Renal outer medullary potassium (ROMK) channel is an important K⁺ excretion channel in the body, and K⁺ secreted by the ROMK channels is most or all source of urinary potassium. Previous studies focused on the ROMK channels of thick ascending limb (TAL) and collecting duct (CD), while there were few studies on the involvement of ROMK channels of the late distal convoluted tubule (DCT2) in K⁺ excretion. The purpose of the present study was mainly to record the ROMK channels current in renal DCT2 and observe the effect of high potassium diet on the ROMK channels by using single channel and whole-cell patch-clamp techniques. The results showed that a small conductance channel current with a conductance of 39 pS could be recorded in the apical membrane of renal DCT2, and it could be blocked by Tertiapin-Q (TPNQ), a ROMK channel inhibitor. The high potassium diet significantly increased the probability of ROMK channel current occurrence in the apical membrane of renal DCT2, and enhanced the activity of ROMK channel, compared to normal potassium diet (P < 0.01). Western blot results also demonstrated that the high potassium diet significantly up-regulated the protein expression levels of ROMK channels and epithelial sodium channel (ENaC), and down-regulated the protein expression level of Na⁺-Cl^- cotransporter (NCC). Moreover, the high potassium diet significantly increased urinary potassium excretion. These results suggest that the high potassium diet may activate the ROMK channels in the apical membrane of renal DCT2 and increase the urinary potassium excretion by up-regulating the expression of renal ROMK channels.
Potassium Channels, Inwardly Rectifying/metabolism* ; Kidney Tubules, Distal/metabolism* ; Potassium/metabolism* ; Epithelial Sodium Channels/metabolism* ; Diet

Virtually all of the dietary potassium intake is absorbed in the intestine, over 90% of which is excreted by the kidneys regarded as the most important organ of potassium excretion in the body. The renal excretion of potassium results primarily from the secretion of potassium by the principal cells in the aldosterone-sensitive distal nephron (ASDN), which is coupled to the reabsorption of Na⁺ by the epithelial Na⁺ channel (ENaC) located at the apical membrane of principal cells. When Na⁺ is transferred from the lumen into the cell by ENaC, the negativity in the lumen is relatively increased. K⁺ efflux, H⁺ efflux, and Cl^- influx are the 3 pathways that respond to Na⁺ influx, that is, all these 3 pathways are coupled to Na⁺ influx. In general, Na⁺ influx is equal to the sum of K⁺ efflux, H⁺ efflux, and Cl^- influx. Therefore, any alteration in Na⁺ influx, H⁺ efflux, or Cl^- influx can affect K⁺ efflux, thereby affecting the renal K⁺ excretion. Firstly, Na⁺ influx is affected by the expression level of ENaC, which is mainly regulated by the aldosterone-mineralocorticoid receptor (MR) pathway. ENaC gain-of-function mutations (Liddle syndrome, also known as pseudohyperaldosteronism), MR gain-of-function mutations (Geller syndrome), increased aldosterone levels (primary/secondary hyperaldosteronism), and increased cortisol (Cushing syndrome) or deoxycorticosterone (hypercortisolism) which also activate MR, can lead to up-regulation of ENaC expression, and increased Na⁺ reabsorption, K⁺ excretion, as well as H⁺ excretion, clinically manifested as hypertension, hypokalemia and alkalosis. Conversely, ENaC inactivating mutations (pseudohypoaldosteronism type 1b), MR inactivating mutations (pseudohypoaldosteronism type 1a), or decreased aldosterone levels (hypoaldosteronism) can cause decreased reabsorption of Na⁺ and decreased excretion of both K⁺ and H⁺, clinically manifested as hypotension, hyperkalemia, and acidosis. The ENaC inhibitors amiloride and Triamterene can cause manifestations resembling pseudohypoaldosteronism type 1b; MR antagonist spironolactone causes manifestations similar to pseudohypoaldosteronism type 1a. Secondly, Na⁺ influx is regulated by the distal delivery of water and sodium. Therefore, when loss-of-function mutations in Na⁺-K⁺-2Cl^- cotransporter (NKCC) expressed in the thick ascending limb of the loop and in Na⁺-Cl^- cotransporter (NCC) expressed in the distal convoluted tubule (Bartter syndrome and Gitelman syndrome, respectively) occur, the distal delivery of water and sodium increases, followed by an increase in the reabsorption of Na⁺ by ENaC at the collecting duct, as well as increased excretion of K⁺ and H⁺, clinically manifested as hypokalemia and alkalosis. Loop diuretics acting as NKCC inhibitors and thiazide diuretics acting as NCC inhibitors can cause manifestations resembling Bartter syndrome and Gitelman syndrome, respectively. Conversely, when the distal delivery of water and sodium is reduced (e.g., Gordon syndrome, also known as pseudohypoaldosteronism type 2), it is manifested as hypertension, hyperkalemia, and acidosis. Finally, when the distal delivery of non-chloride anions increases (e.g., proximal renal tubular acidosis and congenital chloride-losing diarrhea), the influx of Cl^- in the collecting duct decreases; or when the excretion of hydrogen ions by collecting duct intercalated cells is impaired (e.g., distal renal tubular acidosis), the efflux of H⁺ decreases. Both above conditions can lead to increased K⁺ secretion and hypokalemia. In this review, we focus on the regulatory mechanisms of renal potassium excretion and the corresponding diseases arising from dysregulation.
Humans ; Bartter Syndrome/metabolism* ; Pseudohypoaldosteronism/metabolism* ; Potassium/metabolism* ; Aldosterone/metabolism* ; Hypokalemia/metabolism* ; Gitelman Syndrome/metabolism* ; Hyperkalemia/metabolism* ; Clinical Relevance ; Epithelial Sodium Channels/metabolism* ; Kidney Tubules, Distal/metabolism* ; Sodium/metabolism* ; Hypertension ; Alkalosis/metabolism* ; Water/metabolism* ; Kidney/metabolism*

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H or H receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K channels and activation of Na-Ca exchangers were involved in the H receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.
Animals ; Histamine ; pharmacology ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Motor Neurons ; drug effects ; physiology ; Rats ; Receptors, Histamine H2 ; metabolism ; Sodium-Calcium Exchanger ; metabolism

Ion channels mediate ion transport across membranes, and play vital roles in processes of matter exchange, energy transfer and signal transduction in living organisms. Recently, structural studies of ion channels have greatly advanced our understanding of their ion selectivity and gating mechanisms. Structural studies of voltage-gated potassium channels elucidate the structural basis for potassium selectivity and voltage-gating mechanism; structural studies of voltage-gated sodium channels reveal their slow and fast inactivation mechanisms; and structural studies of transient receptor potential (TRP) channels provide complex and diverse structures of TRP channels, and their ligand gating mechanisms. In the article we summarize recent progress on ion channel structural biology, and outlook the prospect of ion channel structural biology in the future.
Ion Channel Gating ; physiology ; Ion Channels ; Voltage-Gated Sodium Channels ; chemistry ; metabolism

Isoflavones are widely consumed by people around the world in the form of soy products, dietary supplements and drugs. Many isoflavones or related crude extracts have been reported to exert pain-relief activities, but the mechanism remains unclear. Voltage-gated sodium channels (VGSCs) play important roles in excitability of pain sensing neurons and many of them are important nociceptors. Here, we report that several isoflavones including 3'-methoxydaidzein (3MOD), genistein (GEN) and daidzein (DAI) show abilities to block VGSCs and thus to attenuate chemicals and heat induced acute pain or chronic constriction injury (CCI) induced pain hypersensitivity in mice. Especially, 3MOD shows strong analgesic potential without inducing addiction through inhibiting subtypes Na1.7, Na1.8 and Na1.3 with the IC of 181 ± 14, 397 ± 26, and 505 ± 46 nmol·L, respectively, providing a promising compound or parent structure for the treatment of pain pathologies. This study reveals a pain-alleviating mechanism of dietary isoflavones and may provide a convenient avenue to alleviate pain.
Analgesics ; administration & dosage ; chemistry ; Animals ; Humans ; Isoflavones ; administration & dosage ; chemistry ; Male ; Mice ; Mice, Inbred C57BL ; Pain ; drug therapy ; genetics ; metabolism ; Voltage-Gated Sodium Channel Blockers ; administration & dosage ; Voltage-Gated Sodium Channels ; genetics ; metabolism

BackgroundLipoxin A4 (LXA4) can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) and acute respiratory distress syndrome through promoting epithelial sodium channel (ENaC) expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4.

MethodsA549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing of A549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis of A549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor (ALX) inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression.

ResultsThe A549 cell models of ALI were constructed and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream-regulated gene-1 (NDRG1) was validated by real-time-PCR and Western blot. NDRG1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG1 expression induced by LXA4. NDRG1 siRNA suppressed viability of LPS-treated A549 cells (treatment vs. control, 0.605 ± 0.063 vs. 0.878 ± 0.083, P = 0.040) and ENaC-α expression (treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008). LY294002 inhibited NDRG1 (treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P = 0.001) and ENaC-α (treatment vs. control, 0.236 ± 0.021 vs. 0.814 ± 0.025, P < 0.001) expressions and serum- and glucocorticoid-inducible kinase 1 phosphorylation (treatment vs. control, 0.442 ± 0.024 vs. 1.046 ± 0.082, P = 0.002), indicating the PI3K signaling pathway was involved in regulating NDRG1 expression induced by LXA4.

ConclusionOur research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.

A549 Cells ; Acute Lung Injury ; metabolism ; Cell Cycle Proteins ; metabolism ; Cell Line ; Epithelial Sodium Channels ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Lipopolysaccharides ; pharmacology ; Lipoxins ; pharmacology ; Signal Transduction ; drug effects

Eukaryota ; Molecular Dynamics Simulation ; Permeability ; Protein Conformation ; Sodium ; metabolism ; Substrate Specificity ; Voltage-Gated Sodium Channels ; chemistry ; metabolism

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.
Animals ; Calcium Channels, L-Type ; chemistry ; genetics ; metabolism ; Channelopathies ; genetics ; metabolism ; Humans ; Mutation ; NAV1.1 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; Protein Domains ; Rabbits ; Structure-Activity Relationship

OBJECTIVETo explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts.

METHODSMultinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts.

RESULTSs Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K.

CONCLUSIONs ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.

Animals ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; metabolism ; Cattle ; Cell Differentiation ; Epithelial Sodium Channels ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Osteoclasts ; cytology ; RANK Ligand ; metabolism ; Rats ; Signal Transduction

OBJECTIVETo investigate the role of interleukin-17 (IL-17) in alveolar fluid clearance in mice with acute lung injury (ALI) and explore the possible mechanism.

METHODSSixteen IL-17-knockout mice and 16 wild-type mice were both randomized for intratracheal instillation of PBS (control) on lipopolysaccharide (LPS) to induce ALI. Forty-eight hours after the treatments, the wet-dry ratio (W/D) of the lungs, IL-8 in the bronchoalveolar lavage fluid (BALF) and histopathological changes of the lung tissues were examined. The expressions of epithelial sodium channel α subunit (α-ENaC) was detected with Western blotting and liver kinase B1 (LKB1) was detected with immunohistochemistry.

RESULTSCompared with wild-type mice treated with LPS, IL-17 knockout mice showed significantly decreased W/D of the lungs (9.739∓3.3 vs 5.351∓0.56) and IL-8 level in the BALF (67.50∓7.33 vs 41.00∓3.16 pg/mL) following LPS challenge. Pathological examination revealed reduced alveolar edema fluid aggregations and lower lung injury score in IL-17 knockout mice with also higher expression levels of ENaC and LKB1 compared with the wild-type mice.

CONCLUSIONKnocking out IL-17 in mice not only alleviates inflammation of the lung tissue following ALI but also reduces the loss of ENaC protein and promotes alveolar fluid clearance, mechanism of which is probably associated with LKB1.

Acute Lung Injury ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Epithelial Sodium Channels ; metabolism ; Gene Knockout Techniques ; Interleukin-17 ; genetics ; metabolism ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; Lung ; pathology ; Mice ; Protein-Serine-Threonine Kinases ; metabolism