal pigment epithelium in which it probably functions as a Cl(-) channel. We sequenced BEST1 in five families, identifying DNA variants in each of ten alleles. These encoded six different missense variants and one nonsense variant. The alleles segregated appropriately for a recessive disorder in each family. No clinical or electrophysiological abnormalities were identified in any heterozygotes. We conducted whole-cell patch-clamping of HEK293 cells transfected with bestrophin-1 to measure the Cl(-) current. Two ARB missense isoforms severely reduced channel activity. However, unlike two other alleles previously associated with Best disease, cotransfection with wild-type bestrophin-1 did not impair the formation of active wild-type bestrophin-1 channels, consistent with the recessive nature of the condition. We propose that ARB is the null phenotype of bestrophin-1 in humans.

Missense Mutations in a Retinal Pigment Epithelium Protein, Bestrophin-1, Cause Retinitis Pigmentosa

Bestrophin-1 is preferentially expressed at the basolateral membrane of the retinal pigmented epithelium (RPE) of the retina. Mutations in the BEST1 gene cause the retinal dystrophies vitelliform macular dystrophy, autosomal-dominant vitreochoroidopathy, and autosomal-recessive bestrophinopathy. Here, we describe four missense mutations in bestrophin-1, three that we believe are previously unreported, in patients diagnosed with autosomal-dominant and -recessive forms of retinitis pigmentosa (RP). The physiological function of bestrophin-1 remains poorly understood although its heterologous expression induces a Cl--specific current. We tested the effect of RP-causing variants on Cl- channel activity and cellular localization of bestrophin-1. Two (p.L140V and p.I205T) produced significantly decreased chloride-selective whole-cell currents in comparison to those of wild-type protein. In a model system of a polarized epithelium, two of three mutations (p.L140V and p.D228N) caused mislocalization of bestrophin-1 from the basolateral membrane to the cytoplasm. Mutations in bestrophin-1 are increasingly recognized as an important cause of inherited retinal dystrophy.

Functional characterization of bestrophin-1 missense mutations associated with autosomal recessive bestrophinopathy.

PURPOSE Autosomal recessive bestrophinopathy (ARB) is a retinal dystrophy affecting macular and retinal pigmented epithelium function resulting from homozygous or compound heterozygous mutations in BEST1. In this study we characterize the functional implications of missense bestrophin-1 mutations that cause ARB by investigating their effect on bestrophin-1s chloride conductance, cellular localization, and stability. METHODS The chloride conductance of wild-type bestropin-1 and a series of ARB mutants were determined by whole-cell patch-clamping of transiently transfected HEK cells. The effect of ARB mutations on the cellular localization of bestrophin-1 was determined by confocal immunofluorescence on transiently transfected MDCK II cells that had been polarized on Transwell filters. Protein stability of wild-type and ARB mutant forms of bestrophin-l was determined by the addition of proteasomal or lysosomal inhibitors to transiently transfected MDCK II cells. Lysates were then analyzed by Western blot analysis. RESULTS All ARB mutants investigated produced significantly smaller chloride currents compared to wild-type bestrophin-1. Additionally, co-transfection of compound heterozygous mutants abolished chloride conductance in contrast to co-transfections of a single mutant with wild-type bestrophin-l, reflecting the recessive nature of the condition. In control experiments, expression of two dominant vitelliform macular dystrophy mutants was shown to inhibit wild-type currents. Cellular localization of ARB mutants demonstrated that the majority did not traffic correctly to the plasma membrane and that five of these seven mutants were rapidly degraded by the proteasome. Two ARB-associated mutants (p.D312N and p.V317M) that were not trafficked correctly nor targeted to the proteasome had a distinctive appearance, possibly indicative of aggresome or aggresome-like inclusion bodies. CONCLUSIONS Differences in cellular processing mechanisms for different ARB associated mutants lead to the same disease phenotype. The existence of distinct pathogenic disease mechanisms has important ramifications for potential gene replacement therapies since we show that missense mutations associated with an autosomal recessive disease have a pathogenic influence beyond simple loss of function.

Ion channel diversity, channel expression and function in the choroid plexuses

Knowledge of the diversity of ion channel form and function has increased enormously over the last 25 years. The initial impetus in channel discovery came with the introduction of the patch clamp method in 1981. Functional data from patch clamp experiments have subsequently been augmented by molecular studies which have determined channel structures. Thus the introduction of patch clamp methods to study ion channel expression in the choroid plexus represents an important step forward in our knowledge understanding of the process of CSF secretion.Two K+ conductances have been identified in the choroid plexus: Kv1 channel subunits mediate outward currents at depolarising potentials; Kir 7.1 carries an inward-rectifying conductance at hyperpolarising potentials. Both K+ channels are localised at the apical membrane where they may contribute to maintenance of the membrane potential while allowing the recycling of K+ pumped in by Na+-K+ ATPase. Two anion conductances have been identified in choroid plexus. Both have significant HCO3- permeability, and may play a role in CSF secretion. One conductance exhibits inward-rectification and is regulated by cyclic AMP. The other is carried by an outward-rectifying channel, which is activated by increases in cell volume. The molecular identity of the anion channels is not known, nor is it clear whether they are expressed in the apical or basolateral membrane. Recent molecular evidence indicates that choroid plexus also expresses the non-selective cation channels such as transient receptor potential channels (TRPV4 and TRPM3) and purinoceptor type 2 (P2X) receptor operated channels. In conclusion, good progress has been made in identifying the channels expressed in the choroid plexus, but determining the precise roles of these channels in CSF secretion remains a challenge for the future.

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 (GQuin). GQuin demonstrated outward rectification and had a relatively low K+ to Na+ selectivity ratio. Loss of GQuin 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 GQuin contributes to the resting membrane potential in the cortical collecting duct and that a fall in GQuin could be an adaptive response in TWIK-1 knockout ducts.

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 (Ba2+)-sensitive whole-cell K+ currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba2+ in a dose- and voltage-dependent fashion, with the Kd decreasing with hyperpolarization. The electrical distance of block by Ba2+ was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba2+ entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba2+ 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.

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 HCO−3 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 HCO−3 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 HCO−3, whereas RVD in the other cell population has an absolute requirement for HCO−3. The HCO−3 dependent RVD requires the normal expression of KCNE1.

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 Henles loop. Mutations in Kir1.1 account for some types of Bartters 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 26°C. 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.

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

Non‐technical summary  The kidney plays a critical role in regulating body fluid volume and blood pressure by conserving ions, solutes and water. Knowing the processes that underpin the handling of ions, solutes and water by the kidney is essential to our understanding of fluid and blood pressure regulation. Movement of ions is mediated by specific transport proteins found in the membranes of kidney cells. These proteins are regulated by additional proteins, called accessory proteins. In the current study, we have examined the role of the accessory protein KCNE1 in regulating a channel, KCNQ1, which is important in kidney function. We have observed that in the absence of KCNE1 the kidney has difficulty conserving sodium, chloride and water. However, by using specific inhibitors of these proteins we have also determined that although KCNE1 has a role in kidney function, the mechanism of its action is unlikely to be by regulating the protein KCNQ1.