Objective This study aims to achieve high-yield functional expression of the human voltage-gated potassium channel K_V3.1 using an Escherichia coli cell-free protein synthesis system, thereby providing a novel synthetic approach for drug screening, structural analysis and functional characterization of K_V3.1. Methods K_V3.1 was expressed in an Escherichia coli cell-free protein synthesis system for 10 hours in the presence of peptide surfactant A₆K. The secondary structure of K_V3.1 was analyzed by circular dichroism spectroscopy. The potassium channel activity of the recombinant protein liposome K_V3.1-A₆K was investigated using fluorescent dyes Oxonol VI as indicators, which are capable of reflecting alterations in membrane potential. Results Soluble K_V3.1 protein was successfully synthesized, achieving a purified yield of up to 1.2 mg/mL via an Escherichia coli cell-free protein synthesis system. Circular dichroism spectroscopy revealed that K_V3.1 exhibited characteristic α-helical secondary structures. Membrane potential fluorescence assays demonstrated that the K_V3.1-A₆K proteoliposomes, which were reconstructed with surfactant peptide A₆K, exhibited remarkable potassium ion permeability. Conclusion This study successfully achieved high-yield expression of human K_V3.1 with activity using an Escherichia coli-based cell-free protein synthesis system. This innovative method not only significantly enhances the expression yield of K_V3.1, but also maintains its functional activity, thereby establishing a novel and efficient synthetic platform for drug screening and advancing our understanding of structure-function relationships in K_V3.1 research.
Humans ; Escherichia coli/metabolism* ; Shaw Potassium Channels/biosynthesis* ; Cell-Free System ; Circular Dichroism ; Protein Biosynthesis ; Recombinant Proteins/metabolism* ; Membrane Potentials ; Shab Potassium Channels

Recent studies have revealed a significant relationship between erectile dysfunction (ED) and lower urinary tract symptoms (LUTS), both of which commonly affect middle-aged and older men. These conditions share underlying causes, particularly endothelial dysfunction, atherosclerosis, and chronic pelvic ischemia (CPI). This study investigated the therapeutic potential of LDD175, a large-conductance Ca 2+ -activated K + channel (BKCa channel) opener, in simultaneously treating both conditions using a CPI animal model of male Sprague Dawley rats. Our study investigated the induction of CPI through surgical endothelial damage combined with a high-cholesterol diet. We assessed erectile and voiding functions by measuring intracavernosal pressure (ICP) and intraurethral pressure (IUP), respectively, after nerve stimulation. We performed histological examinations of vascular changes and western blot analyses of cavernous and prostate tissues to understand the underlying mechanisms. This study evaluated the effectiveness of LDD175 compared to standard treatments, such as sildenafil for ED and tamsulosin for LUTS. Therefore, the CPI model successfully demonstrated ED and LUTS symptoms with decreased ICP and increased IUP. Analysis revealed elevated levels of hypoxia-inducible factor-1α, transforming growth factor-β1 and β2 in cavernous tissue, and increased α1A-adrenoceptor expression in prostate tissue. LDD175 administration showed promising results, with dose-dependent improvements in ICP and IUP, and therapeutic effects comparable to those of established treatments. Our findings suggest a novel therapeutic approach that can simultaneously address ED and LUTS, opening new possibilities for clinical application in the treatment of these interconnected conditions.
Male ; Animals ; Erectile Dysfunction/etiology* ; Rats, Sprague-Dawley ; Lower Urinary Tract Symptoms/etiology* ; Ischemia/drug therapy* ; Rats ; Tamsulosin ; Hypoxia-Inducible Factor 1, alpha Subunit/drug effects* ; Sildenafil Citrate/therapeutic use* ; Penis/blood supply* ; Disease Models, Animal ; Transforming Growth Factor beta1/metabolism* ; Pelvis/blood supply* ; Prostate/metabolism* ; Sulfonamides/therapeutic use* ; Large-Conductance Calcium-Activated Potassium Channels/agonists*

The prelimbic cortex (PL) plays a critical role in processing both the sensory and affective components of pain. However, the underlying molecular mechanisms remain poorly understood. In this study, we observed a reduction in hyperpolarization-activated cation current (I_h) in layer V pyramidal neurons of the contralateral PL in a mouse model of spared nerve injury (SNI). The expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channels was also decreased in the contralateral PL. Conversely, microinjection of fisetin, a partial agonist of HCN2, produced both analgesic and anxiolytic effects. Additionally, we found that cyclin-dependent kinase 5 (CDK5) was activated in the contralateral PL, where it formed a complex with HCN2 and phosphorylated its C-terminus. Knockdown of CDK5 restored HCN2 expression and alleviated both pain hypersensitivity and anxiety-like behaviors. Collectively, these results indicate that CDK5-mediated dysfunction of HCN2 in the PL underlies nerve injury-induced mechanical hypersensitivity and anxiety.
Animals ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Hyperalgesia/metabolism* ; Cyclin-Dependent Kinase 5/metabolism* ; Neuralgia/metabolism* ; Male ; Anxiety/metabolism* ; Mice ; Potassium Channels/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Pyramidal Cells/metabolism*

Two-pore-domain potassium channels (K2P) family is widely expressed in many human cell types and organs, which has important regulatory effect on physiological processes. K2P is sensitive to a variety of chemical and physical stimuli, and they have also been critically implicated in transmission of neural signal, ion homeostasis, cell development and death, and synaptic plasticity. Aberrant expression and dysfunction of K2P channels are involved in a range of diseases, including autoimmune, central nervous system, cardiovascular disease and others. The scope of this review is to give a detailed overview of the structure, function, pharmacological regulation, and related diseases of TREK-1 channels, a member of the K2P family.
Potassium Channels, Tandem Pore Domain/genetics* ; Humans ; Animals ; Cardiovascular Diseases/physiopathology* ; Autoimmune Diseases/metabolism* ; Central Nervous System Diseases/physiopathology*

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*

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*

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.
Osteogenesis ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; Large-Conductance Calcium-Activated Potassium Channels/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Calcium/metabolism* ; Cell Differentiation ; RNA, Messenger/metabolism* ; Cell Proliferation ; Osteoblasts

Arrhythmia is an external manifestation of cardiac electrophysiological disorder. It exists in healthy people and patients with various heart diseases, which is often associated with other cardiovascular diseases. The contraction and diastole of myocardium are inseparable from the movement of ions. There are many ion channels in the membrane and organelle membrane of myocardium. The dynamic balance of myocardial ions is vital in maintaining myocardial electrical homeostasis. Potassium ion channels that have a complex variety and a wide distribution are involved in the whole process of resting potential and action potential of cardiomyocytes. Potassium ion channels play a vital role in maintaining normal electrophysiological activity of myocardium and is one of the pathogenesis of arrhythmia. Traditional Chinese medicine(TCM)has unique advantages in treating arrhythmia for its complex active components and diverse targets. A large number of TCM preparations have definite effect on treating arrhythmia-related diseases, whose antiarrhythmic mechanism may be related to the effect on potassium channel. This article mainly reviewed the relevant studies on the active components in TCM acting on different potassium channels to provide references for clinical drug use and development.
Humans ; Potassium Channels ; Medicine, Chinese Traditional ; Anti-Arrhythmia Agents/therapeutic use* ; Arrhythmias, Cardiac/drug therapy* ; Heart Diseases/drug therapy* ; Ions

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases