Chengbiao Zhang

KCNJ10 determines the expression of the apical Na-Cl cotransporter (NCC) in the early distal convoluted tubule (DCT1)

Significance Loss of function mutations in the potassium channel KCNJ10 causes a salt-wasting syndrome. The phenotype resembles Gitelman syndrome, which results from loss of sodium chloride transport along the distal convoluted tubule (DCT), but the mechanisms involved are not clear. Here, we perform experiments in the kidney from Kcnj10 knockout mice and demonstrate that Kcnj10 is a main contributor to the basolateral potassium conductance in the DCT and that the potassium channel activity regulates the expression of ste20-related proline–alanine-rich kinase (SPAK) and determines Na-Cl cotransporter (NCC) expression. Our study suggests the possibility that the regulation of basolateral Kcnj10 activity in the DCT may be the first step in response to a variety of physiological stimuli for initiating SPAK-WNK-dependent modulation of NCC expression in the kidney. The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10+/+ mice (WT). This 40-pS K channel is absent in homozygous Kcnj10−/− (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline–alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline–alanine-rich kinase-dependent mechanism.

Src Family Protein Tyrosine Kinase Regulates the Basolateral K Channel in the Distal Convoluted Tubule (DCT) by Phosphorylation of KCNJ10 Protein

Background: KCNJ10 is a key component of the basolateral K channels in DCT. Results: SFK phosphorylates KCNJ10 at Tyr9, and inhibition of SFK decreases basolateral K channel activity in DCT. Conclusion: SFK stimulates the basolateral K channels in DCT by phosphorylating KCNJ10. Significance: Tyrosine phosphorylation of KCNJ10 plays a role in regulating membrane transport in DCT. The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba2+-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from −65 to −43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr8 and Tyr9. The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr9 did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr9 on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

WNK4 inhibits Ca2 +-activated big-conductance potassium channels (BK) via mitogen-activated protein kinase-dependent pathway

We used the perforated whole-cell recording technique to examine the effect of with-no-lysine kinase 4 (WNK4) on the Ca(2+) activated big-conductance K channels (BK) in HEK293T cells transfected with BK-α subunit (BK-α). Expression of WNK4 inhibited BK channels and decreased the outward K currents. Coexpression of SGK1 abolished the inhibitory effect of WNK4 on BK channels and restored the outward K currents. Expression of WNK4(S1169D//1196D), in which both SGK1-phosphorylation sites (serine 1169 and 1196) were mutated to aspartate, had no effect on BK channels. Moreover, coexpression of SGK1 had no additional effect on K currents in the cells transfected with BKα+WNK4(S1169D//1196D), suggesting that SGK1 reversed WNK4-induced inhibition of BK channels by stimulating WNK4 phosphorylation. Expression of WNK4 but not WNK4(S1169D//1196D) increased the phosphorylation of ERK and p38 mitogen-activated protein kinase (MAPK); an effect was abolished by coexpression of SGK1. The role of ERK and p38 MAPK in mediating the effect of WNK4 on BK channels was further suggested by the finding that the inhibition of ERK and P38 MAPK completely abolished the inhibitory effect of WNK4 on BK channels. In contrast, inhibition of MAPK failed to abolish the inhibitory effect of WNK4 on ROMK channels in both HEK cells and Xenopus oocytes. Expression of dominant negative dynaminK44A (Dyn(K44A)) or treatment of the cells with dynasore, a dynamin inhibitor, not only increased K currents but also largely abolished the inhibitory effect of WNK4 on BK channels. However, inhibition of MAPK still increased the outward K currents in the cells transfected with BKα+WNK4 and treated with dynasore. Similar results were obtained in experiments performed in the native tissue in which inhibition of ERK and p38 MAPK increased BK channel activity in the cortical collecting duct (CCD) treated with dynasore. We concluded that WNK4 inhibited BK channels by stimulating ERK and p38 MAPK and that activation of MAPK by WNK4 may inhibit BK channels partially via a mechanism other than stimulating endocytosis.

KCNJ10 (Kir4.1) is expressed in the basolateral membrane of the cortical thick ascending limb.

The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10(+/+) wild-type (WT) and Kcnj10(-/-) knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression.

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Disruption of KCNJ10 (Kir4.1) stimulates the expression of ENaC in the collecting duct.

Kcnj10 encodes the inwardly rectifying K(+) channel 4.1 (Kir4.1) and is expressed in the basolateral membrane of late thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). In the present study, we perform experiments in postneonatal day 9 Kcnj10(-/-) or wild-type mice to examine the role of Kir.4.1 in contributing to the basolateral K(+) conductance in the CNT and CCD, and to investigate whether the disruption of Kir4.1 upregulates the expression of the epithelial Na(+) channel (ENaC). Immunostaining shows that Kir4.1 is expressed in the basolateral membrane of CNT and CCD. Patch-clamp studies detect three types of K(+) channels (23, 40, and 60 pS) in the basolateral membrane of late CNT and initial CCD in wild-type (WT) mice. However, only 23- and 60-pS K(+) channels but not the 40-pS K(+) channel were detected in Kcnj10(-/-) mice, suggesting that Kir.4.1 is a key component of the 40-pS K(+) channel in the CNT/CCD. Moreover, the depletion of Kir.4.1 did not increase the probability of finding the 23- and 60-pS K(+) channel in the CNT/CCD. We next used the perforated whole cell recording to measure the K(+) reversal voltage in the CNT/CCD as an index of cell membrane potential. Under control conditions, the K(+) reversal potential was -69 mV in WT mice and -61 mV in Kcnj10(-/-) mice, suggesting that Kir4.1 partially participates in generating membrane potential in the CNT/CCD. Western blotting and immunostaining showed that the expression of ENaCβ and ENaCγ subunits from a renal medulla section of Kcnj10(-/-) mice was significantly increased compared with that of WT mice. Also, the disruption of Kir4.1 increased aquaporin 2 expression. We conclude that Kir4.1 is expressed in the CNT and CCD and partially participates in generating the cell membrane potential. Also, increased ENaC expression in medullary CD of Kcnj10(-/-) mice is a compensatory action in response to the impaired Na(+) transport in the DCT.

Caveolin-1 Deficiency Inhibits the Basolateral K+ Channels in the Distal Convoluted Tubule and Impairs Renal K+ and Mg2+ Transport

Kcnj10 encodes the inwardly rectifying K(+) channel Kir4.1 in the basolateral membrane of the distal convoluted tubule (DCT) and is activated by c-Src. However, the regulation and function of this K(+) channel are incompletely characterized. Here, patch-clamp experiments in Kcnj10-transfected HEK293 cells demonstrated that c-Src-induced stimulation of Kcnj10 requires coexpression of caveolin-1 (cav-1), and immunostaining showed expression of cav-1 in the basolateral membrane of parvalbumin-positive DCT. Patch-clamp experiments detected a 40-pS inwardly rectifying K(+) channel, a heterotetramer of Kir4.1/Kir5.1, in the basolateral membrane of the early DCT (DCT1) in both wild-type (WT) and cav-1-knockout (KO) mice. However, the activity of this basolateral 40-pS K(+) channel was lower in KO mice than in WT mice. Moreover, the K(+) reversal potential (an indication of membrane potential) was less negative in the DCT1 of KO mice than in the DCT1 of WT mice. Western blot analysis demonstrated that cav-1 deficiency decreased the expression of the Na(+)/Cl(-) cotransporter and Ste20-proline-alanine-rich kinase (SPAK) but increased the expression of epithelial Na(+) channel-α. Furthermore, the urinary excretion of Mg(2+) and K(+) was significantly higher in KO mice than in WT mice, and KO mice developed hypomagnesemia, hypocalcemia, and hypokalemia. We conclude that disruption of cav-1 decreases basolateral K(+) channel activity and depolarizes the cell membrane potential in the DCT1 at least in part by suppressing the stimulatory effect of c-Src on Kcnj10. Furthermore, the decrease in Kcnj10 and Na(+)/Cl(-) cotransporter expression induced by cav-1 deficiency may underlie the compromised renal transport of Mg(2+), Ca(2+), and K(+).

Cyp2c44 epoxygenase in the collecting duct is essential for the high K+ intake-induced antihypertensive effect

Cytochrome P-450, family 2, subfamily c, polypeptide 44 (Cyp2c44) epoxygenase metabolizes arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) in kidney and vascular tissues. In the present study, we used real-time quantitative PCR techniques to examine the effect of high salt or high K(+) (HK) intake on the expression of Cyp2c44, a major Cyp2c epoxygenase in the mouse kidney. We detected Cyp2c44 in the proximal convoluted tubule, thick ascending limb, distal convoluted tubule (DCT)/connecting tubule (CNT), and collecting duct (CD). A high-salt diet increased the expression of Cyp2c44 in the thick ascending limb and DCT/CNT but not in the proximal convoluted tubule and CD. In contrast, an increase in dietary K(+) intake augmented Cyp2c44 expression only in the DCT/CNT and CD. Neither high salt nor HK intake had a significant effect on the blood pressure (BP) of wild-type mice. However, HK but not high salt intake increased BP in CD-specific, Cyp2c44 conditional knockout (KO) mice. Amiloride, an epithelial Na(+) channel (ENaC) inhibitor, normalized the BP of KO mice fed HK diets, suggesting that lack of Cyp2c44 in the CD enhances ENaC activity and increases Na(+) absorption in KO mice fed HK diets. This notion was supported by metabolic cage experiments demonstrating that renal Na(+) excretion was compromised in KO mice fed HK diets. Also, patch-clamp experiments demonstrated that 11,12-EET, a major Cyp2c44 product, but not AA inhibited ENaC activity in the cortical CD of KO mice. We conclude that Cyp2c44 in the CD is required for preventing the excessive Na(+) absorption induced by HK intake by inhibition of ENaC and facilitating renal Na(+) excretion.

ENaC and ROMK activity are inhibited in the DCT2/CNT of TgWnk4PHAII mice

Mice transgenic for genomic segments harboring PHAII (pseudohypoaldosteronism type II) mutant Wnk4 (with-No-Lysine kinase 4) (TgWnk4PHAII) have hyperkalemia which is currently believed to be the result of high activity of Na-Cl cotransporter (NCC). This leads to decreasing Na+ delivery to the distal nephron segment including late distal convoluted tubule (DCT) and connecting tubule (CNT). Since epithelial Na+ channel (ENaC) and renal outer medullary K+ channel (ROMK or Kir4.1) are expressed in the late DCT and play an important role in mediating K+ secretion, the aim of the present study is to test whether ROMK and ENaC activity in the DCT/CNT are also compromised in the mice expressing PHAII mutant Wnk4. Western blot analysis shows that the expression of βENaC and γENaC subunits but not αENaC subunit was lower in TgWnk4PHAII mice than that in wild-type (WT) and TgWnk4WT mice. Patch-clamp experiments detected amiloride-sensitive Na+ currents and TPNQ-sensitive K+ currents in DCT2/CNT, suggesting the activity of ENaC and ROMK. However, both Na+ and ROMK currents in DCT2/CNT of TgWnk4PHAII mice were significantly smaller than those in WT and TgWnk4WT mice. In contrast, the basolateral K+ currents in the DCT were similar among three groups, despite higher NCC expression in TgWnk4PHAII mice than those of WT and TgWnk4WTmice. An increase in dietary K+ intake significantly increased both ENaC and ROMK currents in the DCT2/CNT of all three groups. However, high-K+ (HK) intake-induced stimulation of Na+ and K+ currents was smaller in TgWnk4PHAII mice than those in WT and TgWnk4WT mice. We conclude that ENaC and ROMK channel activity in DCT2/CNT are inhibited in TgWnk4PHAII mice and that Wnk4PHAII-induced inhibition of ENaC and ROMK may contribute to the suppression of K+ secretion in the DCT2/CNT in addition to increased NCC activity.

Vasopressin-induced stimulation of the Na+-activated K+ channels is responsible for maintaining the basolateral K+ conductance of the thick ascending limb (TAL) in EAST/SeSAME syndrome

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelmans syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na(+)-activated 80-150pS K(+) channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10(-/-) mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K(+) conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10(-/-) mice. Application of AVP significantly stimulated the 80-150pS K(+) channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80-150pS K(+) channel and the K(+) channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K(+) channel, suggesting that cAMP-PKA pathway stimulates the 80-150pS K(+) channels. The role of the basolateral K(+) conductance in maintaining transcellular Cl(-) transport is further suggested by the finding that the inhibition of basolateral K(+) channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl(-) channels. We conclude that vasopressin stimulates the 80-150pS K(+) channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K(+) channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K(+) conductance which is essential for the transport function in the TAL.