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

Objective: To clarify the phenotypes of the newborns with SLC26A4 single-allele mutation in deafness genetic screening and second variant; to analyze the SLC26A4 genotype and hearing phenotype. Methods: 850 newborns born in Beijing from April 2015 to December 2019 were included and there were 468 males and 382 females. They received genetic deafness screening for 9 or 15 variants, with the result of SLC26A4 single-allele mutation. Firstly, three step deafness gene sequencing was adopted in this work, i.e., the first step was "SLC26A4 gene whole exons and splice sites" sequencing; the second step was "SLC26A4 gene promoter, FOXI1 gene and KCNJ10 gene whole exons" sequencing; and the third step was detection for "SLC26A4 gene copy number variation". Secondly, we collected the results of newborn hearing screening for all patients with the second mutation found in the three step test, and conducted audiological examinations, such as acoustic immittance, auditory brainstem response and auditory steady state response. Thirdly, for novel/VUS mutations, we searched the international deafness gene database or software, such as DVD, ClinVar and Mutation Taster, to predict the pathogenicity of mutations according to the ACMG guideline. Lastly, we analyzed the relationship between genotype and phenotype of newborns with SLC26A4 single allele mutation. Results: Among 850 cases, the median age of diagnosis was 4 months. In the first step, 850 cases were sequenced. A total of 32 cases (3.76%, 32/850) of a second variants were detected, including 18 cases (2.12%, 18/850) with identified pathogenic variants; 832 cases were sequenced and 8 cases of KCNJ10 gene missense variants were detected among the second step. No missense mutations in the FOXI1 gene and abnormal SLC26A4 gene promoter were detected; the third step sequencing results were all negative. Genotypes and hearing phenotypes included 18 cases combined with the second clear pathogenic variant, 16 cases (16/18) referred newborn hearing screening and 2 cases (2/18) passed in both ears; degree of hearing loss consisted of 18 profound ears (18/36), 13 severe ears (13/36) and 5 moderate ears (5/36); audiogram patterns comprised 17 high frequency drop ears (17/36), 14 flat ears (14/36), 3 undistinguished ears (3/36), and 2 U shaped ears (2/36); 11 cases underwent imaging examination, all of which were bilateral enlarged vestibular aqueduct. As for 22 cases of other genotypes, all passed neonatal hearing screening and the hearing diagnosis was normal, including 9 cases with VUS or possibly novel benign variants, 8 cases with KCNJ10 double gene heterozygous variants, and 5 cases with double heterozygous variants. Conclusions: The probability of individuals with SLC26A4 single-allele variant who merge with a second pathogenic variant is 2.12%, all of which are SNV, which can provide scientific basis for the genetic diagnosis and genetic counseling of SLC26A4 variants. Those who have merged with second pathogenic variant are all diagnosed with sensorineural hearing loss. Patients with KCNJ10 gene mutations do not manifest hearing loss during the infancy, suggesting the need for further follow-up.
Female ; Humans ; Male ; Alleles ; Deafness/genetics* ; DNA Copy Number Variations ; Forkhead Transcription Factors/genetics* ; Genotype ; Hearing Loss/genetics* ; Hearing Loss, Sensorineural/genetics* ; Mutation ; Phenotype ; Sulfate Transporters/genetics* ; Vestibular Aqueduct ; Infant, Newborn ; Potassium Channels, Inwardly Rectifying/genetics*

OBJECTIVE: To investigate the clinical characteristics and genetic basis for a child with Keppen-Lubinsky syndrome (KPLBS). METHODS: Trio-whole exome sequencing (Trio-WES) was carried out for the proband and her parents. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child has featured peculiar facies including large eyes, alar hypoplasia, microretrognathia, premature aging appearance in addition with growth delay and mental retardation. Trio-WES has identified that she has carried a de novo variant of the KCNJ6 gene, namely c.460G>C (p.Gly154Arg). The variant has not been recorded in the database. Prediction of protein structure indicated that the variant may affect the potassium ion selective filtration structure channel in the transmembrane region of KCNJ6 protein, which may result in up regulation of the function of the channel. CONCLUSION The de novo c.460G>C (p.Gly154Arg) variant of the KCNJ6 gene probably underlay the KPLBS in this child. Above finding has enriched the genotypic and phenotype spectrum of this syndrome.
Cataract ; China ; Female ; G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics* ; Humans ; Hypogonadism/congenital* ; Intellectual Disability/genetics* ; Mutation ; Whole Exome Sequencing

OBJECTIVES: Epilepsy is a syndrome of central nervous system dysfunction caused by many reasons, which is mainly characterized by abnormal discharge of neurons in the brain. Therefore, finding new targets for epilepsy therapy has always been the focus and hotspot in neurological research field. Studies have found that 2-deoxy-D-glucose (2-DG) exerts anti-epileptic effect by up-regulation of K_ATP channel subunit Kir6.1, Kir6.2 mRNA and protein. By using the database of TargetScan and miRBase to perform complementary pairing analysis on the sequences of miRNA and related target genes, it predicted that miR-194 might be the upstream signaling molecule of K_ATP channel. This study aims to explore the mechanism by which 2-DG exerts its anti-epileptic effect by regulating K_ATP channel subunits Kir6.1 and Kir6.2 via miR-194. METHODS: A magnesium-free epilepsy model was established and randomly divided into a control group, an epilepsy group (EP group), an EP+2-DG group, and miR-194 groups (including EP+miR-194 mimic, EP+miR-194 mimic+2-DG, EP+miR-194 mimic control, EP+miR-194 inhibitor, EP+miR-194 inhibitor+2-DG, and EP+miR-194 inhibitor control groups). The 2-DG was used to intervene miR-194 mimics, patch-clamp method was used to detect the spontaneous recurrent epileptiform discharges, real-time PCR was used to detect neuronal miR-194, Kir6.1, and Kir6.2 expressions, and the protein levels of Kir6.1 and Kir6.2were detected by Western blotting. RESULTS: Compared with the control group, there was no significant difference in the amplitude of spontaneous discharge potential in the EP group (P>0.05), but the frequency of spontaneous discharge was increased (P<0.05). Compared with the EP group, the frequency of spontaneous discharge was decreased (P<0.05). Compared with the EP+miR-194 mimic control group, the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 mimic group were down-regulated (all P<0.05). Compared with the EP+miR-194 inhibitor control group, the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 inhibitor group were up-regulated (all P<0.05). After pretreatment with miR-194 mimics, the mRNA and protein expression levels of K_ATP channel subunits Kir6.1 and Kir6.2 were decreased (all P<0.05). Compared with the EP+2-DG group, the mRNA and protein expression levels of Kir6.1 and Kir6.2 in the EP+miR-194 mimic+2-DG group were down-regulated (all P<0.05) and the mRNA and protein expression levels of Kir6.1 and Kir6.2 in the EP+miR-194 inhibitor+2-DG group were up-regulated (all P<0.05). CONCLUSIONS The 2-DG might play an anti-epilepsy effect by up-regulating K_ATP channel subunits Kir6.1 and Kir6.2via miR-194.
Adenosine Triphosphate ; Anticonvulsants ; Deoxyglucose/pharmacology* ; Epilepsy/genetics* ; Glucose ; Humans ; MicroRNAs/genetics* ; Potassium Channels, Inwardly Rectifying/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

ATP-sensitive potassium channels (K) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.
Adenosine Triphosphate ; metabolism ; Amino Acid Sequence ; Animals ; Binding Sites ; Cryoelectron Microscopy ; Ligands ; Mesocricetus ; Mice ; Models, Molecular ; Nucleotides ; metabolism ; Pancreas ; metabolism ; Potassium Channels, Inwardly Rectifying ; chemistry ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Subunits ; chemistry ; metabolism ; Sf9 Cells ; Spodoptera ; Sulfonylurea Receptors ; chemistry ; metabolism

OBJECTIVE: Corticotropin-releasing hormone (CRH) is a crucial regulator of human pregnancy and parturition. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are important for regulating myometrial quiescence during pregnancy. We investigated regulatory effects of different concentrations of CRH on KATP channel expression in human myometrial smooth muscle cells (HSMCs) in in vitro conditions. METHODS: After treating HSMCs with different concentrations of CRH (1, 10, 102, 103, 104 pmol/L), mRNA and protein expression of KATP channel subunits (Kir6.1 and SUR2B) was analyzed by reverse transcription-polymerase chain reaction and western blot. We investigated which CRH receptor was involved in the reaction and measured the effects of CRH on intracellular Ca2+ concentration when oxytocin was administered in HSMCs using Fluo-8 AM ester. RESULTS: When HSMCs were treated with low (1 pmol/L) and high (103, 104 pmol/L) CRH concentrations, KATP channel expression significantly increased and decreased, respectively. SUR2B mRNA expression at low and high CRH concentrations was significantly antagonized by antalarmin (CRH receptor-1 antagonist) and astressin 2b (CRH receptor-2 antagonist), respectively; however, Kir6.1 mRNA expression was not affected. After oxytocin treatment, the intracellular Ca2+ concentration in CRH-treated HSMCs was significantly lowered in low concentration of CRH (1 pmol/L), but not in high concentration of CRH (103 pmol/L), compared to control. CONCLUSION: Our data demonstrated the regulatory effect was different when HSMCs were treated with low (early pregnancy-like) and high (labor-like) CRH concentrations and the KATP channel expression showed significant increase and decrease. This could cause inhibition and activation, respectively, of uterine muscle contraction, demonstrating opposite dual actions of CRH.
Adenosine Triphosphate ; Adenosine ; Animals ; Blotting, Western ; Corticotropin-Releasing Hormone ; Female ; Humans ; In Vitro Techniques ; KATP Channels ; Mice ; Myocytes, Smooth Muscle ; Myometrium ; Oxytocin ; Parturition ; Potassium Channels ; Potassium ; Pregnancy ; Receptors, Corticotropin-Releasing Hormone ; RNA, Messenger

Basolateral inwardly-rectifying K channels (Kir) play an important role in the control of resting membrane potential and transepithelial voltage, thereby modulating water and electrolyte transport in the distal part of nephron. Kir4.1 and Kir4.1/Kir5.1 heterotetramer are abundantly expressed in the basolateral membrane of late thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD). Loss-of-function mutations in KCNJ10 cause EAST/SeSAME syndrome in humans associated with epilepsy, ataxia, sensorineural deafness and water-electrolyte metabolism imbalance, which is characterized by salt wasting, hypomagnesaemia, hypokalaemia and metabolic alkalosis. In contrast, mice lacking Kir5.1 have severe renal phenotype apart from hypokalaemia such as high chlorine metabolic acidosis and hypercalcinuria. The genetic knockout or functional inhibition of Kir4.1 suppresses Na-Cl cotransporter (NCC) expression and activity in the DCT. However, the downregulation of Kir4.1 increases epithelial Na channel (ENaC) expression in the collecting duct. Recently, factors regulating expression and activity of Kir4.1 and Kir4.1/Kir5.1 were identified, such as cell acidification, dopamine, insulin and insulin-like growth factor-1. The involved mechanisms include PKC, PI3K, Src family protein tyrosine kinases and WNK-SPAK signal transduction pathways. Here we review the progress of renal tubule basolateral Kir, and mainly discuss the function and regulation of Kir4.1 and Kir4.1/Kir5.1.
Animals ; Cell Membrane ; Humans ; Kidney Tubules ; metabolism ; Kidney Tubules, Distal ; Membrane Potentials ; Mice ; Potassium Channels, Inwardly Rectifying ; metabolism

OBJECTIVE: To observe the mechanism of "" acupuncture for the opening of ATP sensitive potassium channel (K) against cerebral ischemia reperfusion injury in rats. METHODS: Eighty-four rats were randomly divided into a sham-operation group, a model group, an electroacupuncture (EA) group, an EA+K blocker group, 21 rats in each group. 10 μL intracerebral injection with glipizide (1 μmol/5 μL) was used in the EA+K blocker group. The cerebral ischemia reperfusion model was established by Zea Longa's suture method in the model group, the EA group and the EA+K blocker group. Rats in the sham-operation group were received the same surgery but without nylon filament insertion. Acupuncture (20 min a time) was performed at "Neiguan" (PC 6), "Shuigou" (GV 26) and "Sanyinjiao" (SP 6) in the EA group and the EA+K blocker group at 10:00 and 16:00 for 3 days, firstly 90 min after model establishment. EA (2 Hz/15 Hz, 1 mA) was connected at the affected "Neiguan" (PC 6) and "Sanyinjiao" (SP 6). The same fixation was used in the sham-operation group and the model group, without EA. Neurological function was assessed by Zausinger's neurologic assessment scale. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining was used to detect infarct volume. Neurocyte apoptosis in the hippocampus was detected by flow cytometry and the protein expressions of B lymphocytoma-2 gene (Bcl-2) and B cell lymphoma factor-associated X protein (Bax) were measured by Western-blot. RESULTS: In comparison with the model group, the neurological score of the EA group increased (<0.01); the infarction volume and the hippocampal neuron's total apoptosis rate of the EA group decreased (both <0.05); the protein expression of Bcl-2 and Bcl-2/Bax of the EA group increased (<0.05, <0.01); and the protein expression of Bax of the EA group decreased (<0.01). Compared with the EA group, the neurological score of the EA+K blocker group decreased (<0.05); the total apoptosis rate of hippocampus neurons of the EA+K blocker group increased (<0.05); the expression of Bcl-2 protein of the EA+K blocker group reduced (<0.05); the expression of Bax protein of the EA+K blocker group increased (<0.05). CONCLUSION "" acupuncture has brain protective effect on rats with focal cerebral ischemia reperfusion injury. The mechanism may be related to regulating the opening of K channels and decreasing the apoptosis of neurons.
Acupuncture Points ; Animals ; Brain Ischemia ; Electroacupuncture ; KATP Channels ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury

OBJECTIVETo analyze the clinical characteristics of an infant with neonatal diabetes mellitus (NDM) and to sequence the ABCC8 gene of this family in order to provide a theoretical basis for the diagnosis and treatment.

METHODSThe clinical data of the patient was collected, and the proband and his direct relatives within three generations were sequenced.

RESULTSThe patient was 1-month-old, random blood glucose was more than 27.8 mmol/L, C-peptide was 33.8 pmol/L, blood gas analysis was pH 7.16, HCO3.9 mmol/L and urine alkone was 3+. Genetic testing revealed that the patient, his father, elder brother and grandmother have carried heterozygous mutation c.2690A>T(p.D897V) of the ABCC8 gene. Fluid infusion, intravenous administration of insulin and other supportive therapies were provided. After the correction of acidosis, subcutaneous insulin injection were uesd to control the blood glucose. Eight months later, blood glucose was pooly controlled. After combined with glibenclamide, blood glucose was under control.

CONCLUSIONThe patient carries a heterozygous mutation c.2690A>T(p.D897V) of ABCC8 gene, which is a novel mutation. Glibenclamide was partly effective for the patient.

Blood Glucose ; genetics ; Diabetes Mellitus ; genetics ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Infant, Newborn, Diseases ; genetics ; Male ; Mutation ; genetics ; Sulfonylurea Receptors ; genetics