The apical 70-pS potassium K channel in the thick ascending limb of Henle's loop is a large-conductance Na+-and Cl--activated, KNa1.1-like, channel.

Apical potassium channels are crucial for thick ascending limb (TAL) of Henle's loop transport function. The ROMK (KNCJ1) gene encodes a 30-pS K channel whose loss of function causes the reduced NaCl reabsorption in the TAL associated with Type 2 Bartter's syndrome. In contrast, the molecular basis of a functionally ROMK-related 70-pS K channel is still unclear. The aim of this study was to highlight new specific channel properties that may give insights on its molecular identity. Using the patch-clamp technique on the apical membrane of mouse split-open TAL tubules, we observed that 70-pS K channel activity, but not ROMK channel activity, increases with the internal Na+ and Cl- concentrations, with relative 50 % effective concentrations (EC50) and Hill coefficients (nH) of 40.6 mM (SD 1.65) and 2.4 (SD 0.28) for Na+, and of 29.3 mM (SD 2.35) and 2.2 (SD 0.39) for Cl-. Conversely, 70-pS K channel activity was inhibited by internal K+ with a relative EC50 of 64 mM (SD 13.5) and a nH of 3.5 (SD 2.3), and by internal NH4+ and Ca2+. The reevaluation of channel conductive properties revealed an actual inward conductance of ~ 170 pS, with multiple subconductance levels and an inward rectification, and a substantial permeability to NH4+ ( = 0.2). We conclude that the apical 70-pS K channel in TAL cells is a large-conductance Na+- and Cl--activated potassium channel functionally resembling a KNa1.1 channel and propose that ROMK determines its functional expression possibly at the level of channel protein synthesis or trafficking.

Diversity of functional alterations of the ClC-5 exchanger in the region of the proton glutamate in patients with Dent disease 1.

Mutations in the CLCN5 gene encoding the 2Cl- /1H+ exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low-molecular-weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells, where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the "proton glutamate" that serves as a crucial H+ -binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for the second group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations might alter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.

Defective bicarbonate reabsorption in Kir4.2 potassium channel deficient mice impairs acid-base balance and ammonia excretion.

The kidneys excrete the daily acid load mainly by generating and excreting ammonia but the underlying molecular mechanisms are not fully understood. Here we evaluated the role of the inwardly rectifying potassium channel subunit Kir4.2 (Kcnj15 gene product) in this process. In mice, Kir4.2 was present exclusively at the basolateral membrane of proximal tubular cells and disruption of Kcnj15 caused a hyperchloremic metabolic acidosis associated with a reduced threshold for bicarbonate in the absence of a generalized proximal tubule dysfunction. Urinary ammonium excretion rates in Kcnj15- deleted mice were inappropriate to acidosis under basal and acid-loading conditions, and not related to a failure to acidify urine or a reduced expression of ammonia transporters in the collecting duct. In contrast, the expression of key proteins involved in ammonia metabolism and secretion by proximal cells, namely the glutamine transporter SNAT3, the phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase enzymes, and the sodium-proton exchanger NHE-3 was inappropriate in Kcnj15-deleted mice. Additionally, Kcnj15 deletion depolarized the proximal cell membrane by decreasing the barium-sensitive component of the potassium conductance and caused an intracellular alkalinization. Thus, the Kir4.2 potassium channel subunit is a newly recognized regulator of proximal ammonia metabolism. The kidney consequences of its loss of function in mice support the proposal for KCNJ15 as a molecular basis for human isolated proximal renal tubular acidosis.

A novel CLCN5 pathogenic mutation supports Dent disease with normal endosomal acidification.

Dent disease is an X-linked recessive renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. Inactivating mutations of CLCN5, the gene encoding the 2Cl- /H+ exchanger ClC-5, have been reported in patients with Dent disease 1. In vivo studies in mice harboring an artificial mutation in the "gating glutamate" of ClC-5 (c.632A > C, p.Glu211Ala) and mathematical modeling suggest that endosomal chloride concentration could be an important parameter in endocytosis, rather than acidification as earlier hypothesized. Here, we described a novel pathogenic mutation affecting the "gating glutamate" of ClC-5 (c.632A>G, p.Glu211Gly) and investigated its molecular consequences. In HEK293T cells, the p.Glu211Gly ClC-5 mutant displayed unaltered N-glycosylation and normal plasma membrane and early endosomes localizations. In Xenopus laevis oocytes and HEK293T cells, we found that contrasting with wild-type ClC-5, the mutation abolished the outward rectification, the sensitivity to extracellular H+ and converted ClC-5 into a Cl- channel. Investigation of endosomal acidification in HEK293T cells using the pH-sensitive pHluorin2 probe showed that the luminal pH of cells expressing a wild-type or p.Glu211Gly ClC-5 was not significantly different. Our study further confirms that impaired acidification of endosomes is not the only parameter leading to defective endocytosis in Dent disease 1.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1.

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation.

Characterization of the mouse ClC-K1/Barttin chloride channel.

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca2+ dependence of ClC-Kb channels.

ClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50 %. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome.

Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K+ channel subunit contrasts with that observed in SeSAME/EAST syndrome.

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K(+) channel underlies the basolateral K(+) conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene (KCNJ10) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene (Kcnj16). The Kir5.1(-/-) mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na(+) absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na(+) and Ca(2+), and abolished acidosis in Kir5.1(-/-) mice. Finally, in contrast to WT mice, electrophysiological recording of K(+) channels in the DCT basolateral membrane of Kir5.1(-/-) mice revealed that, even though Kir5.1 is absent, there is an increased K(+) conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.

A novel transgenic mouse model highlights molecular disruptions involved in the pathogenesis of Dent disease 1.

Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl-/H+exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at finding therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease.

Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression.

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance.

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney.

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but little is known about functional channels on the apical side. Apical membranes of microdissected fragments of mouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of the patch-clamp technique. A cation channel with a linear current-voltage relationship (19pS) that was permeable both to monovalent cations [P(NH4)(1.7)>P(Na) (1.0)=P(K) (1.0)] and to Ca(2+) (P(Ca)/P(Na)≈0.3) was detected. Unlike the basolateral TRPM4 Ca(2+)-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca(2+), pH and ATP. The channel was already active after patch excision, and its activity increased after reduced pressure was applied via the pipette. External gadolinium (10(-5)M) decreased the channel-open probability by 70% in outside-out patches, whereas external amiloride (10(-4)M) had no effect. Internal flufenamic acid (10(-4)M) inhibited the channel in inside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein that is expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involved in Ca(2+) signaling.

ClC-5 mutations associated with Dent's disease: a major role of the dimer interface.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules. Mutations in the 2Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent's disease. Functional characterization of mutations of CLCN5 have helped to elucidate the physiopathology of Dent's disease and provided evidence that several different mechanisms underlie the ClC-5 dysfunction in Dent's disease. Modeling studies indicate that many CLCN5 mutations are located at the interface between the monomers of ClC-5, demonstrating that this protein region plays an important role in Dent's disease. On the basis of functional data, CLCN5 mutations can be divided into three different classes. Class 1 mutations impair processing and folding, and as a result, the ClC-5 mutants are retained within the endoplasmic reticulum and targeted for degradation by quality control mechanisms. Class 2 mutations induce a delay in protein processing and reduce the stability of ClC-5. As a consequence, the cell surface expression and currents of the ClC-5 mutants are lower. Class 3 mutations do not alter the trafficking of ClC-5 to the cell surface and early endosomes but induce altered electrical activity. Here, we discuss the functional consequences of the three classes of CLCN5 mutations on ClC-5 structure and function.

Heterogeneity in the processing of CLCN5 mutants related to Dent disease.

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease.

Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins.

Eukaryotic members of the CLC gene family function as plasma membrane chloride channels, or may provide neutralizing anion currents for V-type H(+)-ATPases that acidify compartments of the endosomal/lysosomal pathway. Loss-of-function mutations in the endosomal protein ClC-5 impair renal endocytosis and lead to kidney stones, whereas loss of function of the endosomal/lysosomal protein ClC-7 entails osteopetrosis and lysosomal storage disease. Vesicular CLCs have been thought to be Cl- channels, in particular because ClC-4 and ClC-5 mediate plasma membrane Cl- currents upon heterologous expression. Here we show that these two mainly endosomal CLC proteins instead function as electrogenic Cl-/H+ exchangers (also called antiporters), resembling the transport activity of the bacterial protein ClC-e1, the crystal structure of which has already been determined. Neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion flux to proton counter-transport. ClC-4 and ClC-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to couple directly vesicular pH gradients to Cl- gradients.

Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules and is frequently associated with mutations in CLCN5, which encodes the electrogenic chloride-proton exchanger ClC-5. To better understand the functional consequences of CLCN5 mutations in this disease, we screened four newly identified missense mutations (G179D, S203L, G212A, L469P), one new nonsense mutation (R718X), and three known mutations (L200R, C219R, and C221R), in Xenopus laevis oocytes and HEK293 cells expressing either wild-type or mutant exchanger. A type-I mutant (G212A) trafficked normally to the cell surface and to early endosomes, underwent complex glycosylation at the cell surface like wild-type ClC-5, but exhibited significant reductions in outwardly rectifying ion currents. The type-II mutants (G179D, L200R, S203L, C219R, C221R, L469P, and R718X) were improperly N-glycosylated and were non-functional due to retention in the endoplasmic reticulum. Thus these mutations have distinct mechanisms by which they could impair ClC-5 function in Dent's disease.

Renal Chloride Channels in Relation to Sodium Chloride Transport.

The many mechanisms governing NaCl absorption in the diverse parts of the renal tubule have been largely elucidated, although some of them, as neutral NaCl absorption across the cortical collecting duct or regulation through with-no-lysine (WNK) kinases have emerged only recently. Chloride channels, which are important players in these processes, at least in the distal nephron, are the focus of this review. Over the last 20-year period, experimental studies using molecular, electrophysiological, and physiological/functional approaches have deepened and renewed our views on chloride channels and their role in renal function. Two chloride channels of the ClC family, named as ClC-Ka and ClC-Kb in humans and ClC-K1 and ClC-K2 in other mammals, are preponderant and play complementary roles: ClC-K1/Ka is mainly involved in the building of the interstitial cortico-medullary concentration gradient, while ClC-K2/Kb participates in NaCl absorption in the thick ascending limb, distal convoluted tubule and the intercalated cells of the collecting duct. The two ClC-Ks might also be involved indirectly in proton secretion by type A intercalated cells. Other chloride channels in the kidneys include CFTR, TMEM16A, and probably volume-regulated LRRC8 chloride channels, whose function and molecular identity have not as yet been established. © 2019 American Physiological Society. Compr Physiol 9:301-342, 2019.

[Blood pressure regulation: A crucial role of potassium intake]. / Régulation de la pression artérielle - Un rôle majeur de l'apport en potassium.

Title: Régulation de la pression artérielle - Un rôle majeur de l'apport en potassium. Abstract: Le Master de Biologie intégrative et physiologie (BIP) de Sorbonne Université recouvre un large champ disciplinaire, allant de la biologie du vieillissement à la physiopathologie, à la nutrition, aux bioressources marines et aux neurosciences, dont les enjeux sont essentiels aussi bien sur le plan fondamental qu'appliqué. L'objectif de la mention est de former des spécialistes de haut niveau aux concepts les plus récents de la biologie intégrative. La mention forme les étudiants à appréhender les mécanismes qui sous-tendent les différentes fonctions physiologiques et pathologiques depuis la cellule jusqu'à l'organisme, chez les animaux ou dans l'espèce humaine.

How Bartter's and Gitelman's syndromes, and Dent's disease have provided important insights into the function of three renal chloride channels: ClC-Ka/b and ClC-5.

Chloride channels are expressed in almost all cell membranes and are potentially involved in a wide variety of functions. The kidney expresses 8 of the 9 chloride channels of the ClC family that have been cloned so far to date in mammals. This review focuses on the pathophysiology of two renal disorders that have contributed recently to our understanding of the physiological role of chloride channels belonging to the ClC family. First are the related syndromes of Bartter's and Gitelman's, in which inactivating mutations of the genes encoding either of the ClC-Ks, or their regulatory beta-subunit barttin, have shown the important contribution of these chloride channels to renal tubular sodium and chloride (NaCl) transport along the loop of Henle and distal tubule. Second is the renal Fanconi syndrome known as Dent's disease, in which ClC-5 disruption has revealed the key role of this endosomal chloride channel in the megalin-mediated endocytotic pathway in the proximal tubule. The underlying pathophysiology of this inherited disorder demonstrates how ClC-5 is directly or indirectly required for the reabsorption of filtered low-molecular-weight proteins and bioactive peptides, also expression of membrane transporters, and clearance of calcium-based stone-forming crystals.

Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells.