In this study, K⁺ channels present in the basolateral membrane of the distal convoluted tubule (DCT) were investigated using patch‐clamp methods. In addition, Kir4.1, Kir4.2 and Kir5.1 inward rectifier channels were investigated using RT‐PCR and immunohistochemistry (Kir4.1). DCTs were microdissected from collagenase‐treated mouse kidneys. One type of K⁺ channel was detected in about 50 % of cell‐attached patches from the DCT basolateral membrane; this channel was inwardly rectifying and had an inward conductance (g_in) of ∼40 pS at an external [K⁺] of 145 mm. The current‐voltage relationship was linear when inside‐out patches were exposed to a Mg²⁺‐free medium. Mg²⁺ at a concentration of 1.2 mm considerably reduced the outward conductance (g_out), yielding a g_in/g_out ratio of ∼4.7. The polycation spermine (5 × 10⁻⁷m) reduced the open probability (P_o) by 50 %. Channel activity was dependent upon the intracellular pH, with acid pH decreasing, and basic pH increasing, P_o. Internal ATP (2 mm) and Ca²⁺ (up to 10⁻³m) had no effect. Channel activity declined irreversibly when the inner side of the patch was exposed to Mg²⁺. Kir4.1, Kir4.2 and Kir5.1 mRNAs were all detected in the DCT. The Kir4.1 protein co‐localised with the Na⁺‐Cl⁻ cotransporter, which is specific to the DCT, and was located on basolateral membranes. The DCT K⁺ channel differs from other functionally identified renal K⁺ channels with regard to its inhibition by spermine and insensitivity to internal ATP and Ca²⁺. At the current state of knowledge, the channel is similar to Kir4.1‐Kir5.1 and Kir4.2‐Kir5.1 heteromeric channels, but not to Kir4.1 or Kir4.2 homomeric channels.

K⁺channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K⁺channels with conductances of ∼75, 40, and 20 pS were observed, but the K⁺channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg²⁺-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg²⁺had no influence on the inward conductance ( G_in= 35 pS) but reduced outward conductance ( G_out) to 13 pS, yielding a G_in/ G_outof 3.2. The polycation spermine (6 × 10⁻⁷M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pH_i): a sigmoid relationship between pH_iand channel normalized current ( NP_o) was observed with a p K of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K⁺(Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K⁺had no influence on the properties of the intermediate-conductance channel, but a Na⁺-depleted diet increased its open probability by ∼25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K⁺conductance in the basolateral membrane of mouse CCD principal cells.

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.

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.

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na⁺-Cl⁻ cotransporter located at the apical membrane, and Na⁺, K⁺ ATPase at the basolateral side. Cl⁻ transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl⁻ channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP_o in cell-attached patches was not modified when tubules were preincubated in the presence of 10⁻⁵ M forskolin, but the channel was inhibited by phorbol ester (10⁻⁶ M). In addition, NP_o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NP_o increased threefold), or pH increased from 6.4 to 8.0 (NP_o increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na⁺ to Cl⁻ relative permeability was 0.09, and the anion selectivity sequence Cl⁻ ∼ I⁻> Br⁻ ∼ NO₃⁻ > F⁻. Intracellular NPPB (10⁻⁴ M) and DPC (10⁻³ M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl⁻ channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.