The Fusicoccin story revisited.

In plant biology Fusicoccin (FC) is one of the most studied fungal metabolites to date. Since the structural identification in 1964, much has been learned about its effects on the physiology of plants, about the interference with the action of plant hormones, the molecular nature of the plant receptor(s) for FC and the biosynthetic pathway for FC in the fungus. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase|14-3-3 receptor has taken center stage in explaining all FC induced physiological effects. However, a more critical review shows that this is not at all evident for a number of FC induced effects. Examples of this are: the inhibition of outward rectifying K+-channels in guard cells, the phosphorylation/activation of PEP-carboxylase and malate accumulation, the antagonism with ABA induced production of H2O2 / NO and the effect on ethylene production. In addition, recently two other physiological processes were shown to be targeted by FC, viz. the activation of TORC1 and the interference of FC with the immune response to fungal elicitors. In this review, the notion will be challenged that all FC affected processes start with the binding to and activation of the PM-ATPase and the question is raised whether may be other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule; in analogy to the fungal product and plant hormone gibberellic acid. A relevant question in this respect is whether it is a coincidence that proteins that act in a coordinated fashion during stomatal opening (the ATPases and K+-channels) are targeted by FC? Or are the sites where FC binds in the plant, conserved during evolution because they serve a physiological role, namely the accommodation of a plant produced molecule? In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile to make a renewed attempt with current day improved technology to answer this question and may be upgrade FC or structural analogue(s) to a new level, the level of plant hormone.

Alignment of lyotropic liquid crystals using magnetic nanoparticles improves ionic transport through built-in peptide ion channels.

HYPOTHESIS: We hypothesize that simultaneous incorporation of ion channel peptides (in this case, potassium channel as a model) and hydrophobic magnetite Fe3O4 nanoparticles (hFe3O4NPs) within lipidic hexagonal mesophases, and aligning them using an external magnetic field can significantly enhance ion transport through lipid membranes. EXPERIMENTS: In this study, we successfully characterized the incorporation of gramicidin membrane ion channels and hFe3O4NPs in the lipidic hexagonal structure using SAXS and cryo-TEM methods. Additionally, we thoroughly investigated the conductive characteristics of freestanding films of lipidic hexagonal mesophases, both with and without gramicidin potassium channels, utilizing a range of electrochemical techniques, including impedance spectroscopy, normal pulse voltammetry, and chronoamperometry. FINDINGS: Our research reveals a state-of-the-art breakthrough in enhancing ion transport in lyotropic liquid crystals as matrices for integral proteins and peptides. We demonstrate the remarkable efficacy of membranes composed of hexagonal lipid mesophases embedded with K+ transporting peptides. This enhancement is achieved through doping with hFe3O4NPs and exposure to a magnetic field. We investigate the intricate interplay between the conductive properties of the lipidic hexagonal structure, hFe3O4NPs, gramicidin incorporation, and the influence of Ca2+ on K+ channels. Furthermore, our study unveils a new direction in ion channel studies and biomimetic membrane investigations, presenting a versatile model for biomimetic membranes with unprecedented ion transport capabilities under an appropriately oriented magnetic field. These findings hold promise for advancing membrane technology and various biotechnological and biomedical applications of membrane proteins.

In vivo and in silico elucidation of possible potential and mechanisms involved in the analgesic action of ethanolic extract of Lavandula Stoechas.

OBJECTIVES: Our research focused on plant's ethanolic extract Lavandula stoechas flower part to investigate the potential analgesic effects and possible pathways involvements. METHODS: Four experimental tests were performed on Swiss albino mice with five animals in each group at different doses (50, 100, and 200mg/kg); formalin test, tail-flick test, acetic acid-induced writhing, and hot-plate test. The opioidergic, noradrenergic, cholinergic, and K channel blockers in the analgesic actions were also carried out for the potential route involvement. KEY FINDING: The percentage inhibition for abdominal writhing's and formalin activity showed a dose-dependent manner for early and late phases reducing abdominal writhing's and time period of licking, respectively. Tail immersion and hot-plate test demonstrated a substantial and dose-dependent increase in the latency time and time period of paw liking and jumping response respectively. GC-MS showed the abundantly present compounds were octadecatrienoic acid (34.35%), n-hexadecanoic acid (12.98%). In silico analyses have revealed three compounds that had good interactions with 6y3c receptor proteins, demonstrating strong binding affinities and satisfying docking parameters. CONCLUSIONS: Overall, these studies showed that ethanolic extract of L. stoechas is an important medicinal plant, with both central and peripheral antinociceptive and analgesic activities supporting its traditional use for therapeutic purposes.

Expression levels of KATP channel subunits and morphological changes in the mouse liver after exposure to radiation.

BACKGROUND: ATP sensitive K+ (KATP) channels are ubiquitously distributed in various of cells and tissues, including the liver. They play a role in the pathogenesis of myocardial and liver ischemia. AIM: To evaluate the radiation-induced changes in the expression of KATP channel subunits in the mouse liver to understand the potential role of KATP channels in radiation injury. METHODS: Adult C57BL/6 mice were randomly exposed to γ-rays at 0 Gy (control, n = 2), 0.2 Gy (n = 6), 1 Gy (n = 6), or 5 Gy (n = 6). The livers were removed 3 and 24 h after radiation exposure. Hematoxylin and eosin staining was used for morphological observation; immunohistochemical staining was applied to determine the expression of KATP channel subunits in the liver tissue. RESULTS: Compared with the control group, the livers exposed to 0.2 Gy γ-ray showed an initial increase in the expression of Kir6.1 at 3 h, followed by recovery at 24 h after exposure. Exposure to a high dose of 5.0 Gy resulted in decreased expression of Kir6.1 and increased expression of SUR2B at 24 h. However, the expression of Kir6.2, SUR1, or SUR2A had no remarkable changes at 3 and 24 h after exposure to any of these doses. CONCLUSION: The expression levels of Kir6.1 and SUR2B in mouse liver changed differently in response to different radiation doses, suggesting a potential role for them in radiation-induced liver injury.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Analysis of cardiohemodynamic and electrophysiological effects of morphine along with its toxicokinetic profile using the halothane-anesthetized dogs.

Although morphine has been used for treatment-resistant dyspnea in end-stage heart failure patients, information on its cardiovascular safety profile remains limited. Morphine was intravenously administered to halothane-anesthetized dogs (n=4) in doses of 0.1, 1 and 10 mg/kg/10 min with 20 min of observation period. The low and middle doses attained therapeutic (0.13 µg/mL) and supratherapeutic (0.97 µg/mL) plasma concentrations, respectively. The low dose hardly altered any of the cardiovascular variables except that the QT interval was prolonged for 10-15 min after its start of infusion. The middle dose reduced the preload and afterload to the left ventricle for 5-15 min, then decreased the left ventricular contractility and mean blood pressure for 10-30 min, and finally suppressed the heart rate for 15-30 min. Moreover, the middle dose gradually but progressively prolonged the atrioventricular conduction time, QT interval/QTcV, ventricular late repolarization period and ventricular effective refractory period without altering the intraventricular conduction time, ventricular early repolarization period or terminal repolarization period. A reverse-frequency-dependent delay of ventricular repolarization was confirmed. The high dose induced cardiohemodynamic collapse mainly due to vasodilation in the initial 2 animals by 1.9 and 3.3 min after its start of infusion, respectively, which needed circulatory support to treat. The high dose was not tested further in the remaining 2 animals. Thus, intravenously administered morphine exerts a rapidly appearing vasodilator action followed by slowly developing cardiosuppressive effects. Morphine can delay the ventricular repolarization possibly through IKr inhibition in vivo, but its potential to develop torsade de pointes will be small.

Loss of protein phosphatase 2A regulatory subunit PPP2R5A is associated with increased incidence of stress-induced proarrhythmia.

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. Methods: The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). Results: ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. Conclusion: These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

Electrophysiological Effects of the Sodium-Glucose Co-Transporter-2 (SGLT2) Inhibitor Dapagliflozin on Human Cardiac Potassium Channels.

The sodium-glucose co-transporter-2 (SGLT2) inhibitor dapagliflozin is increasingly used in the treatment of diabetes and heart failure. Dapagliflozin has been associated with reduced incidence of atrial fibrillation (AF) in clinical trials. We hypothesized that the favorable antiarrhythmic outcome of dapagliflozin use may be caused in part by previously unrecognized effects on atrial repolarizing potassium (K+) channels. This study was designed to assess direct pharmacological effects of dapagliflozin on cloned ion channels Kv11.1, Kv1.5, Kv4.3, Kir2.1, K2P2.1, K2P3.1, and K2P17.1, contributing to IKur, Ito, IKr, IK1, and IK2P K+ currents. Human channels coded by KCNH2, KCNA5, KCND3, KCNJ2, KCNK2, KCNK3, and KCNK17 were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using the voltage clamp technique. Dapagliflozin (100 µM) reduced Kv11.1 and Kv1.5 currents, whereas Kir2.1, K2P2.1, and K2P17.1 currents were enhanced. The drug did not significantly affect peak current amplitudes of Kv4.3 or K2P3.1 K+ channels. Biophysical characterization did not reveal significant effects of dapagliflozin on current-voltage relationships of study channels. In conclusion, dapagliflozin exhibits direct functional interactions with human atrial K+ channels underlying IKur, IKr, IK1, and IK2P currents. Substantial activation of K2P2.1 and K2P17.1 currents could contribute to the beneficial antiarrhythmic outcome associated with the drug. Indirect or chronic effects remain to be investigated in vivo.

Research Progress on Plant Shaker K+ Channels.

Plant growth and development are driven by intricate processes, with the cell membrane serving as a crucial interface between cells and their external environment. Maintaining balance and signal transduction across the cell membrane is essential for cellular stability and a host of life processes. Ion channels play a critical role in regulating intracellular ion concentrations and potentials. Among these, K+ channels on plant cell membranes are of paramount importance. The research of Shaker K+ channels has become a paradigm in the study of plant ion channels. This study offers a comprehensive overview of advancements in Shaker K+ channels, including insights into protein structure, function, regulatory mechanisms, and research techniques. Investigating Shaker K+ channels has enhanced our understanding of the regulatory mechanisms governing ion absorption and transport in plant cells. This knowledge offers invaluable guidance for enhancing crop yields and improving resistance to environmental stressors. Moreover, an extensive review of research methodologies in Shaker K+ channel studies provides essential reference solutions for researchers, promoting further advancements in ion channel research.

Second-generation antipsychotic quetiapine blocks voltage-dependent potassium channels in coronary arterial smooth muscle cells.

Voltage-dependent K+ (Kv) channels play an important role in restoring the membrane potential to its resting state, thereby maintaining vascular tone. In this study, native smooth muscle cells from rabbit coronary arteries were used to investigate the inhibitory effect of quetiapine, an atypical antipsychotic agent, on Kv channels. Quetiapine showed a concentration-dependent inhibition of Kv channels, with an IC50 of 47.98 ± 9.46 µM. Although quetiapine (50 µM) did not alter the steady-state activation curve, it caused a negative shift in the steady-state inactivation curve. The application of 1 and 2 Hz train steps in the presence of quetiapine significantly increased the inhibition of Kv current. Moreover, the recovery time constants from inactivation were prolonged in the presence of quetiapine, suggesting that its inhibitory action on Kv channels is use (state)-dependent. The inhibitory effects of quetiapine were not significantly affected by pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors. Based on these findings, we conclude that quetiapine inhibits Kv channels in both a concentration- and use (state)-dependent manner. Given the physiological significance of Kv channels, caution is advised in the use of quetiapine as an antipsychotic due to its potential side effects on cardiovascular Kv channels.

Genome-wide identification of Shaker K+ channel family in Nicotiana tabacum and functional analysis of NtSKOR1B in response to salt stress.

Soil salinization poses a mounting global ecological and environmental threat. The identification of genes responsible for negative regulation of salt tolerance and their utilization in crop improvement through gene editing technologies emerges as a swift strategy for the effective utilization of saline-alkali lands. One efficient mechanism of plant salt tolerance is maintaining the proper intracellular K+/Na+ ratio. The Shaker K+ channels play a crucial role in potassium absorption, transport, and intracellular potassium homeostasis in plant cells. Here, the study presents the first genome-wide identification of Shaker K+ channels in Nicotiana tabacum L., along with a detailed bioinformatic analysis of the 20 identified members. Transcriptome analysis revealed a significant up-regulation of NtSKOR1B, an outwardly-rectifying member predominantly expressed in the root tissue of tobacco seedlings, in response to salt stress. This finding was then confirmed by GUS staining of ProNtSKOR1B::GUS transgenic lines and RT-qPCR analysis. Subsequently, NtSKOR1B knockout mutants (ntskor1) were then generated and subjected to salt conditions. It was found that ntskor1 mutants exhibit enhanced salt tolerance, characterized by increased biomass, higher K+ content and elevated K+/Na+ ratios in both leaf and root tissues, compared to wild-type plants. These results indicate that NtSKOR1B knockout inhibits K+ efflux in root and leaf tissues of tobacco seedlings under salt stress, thereby maintaining higher K+/Na+ ratios within the cells. Thus, our study identifies NtSKOR1B as a negative regulator of salt tolerance in tobacco seedlings.

FLRT3 and TGF-ß/SMAD4 signalling: Impacts on apoptosis, autophagy and ion channels in supraventricular tachycardia.

To explore the underlying molecular mechanisms of supraventricular tachycardia (SVT), this study aimed to analyse the complex relationship between FLRT3 and TGF-ß/SMAD4 signalling pathway, which affects Na+ and K+ channels in cardiomyocytes. Bioinformatics analysis was performed on 85 SVT samples and 15 healthy controls to screen overlapping genes from the key module and differentially expressed genes (DEGs). Expression profiling of overlapping genes, coupled with Receiver Operating Characteristic (ROC) curve analyses, identified FLRT3 as a hub gene. In vitro studies utilizing Ang II-stimulated H9C2 cardiomyocytes were undertaken to elucidate the consequences of FLRT3 silencing on cardiomyocyte apoptosis and autophagic processes. Utilizing a combination of techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting (WB), flow cytometry, dual-luciferase reporter assays and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were conducted to decipher the intricate interactions between FLRT3, the TGF-ß/SMAD4 signalling cascade and ion channel gene expression. Six genes (AADAC, DSC3, FLRT3, SYT4, PRR9 and SERTM1) demonstrated reduced expression in SVT samples, each possessing significant clinical diagnostic potential. In H9C2 cardiomyocytes, FLRT3 silencing mitigated Ang II-induced apoptosis and modulated autophagy. With increasing TGF-ß concentration, there was a dose-responsive decline in FLRT3 and SCN5A expression, while both KCNIP2 and KCND2 expressions were augmented. Moreover, a direct interaction between FLRT3 and SMAD4 was observed, and inhibition of SMAD4 expression resulted in increased FLRT3 expression. Our results demonstrated that the TGF-ß/SMAD4 signalling pathway plays a critical role by regulating FLRT3 expression, with potential implications for ion channel function in SVT.

Exploring the Impact of BKCa Channel Function in Cellular Membranes on Cardiac Electrical Activity.

This review paper delves into the current body of evidence, offering a thorough analysis of the impact of large-conductance Ca2+-activated K+ (BKCa or BK) channels on the electrical dynamics of the heart. Alterations in the activity of BKCa channels, responsible for the generation of the overall magnitude of Ca2+-activated K+ current at the whole-cell level, occur through allosteric mechanisms. The collaborative interplay between membrane depolarization and heightened intracellular Ca2+ ion concentrations collectively contribute to the activation of BKCa channels. Although fully developed mammalian cardiac cells do not exhibit functional expression of these ion channels, evidence suggests their presence in cardiac fibroblasts that surround and potentially establish close connections with neighboring cardiac cells. When cardiac cells form close associations with fibroblasts, the high single-ion conductance of these channels, approximately ranging from 150 to 250 pS, can result in the random depolarization of the adjacent cardiac cell membranes. While cardiac fibroblasts are typically electrically non-excitable, their prevalence within heart tissue increases, particularly in the context of aging myocardial infarction or atrial fibrillation. This augmented presence of BKCa channels' conductance holds the potential to amplify the excitability of cardiac cell membranes through effective electrical coupling between fibroblasts and cardiomyocytes. In this scenario, this heightened excitability may contribute to the onset of cardiac arrhythmias. Moreover, it is worth noting that the substances influencing the activity of these BKCa channels might influence cardiac electrical activity as well. Taken together, the BKCa channel activity residing in cardiac fibroblasts may contribute to cardiac electrical function occurring in vivo.

Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Anji white tea relaxes precontracted arteries, represses voltage-gated Ca2+ channels and voltage-gated K+ channels in the arterial smooth muscle cells: Comparison with green tea main component (-)-epigallocatechin gallate.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea (Camellia sinensis) is a favorite drink worldwide. Tea extracts and green tea main component (-)-epigallocatechin gallate (EGCG) are recommended for various vascular diseases. Anji white tea is a very popular green tea. Its vascular effect profile, the mechanisms, and the contribution of EGCG to its integrated effect need elucidation. AIM: To characterize the vasomotion effects of Anji white tea and EGCG, and to explore possible involvement of voltage-gated Ca2+ channels (VGCCs) and voltage-gated K+ (Kv) channels in their vasomotion effects. MATERIALS AND METHODS: Anji white tea water soaking solution (AJWT) was prepared as daily tea-making process and concentrated to a concentration amounting to 200 mg/ml of dry tea leaves. The tension of rat arteries including aorta, coronary artery (RCA), cerebral basilar artery (CBA), intrarenal artery (IRA), intrapulmonary artery (IPA) and mesenteric artery (MA) was recorded with myographs. In arterial smooth muscle cells (ASMCs) freshly isolated from RCA, the levels of intracellular Ca2+ were measured with Ca2+-sensitive fluorescent probe fluo 4-AM, and Kv currents were recorded with patch clamp. The expressions of VGCCs and Kv channels were assayed with RT-qPCR and immunofluorescence staining. RESULTS: At 0.4-12.8 mg/ml of dry tea leaves, AJWT profoundly relaxed all tested arteries precontracted with various vasoconstrictors about half with a small transient potentiation on the precontractions before the relaxation. KCl-induced precontraction was less sensitive than precontractions induced by phenylephrine (PE), U46619 and serotonin (5-HT). IPA was less sensitive to the relaxation compared with other arteries. AJWT pretreatment for 1 h, 24 h and 72 h time-dependently inhibited the contractile responses of RCAs. In sharp contrast, at equivalent concentrations according to its content in AJWT, EGCG intensified the precontractions in most small arteries, except that it induced relaxation in PE-precontracted aorta and MA, U46619-precontracted aorta and CBA. EGCG pretreatment for 1 h and 24 h did not significantly affect RCA contractile responses. In RCA ASMCs, AJWT reduced, while EGCG enhanced, intracellular Ca2+ elevation induced by depolarization which activates VGCCs. Patch clamp study showed that both AJWT and EGCG reduced Kv currents. RT-qPCR and immunofluorescence staining demonstrated that both AJWT and EGCG reduced the expressions of VGCCs and Kv channels. CONCLUSION: AJWT, but not EGCG, consistently induces vasorelaxation. The vasomotion effects of either AJWT or EGCG vary with arterial beds and vasoconstrictors. Modulation of VGCCs, but not Kv channels, contributes to AJWT-induced vasorelaxation. It is suggested that Anji white tea water extract instead of EGCG may be a promising food supplement for vasospastic diseases.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Kinetics and functional consequences of BK channels activation by N-type Ca2+ channels in the dendrite of mouse neocortical layer-5 pyramidal neurons.

The back-propagation of an action potential (AP) from the axon/soma to the dendrites plays a central role in dendritic integration. This process involves an intricate orchestration of various ion channels, but a comprehensive understanding of the contribution of each channel type remains elusive. In this study, we leverage ultrafast membrane potential recordings (Vm) and Ca2+ imaging techniques to shed light on the involvement of N-type voltage-gated Ca2+ channels (VGCCs) in layer-5 neocortical pyramidal neurons' apical dendrites. We found a selective interaction between N-type VGCCs and large-conductance Ca2+-activated K+ channels (BK CAKCs). Remarkably, we observe that BK CAKCs are activated within a mere 500 µs after the AP peak, preceding the peak of the Ca2+ current triggered by the AP. Consequently, when N-type VGCCs are inhibited, the early broadening of the AP shape amplifies the activity of other VGCCs, leading to an augmented total Ca2+ influx. A NEURON model, constructed to replicate and support these experimental results, reveals the critical coupling between N-type and BK channels. This study not only redefines the conventional role of N-type VGCCs as primarily involved in presynaptic neurotransmitter release but also establishes their distinct and essential function as activators of BK CAKCs in neuronal dendrites. Furthermore, our results provide original functional validation of a physical interaction between Ca2+ and K+ channels, elucidated through ultrafast kinetic reconstruction. This insight enhances our understanding of the intricate mechanisms governing neuronal signaling and may have far-reaching implications in the field.

Enhancing Human Cutaneous Wound Healing through Targeted Suppression of Large Conductance Ca2+-Activated K+ Channels.

The modulation of K+ channels plays a crucial role in cell migration and proliferation, but the effect of K+ channels on human cutaneous wound healing (CWH) remains underexplored. This study aimed to determine the necessity of modulating K+ channel activity and expression for human CWH. The use of 25 mM KCl as a K+ channel blocker markedly improved wound healing in vitro (in keratinocytes and fibroblasts) and in vivo (in rat and porcine models). K+ channel blockers, such as quinine and tetraethylammonium, aided in vitro wound healing, while Ba2+ was the exception and did not show similar effects. Single-channel recordings revealed that the Ba2+-insensitive large conductance Ca2+-activated K+ (BKCa) channel was predominantly present in human keratinocytes. NS1619, an opener of the BKCa channel, hindered wound healing processes like proliferation, migration, and filopodia formation. Conversely, charybdotoxin and iberiotoxin, which are BKCa channel blockers, dramatically enhanced these processes. The downregulation of BKCa also improved CWH, whereas its overexpression impeded these healing processes. These findings underscore the facilitative effect of BKCa channel suppression on CWH, proposing BKCa channels as potential molecular targets for enhancing human cutaneous wound healing.

The apical 70-pS potassium K channel in the thick ascending limb of Henle's loop is a large-conductance Na+-and Cl--activated, KNa1.1-like, channel.

Apical potassium channels are crucial for thick ascending limb (TAL) of Henle's loop transport function. The ROMK (KNCJ1) gene encodes a 30-pS K channel whose loss of function causes the reduced NaCl reabsorption in the TAL associated with Type 2 Bartter's syndrome. In contrast, the molecular basis of a functionally ROMK-related 70-pS K channel is still unclear. The aim of this study was to highlight new specific channel properties that may give insights on its molecular identity. Using the patch-clamp technique on the apical membrane of mouse split-open TAL tubules, we observed that 70-pS K channel activity, but not ROMK channel activity, increases with the internal Na+ and Cl- concentrations, with relative 50 % effective concentrations (EC50) and Hill coefficients (nH) of 40.6 mM (SD 1.65) and 2.4 (SD 0.28) for Na+, and of 29.3 mM (SD 2.35) and 2.2 (SD 0.39) for Cl-. Conversely, 70-pS K channel activity was inhibited by internal K+ with a relative EC50 of 64 mM (SD 13.5) and a nH of 3.5 (SD 2.3), and by internal NH4+ and Ca2+. The reevaluation of channel conductive properties revealed an actual inward conductance of ~ 170 pS, with multiple subconductance levels and an inward rectification, and a substantial permeability to NH4+ ( = 0.2). We conclude that the apical 70-pS K channel in TAL cells is a large-conductance Na+- and Cl--activated potassium channel functionally resembling a KNa1.1 channel and propose that ROMK determines its functional expression possibly at the level of channel protein synthesis or trafficking.

Inhibition of voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells by the atypical antipsychotic agent sertindole.

The regulation of membrane potential and the contractility of vascular smooth muscle cells (VSMCs) by voltage-dependent K+ (Kv) potassium channels are well-established. In this study, native VSMCs from rabbit coronary arteries were used to investigate the inhibitory effect of sertindole, an atypical antipsychotic agent, on Kv channels. Sertindole induced dose-dependent inhibition of Kv channels, with an IC50 of 3.13 ± 0.72 µM. Although sertindole did not cause a change in the steady-state activation curve, it did lead to a negative shift in the steady-state inactivation curve. The application of 1- or 2-Hz train pulses failed to alter the sertindole-induced inhibition of Kv channels, suggesting use-independent effects of the drug. The inhibitory response to sertindole was significantly diminished by pretreatment with a Kv1.5 inhibitor but not by Kv2.1 and Kv7 subtype inhibitors. These findings demonstrate the sertindole dose-dependent and use-independent inhibition of vascular Kv channels (mainly the Kv1.5 subtype) through a mechanism that involves altering steady-state inactivation curves. Therefore, the use of sertindole as an antipsychotic drug may have adverse effects on the cardiovascular system.