Potassium Channel Function Research Articles

Exercise augments small conductance Ca2+ -activated potassium channel (SK) function in the paraventricular nucleus (PVN) of Sprague Dawley rats to reduce sympathetic outflow

Elevated sympathetic outflow is a key feature of cardiovascular disease (CVD) that worsens disease progression. Our lab has shown that SK channels expressed in the PVN play a crucial role in regulating neuronal activity and sympathetic outflow, and that SK channels become dysfunctional in rats fed a high salt diet. Exercise has been shown to be an effective treatment for reducing sympathoexcitation in CVD including hypertension and heart failure, but the underlying mechanisms are not fully understood. We hypothesized that aerobic exercise would upregulate SK channel function in the PVN to reduce sympathetic nerve activity (SNA). To test this, 5–6 week old Sprague Dawley rats were randomly divided into sedentary (SED) and exercise (EXT) two groups and fed a 0.4% NaCl normal salt diet. Following acclimation, EXT groups ran on a motorized treadmill 5 days/week for 8-10 weeks. Conscious blood pressure was measured weekly via tail plethysmography. After 8-10 weeks, animals were anesthetized and underwent in vivo surgery to record the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) following PVN microinjection of the SK channel blocker, apamin (0.25mM, 60nL/side). The data showed that the RSNA response to PVN apamin was significantly enhanced in EXT rats compared with SED rats (320.8 ± 174.6 % baseline, n=9 vs 184.8 ± 143.1 % baseline, n=9; p = 0.02). The corresponding ABP response to apamin was not significantly different in EXT rats compared with SED rats (20.40 ± 9.98 mmHg, n=9 vs 25.27 ± 9.97 mmHg, n=8; p = 0.1658). Our data indicates exercise enhances PVN SK channel function to reduce sympathetic outflow. This improvement of SK channel function may be one mechanism by which exercise reduces SNA in CVD including hypertension and heart failure. Support: 1R15HL145655 (Chen); 1R15 HL150703 (Shan); MTU Health Research Institute. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potassium Ion Channels in Glioma: From Basic Knowledge into Therapeutic Applications.

Ion channels, specifically those controlling the flux of potassium across cell membranes, have recently been shown to exhibit an important role in the pathophysiology of glioma, the most common primary central nervous system tumor with a poor prognosis. Potassium channels are grouped into four subfamilies differing by their domain structure, gating mechanisms, and functions. Pertinent literature indicates the vital functions of potassium channels in many aspects of glioma carcinogenesis, including proliferation, migration, and apoptosis. The dysfunction of potassium channels can result in pro-proliferative signals that are highly related to calcium signaling as well. Moreover, this dysfunction can feed into migration and metastasis, most likely by increasing the osmotic pressure of cells allowing the cells to initiate the "escape" and "invasion" of capillaries. Reducing the expression or channel blockage has shown efficacy in reducing the proliferation and infiltration of glioma cells as well as inducing apoptosis, priming several approaches to target potassium channels in gliomas pharmacologically. This review summarizes the current knowledge on potassium channels, their contribution to oncogenic transformations in glioma, and the existing perspectives on utilizing them as potential targets for therapy.

Novel Loss of Function KCNA5 Variants in Pulmonary Arterial Hypertension.

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in PAH patients, but their functional consequences and potential impact on the disease are largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC). KCNA5 variants found in PAH patients (namely, p.Arg184Pro and p.Gly384Arg) resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modelling analyses. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1 %. Our data indicate that some KCNA5 variants found PAH patients have critical consequences for channel function supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

Iberiotoxin and clofilium regulate hyperactivation, acrosome reaction, and ion homeostasis synergistically during human sperm capacitation.

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K+ currents in human sperm (IKSper ) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca2+ , K+ , Cl- , and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K+ ]i , [Cl- ]i , and pHi , but a decrease in [Ca2+ ]i . Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K+ ]i , [Cl- ]i , and pHi , and the decrease in [Ca2+ ]i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.

Electrochemical studies of the mitochondrial ROMK2 potassium channel activity reconstituted into the free-standing and tethered bilayer lipid membranes

The renal-outer-medullary‑potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K+ ions in the presence of channel modulators.

Functional Potassium Channels in Macrophages.

Macrophages are the predominant component of innate immunity, which is an important protective barrier of our body. Macrophages are present in all organs and tissues of the body, their main functions include immune surveillance, bacterial killing, tissue remodeling and repair, and clearance of cell debris. In addition, macrophages can present antigens to T cells and facilitate inflammatory response by releasing cytokines. Macrophages are of high concern due to their crucial roles in multiple physiological processes. In recent years, new advances are emerging after great efforts have been made to explore the mechanisms of macrophage activation. Ion channel is a class of multimeric transmembrane protein that allows specific ions to go through cell membrane. The flow of ions through ion channel between inside and outside of cell membrane is required for maintaining cell morphology and intracellular signal transduction. Expressions of various ion channels in macrophages have been detected. The roles of ion channels in macrophage activation are gradually caught attention. K+ channels are the most studied channels in immune system. However, very few of published papers reviewed the studies of K+ channels on macrophages. Here, we will review the four types of K+ channels that are expressed in macrophages: voltage-gated K+ channel, calcium-activated K+ channel, inwardly rectifying K+ channel and two-pore domain K+ channel.

Identification of cardiac-related serum miRNA in patients with type 2 diabetes mellitus and heart failure: Preliminary report

Diabetic patients present an increased risk for heart failure (HF) independently of the presence of coronary artery disease (CAD) and hypertension. However, little is known about circulatory microRNA (miRNA), an important regulatory RNA in this population. To evaluate serum miRNA profile of patients with diabetes mellitus (DM) and HF and analyze its relationship with pathophysiological pathways involved. The accumulation of 179 miRNAs was measured in serum of diabetic patients with HF and compared to the same measurements in healthy control subjects. The miRNAs were assayed using quantitative polymerase chain reaction (qPCR) on the Serum/Plasma Focus microRNA PCR panel (Qiagen) with LightCycler® 96 Real-Time PCR System (Roche). A pairwise comparison of mean relative miRNA accumulation levels was performed to establish those miRNAs that are differently expressed in patients with: 1) HF; 2) HF and chronic coronary syndrome (HF-CAD); and 3) HF without chronic coronary syndrome (HF-nonCAD) compared to healthy controls. To gain insight into these functions of miRNAs, we applied Gene Ontology (GO) enrichment analysis of Biological Processes and Molecular Functions of their predicted targets. The pairwise comparison revealed that 12 miRNAs were significantly downregulated in HF-CAD patients compared to controls, whereas 4 miRNAs were considerably deregulated in HF-nonCAD patients, with miRNA-15b-5p being downregulated in both groups. The GO analysis revealed that differentially accumulated targets of miRNAs include genes involved in potassium channel function, MAPK kinase activity and DNA transcription regulation, with similar alterations observed in the whole HF group and HF-CAD subgroup as well as a response to stress and apoptosis (in HF group), and genes involved in the development (in HF-CAD group). No oriented specialization of deregulated miRNA targets was observed in the HF-nonCAD subgroup. We observed a significant downregulation of 13 miRNAs in diabetic HF patients, which was not reported previously either in HF or diabetic patients. Downregulated miRNAs regulate angiogenesis and apoptosis.

Prenatal exposure to alcohol impairs responses of cerebral arterioles to activation of potassium channels: Role of oxidative stress.

Potassium channels play an important role in the basal tone and dilation of cerebral resistance arterioles in response to many stimuli. However, the effect of prenatal alcohol exposure (PAE) on specific potassium channel function remains unknown. The first goal of this study was to determine the influence of PAE on the reactivity of cerebral arterioles to activation of ATP-sensitive potassium (KATP ) and BK channels. Our second goal was to determine whether oxidative stress contributed to potassium channel dysfunction of cerebral arterioles following PAE. We fed Sprague-Dawley dams a liquid diet with or without alcohol (3% EtOH) for the duration of their pregnancy (21 to 23 days). We examined in vivo responses of cerebral arterioles in control and PAE male and female offspring (14 to 16 weeks after birth) to activators of potassium channels (Iloprost [BK channels] and pinacidil [KATP channels]), before and following inhibition of oxidative stress with apocynin. We found that PAE impaired dilation of cerebral arterioles in response to activation of potassium channels with iloprost and pinacidil, and this impairment was similar in male and female rats. In addition, treatment with apocynin reversed the impaired vasodilation to iloprost and pinacidil in PAE rats to levels observed in control rats. This effect of apocynin also was similar in male and female rats. PAE induces dysfunction in the ability of specific potassium channels to dilate cerebral arterioles which appears to be mediated by an increase in oxidative stress. We suggest that these alterations in potassium channel function may contribute to the pathogenesis of cerebral vascular abnormalities and/or behavioral/cognitive deficits observed in fetal alcohol spectrum disorders.

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction.

A large family of prototoxin-like molecules endogenous to mammals, Ly6 proteins have been implicated in the regulation of cell signaling processes across multiple species. Previous work has shown that certain members of the Ly6 family are expressed in the brain and target nicotinic acetylcholine receptor and potassium channel function. Structural similarities between Ly6 proteins and alpha-neurotoxins suggest the possibility of additional ionotropic receptor targets. Here, we investigated the possibility of lypd2 as a novel regulator of AMPA receptor (AMPAR) function. In particular, we focused on potential interactions with the Q/R isoforms of the GluR2 subunit, which have profound impacts on AMPAR permeability to calcium during neuronal stimulation. We find that although lypd2 and GluR2 share overlapping expression patterns in the mouse hippocampus, there was no interaction between lypd2 and either GluR2Q or GluR2R isoform. These results underscore the importance of continuing to investigate novel targets for Ly6 interaction and regulation.

Genetic and genomic signatures in ethanol withdrawal seizure-prone and seizure-resistant mice implicate genes involved in epilepsy and neuronal excitability.

Alcohol withdrawal is a clinically important consequence and potential driver of Alcohol Use Disorder. However, susceptibility to withdrawal symptoms, ranging from craving and anxiety to seizures and delirium, varies greatly. Selectively bred Withdrawal Seizure-Prone (WSP) and Seizure-Resistant (WSR) mice are an animal model of differential susceptibility to withdrawal and phenotypes with which withdrawal severity correlates. To identify innate drivers of alcohol withdrawal severity, we performed a multi-omic study of the WSP and WSR lines and F2 mice derived from them, using genomic, genetic, and transcriptomic analyses. Genes implicated in seizures and epilepsy were over-represented among those that segregated between WSP and WSR mice and that displayed differential expression in F2 mice high and low in withdrawal. Quantitative trait locus (QTL) analysis of ethanol withdrawal convulsions identified several genome-wide significant loci and pointed to genes that modulate potassium channel function and neural excitability. Perturbations of expression of genes involved in synaptic transmission, including GABAergic and glutamatergic genes, were prominent in prefrontal cortex transcriptome. Expression QTL (eQTL) analysis fine mapped genes within the peak ethanol withdrawal QTL regions. Genetic association analysis in human subjects provided converging evidence for the involvement of those genes in severity of alcohol withdrawal and dependence. Our results reveal a polygenic network and neural signaling pathways contributing to ethanol withdrawal seizures and related phenotypes that overlap with genes modulating epilepsy and neuronal excitability.

Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice.

DYT1 dystonia is an inherited early-onset movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, and abnormal postures. Most DYT1 patients have a heterozygous trinucleotide GAG deletion mutation (ΔGAG) in DYT1/TOR1A, coding for torsinA. Dyt1 heterozygous ΔGAG knock-in (KI) mice show motor deficits and reduced striatal dopamine receptor 2 (D2R). Striatal cholinergic interneurons (ChIs) are essential in regulating striatal motor circuits. Multiple dystonia rodent models, including KI mice, show altered ChI firing and modulation. However, due to the errors in assigning KI mice, it is essential to replicate these findings in genetically confirmed KI mice. Here, we found irregular and decreased spontaneous firing frequency in the acute brain slices from Dyt1 KI mice. Quinpirole, a D2R agonist, showed less inhibitory effect on the spontaneous ChI firing in Dyt1 KI mice, suggesting decreased D2R function on the striatal ChIs. On the other hand, a muscarinic receptor agonist, muscarine, inhibited the ChI firing in both wild-type (WT) and Dyt1 KI mice. Trihexyphenidyl, a muscarinic acetylcholine receptor M1 antagonist, had no significant effect on the firing. Moreover, the resting membrane property and functions of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, μ-opioid receptors, and large-conductance calcium-activated potassium (BK) channels were unaffected in Dyt1 KI mice. The results suggest that the irregular and low-frequency firing and decreased D2R function are the main alterations of striatal ChIs in Dyt1 KI mice. These results appear consistent with the reduced dopamine release and high striatal acetylcholine tone in the previous reports.

УРОВЕНЬ ТЕСТОСТЕРОНА СЫВОРОТКИ КРОВИ КАК ПРЕДИКТОР ЛЕКАРСТВЕННО-ИНДУЦИРОВАННОГО УДЛИНЕНИЯ ИНТЕРВАЛА QT И ПОЛИМОРФНОЙ ЖЕЛУДОЧКОВОЙ ТАХИКАРДИИ У ПАЦИЕНТОВ МУЖСКОГО ПОЛА, ПРИНИМАЮЩИХ АНТИАРИТМИЧЕСКИЕ СРЕДСТВА III КЛАССА

Background. A decrease in serum testosterone level in men is associated with QT interval prolongation on the standard ECG due to changes in the functioning of potassium and calcium ion channels. The scientific literature provides relatively little information on the combined effects of testosterone deficiency and intake of class III antiarrhythmic drugs on the process of myocardial repolarization and development of polymorphic ventricular arrhythmias associated with drug-induced QT prolongation (LQTS). The aim of this study was to establish the prognostic value of serum testosterone levels for the development of drug- induced QT interval prolongation and polymorphic ventricular tachycardia (PVT). Material and methods. To achieve this goal, 59 male patients were examined, mainly those with coronary heart disease, arterial hypertension and cardiac arrhythmias, 29 (49.2%) of which had drug-induced LQTS and 30 (50.8%) were with normal values of the QT interval while taking antiarrhythmic therapy. All patients underwent clinical, laboratory and instrumental studies, including the determination of serum testosterone levels. Results. The patients with drug-induced LQTS had lower testosterone levels compared to patients without LQTS (p<0.001), especially in the middle and older age groups according to the WHO age classification (p=0.009). An inverse correlation relationship (p<0.01) was found between the serum testosterone level and a number of electrocardiographic parameters, including the duration of the corrected QT interval (R=-0.56), the interval T peak - T end (R=-0.58) and corrected cardioelectrophysiological balance index (R=-0.43). Testosterone levels were lower in patients with drug-induced LQTS and PVT than in patients without PVT (p=0.031). The testosterone value ≤13.43 nmol/ml demonstrated high sensitivity (100%) and specificity (78.43%), as well as a fairly high area under the ROC curve (0.917) and can be used to predict non-sustained PVT in male patients taking amiodarone and sotalol (OR=5.50 [95% CI 3.14; 9.63]). Conclusions. Our data indicate an important pathophysiological role of testosterone in the genesis of drug-induced LQTS and PVT development in male patients. Given the small size of the study sample, the applicability of this indicator needs to be tested on a larger group of patients.

Comparing the structural dynamics of the human KCNE3 in reconstituted micelle and lipid bilayered vesicle environments

KCNE3 is a single transmembrane protein of the KCNE family that modulates the function and trafficking of several voltage-gated potassium channels, including KCNQ1. Structural studies of KCNE3 have been previously conducted in a wide range of model membrane mimics. However, it is important to assess the impact of the membrane mimics used on the observed conformation and dynamics. In this study, we have optimized a method for the reconstitution of the KCNE3 into POPC/POPG lipid bilayer vesicles for electron paramagnetic resonance (EPR) spectroscopy. Our CD spectroscopic data suggested that the degree of regular secondary structure for KCNE3 protein reconstituted into lipid bilayered vesicle is significantly higher than in DPC detergent micelles. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) was used to probe the structural dynamics of S49C, M59C, L67C, V85C, and S101C mutations of KCNE3 in both DPC micelles and in POPC/POPG lipid bilayered vesicles. Our CW-EPR power saturation data suggested that the site S74C is buried inside the lipid bilayered membrane while the site V85C is located outside the membrane, in contrast to DPC micelle results. These results suggest that the KCNE3 micelle structures need to be refined using data obtained in the lipid bilayered vesicles in order to ascertain the native structure of KCNE3. This work will provide guidelines for detailed structural studies of KCNE3 in a more native membrane environment and comparing the lipid bilayer results to the isotropic bicelle structure and to the KCNQ1-bound cryo-EM structure.

Large-conductance calcium-activated potassium channel haploinsufficiency leads to sensory deficits in the visual system: a case report

BackgroundMutations in the genes encoding the large-conductance calcium-activated potassium channel, especially KCNMA1 encoding its α-subunit, have been linked to several neurological features, including intellectual disability or autism. Associated with neurodevelopmental phenotypes, sensory function disturbances are considered to be important clinical features contributing to a variety of behavioral impairments. Large-conductance calcium-activated potassium channels are important in regulating neurotransmission in sensory circuits, including visual pathways. Deficits in visual function can contribute substantially to poor quality of life, while therapeutic approaches aimed at addressing such visual deficits represent opportunities to improve neurocognitive and neurobehavioral outcomes.Case presentationWe describe the case of a 25-year-old Caucasian male with autism spectrum disorder and severe intellectual disability presenting large-conductance calcium-activated potassium channel haploinsufficiency due to a de novo balanced translocation (46, XY, t [9; 10] [q23;q22]) disrupting the KCNMA1 gene. The visual processing pathway of the subject was evaluated using both electroretinography and visual contrast sensitivity, indicating that both retinal bipolar cell function and contrast discrimination performance were reduced by approximately 60% compared with normative control values. These findings imply a direct link between KCNMA1 gene disruption and visual dysfunction in humans. In addition, the subject reported photophobia but did not exhibit strabismus, nystagmus, or other visual findings on physical examination.ConclusionsThis case study of a subject with large-conductance calcium-activated potassium channel haploinsufficiency and photophobia revealed a visual pathway deficit at least at the retinal level, with diminished retinal light capture likely due to bipolar cell dysfunction and an associated loss of contrast sensitivity. The data suggest that large-conductance calcium-activated potassium channels play an important role in the normal functioning of the visual pathway in humans, and that their disruption may play a role in visual and other sensory system symptomatology in large-conductance calcium-activated potassium channelopathies or conditions where disruption of large-conductance calcium-activated potassium channel function is a relevant feature of the pathophysiology, such as fragile X syndrome. This work suggests that the combined use of physiological (electroretinography) and functional (contrast sensitivity) approaches may have utility as a biomarker strategy for identifying and characterizing visual processing deficits in individuals with large-conductance calcium-activated potassium channelopathy.Trial registration ID-RCB number 2019-A01015-52, registered 17/05/2019.

Role of Potassium Channel Function and Long‐term Exercise Training in Soleus Resistance Arterioles

IntroductionNuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) expression has been linked to potassium (K+) channel function. We hypothesized that myogenic responses are enhanced by exercise training and also modulated by K+ channel function but would be impaired in arterioles from skeletal muscle of trained rats in which Nrf2 (Nrf2 KO) is deleted.MethodsExercise training was performed in male wildtype (WT) Sprague‐Dawley rats, and their littermates lacking the Nrf2 gene (Nrf2 KO). Rats were exercise trained using a mixed moderate and high‐intensity interval treadmill program for 10‐12 weeks. A WT cage‐confined control group was also studied to compare the impact of exercise training. After exercise training or sedentary cage‐confinement, soleus resistance arterioles were isolated and cannulated for the assessment of vascular reactivity. Vasodilatory responses to low concentrations of KCl [10 and 20mM} were assessed. Vasoconstrictor responses to higher concentrations of KCl [30–100mM] and myogenic responses to increasing intraluminal pressure (pressure 0cm H2O – 130cm H2O) were also assessed. The contribution of inward rectifying K+ channels (Kir) to vasodilation during low concentrations of KCl was assessed by treatment with barium chloride (BaCl2, 100mM).ResultsSoleus resistance arteriole myogenic responsiveness was reduced in trained Nrf2 KO as compared to WT (p<0.01). Trained WT rats produced a significantly higher myogenic responsiveness than sedentary WT controls (p<0.05). Vasodilation to low concentrations of KCl was reduced in soleus arterioles from trained Nrf2 KO rats as compared to arterioles from trained WT rats (p<0.01). Exercise training did not alter vasodilation to low KCl concentrations in WT rats (p>0.05). Constriction to higher concentrations of KCl was similar in arterioles from trained WT and Nrf2 KO rats. Inhibiting Kir channels with BaCl2 eliminated the genotype‐ and training‐related differences in myogenic responses and vasodilation to KCl.ConclusionOur data indicate that Nrf2 may be a critical regulator of vascular adaptation that occurs in skeletal muscle arterioles in response to long‐term exercise training. Elimination of differences in myogenic responsiveness by BaCl2 suggest that Nrf2‐mediated adaptation of the myogenic responsiveness may involve Kir channels.

Cardiovascular toxic effects of antitumor agents: Pathogenetic mechanisms

Cancer treatment is associated with various side effects of antitumor agents, which increase the morbidity and mortality of these patients. Cardiovascular complications are considered to be one of the most important side effect of the anticancer drugs. The antitumor drugs that express cardiovascular effects include anthracyclines, tyrosine kinase inhibitors, taxanes, fluoropyrimidines, alkylating agents, vascular endothelial growth factor inhibitors, immune checkpoint inhibitors, proteasome inhibitors and human epidermal growth receptor type 2 antibodies. The spectrum of cardiovascular effects of anticancer drugs is broad and include, among others, heart failure, arrhythmias such as atrial fibrillation and ventricular tachyarrhythmias, hypertension (systemic or pulmonary), cardiomyopathy, myocarditis, valve disease, pericardial disease, vascular events (arterial thrombosis, venous thromboembolism) and myocardial ischemia (acute coronary syndrome, angina). The molecular mechanisms by which anti-cancer therapies lead to cardiotoxicity are diverse and vary according to the specific type of agent used. They include oxidative stress, topoisomerase 2-β inhibition in cardiomyocytes, inflammation, endothelial dysfunction, apoptosis, disruption of Ca2+ homeostasis, mitochondrial dysfunction, DNA damage, increase in various circulating microRNAs levels, alterations in the function of voltage-gated potassium channels. The management of cardiovascular complications in cancer patients is a new challenge for oncologists and cardiologists. Thus the cardio-oncology field has developed the last decade in order to precisely predict and efficiently treat the cancer treatment-related cardiovascular diseases.

A dual potassium channelopathy underlies small vessel disease of the brain in a mouse model of Alzheimer's Disease

Mounting clinical and basic evidence has suggested that cerebral microcirculatory dysfunction is key to the pathogenesis of Alzheimer’s disease (AD), thus posing a potential new therapeutic target for AD treatment. However, despite observations of altered cerebral blood flow and autoregulatory capacity in AD patients, the underlying pathogenic mechanisms behind these defects are currently unknown. Here we present the first detailed study in to vascular ion channel function in the APP23 mouse model of AD. This model has a seven‐fold increase in amyloid precursor protein leading to substantial amyloid‐β plaque accumulation within the brain, as is described in AD patients. Using a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high‐speed spinning disc confocal microscopy, we show that attenuation of large conductance calcium–activated potassium (BK) channel function in this model, due to a reduced frequency of calcium sparks, is responsible for the reduction in cerebral blood flow described. Further investigation into the role of amyloid‐β 1‐40, in particular, showed that vessels exposed to the peptide for 30 minutes exhibited significantly reduced BK channel function at both the cellular and vascular level. However, disparate from the APP23 mouse model, acute exposure to amyloid‐β 1‐40 resulted in an increase in calcium waves. Both a decrease in calcium sparks (as seen in the APP23 model) and an increase in calcium waves (as seen with acute application of the amyloid‐β 1‐40 peptide) results in contraction of cerebral arteries and a subsequent reduction of cerebral blood flow. The data directs future research in to preventative strategies that may help to stop alterations in calcium event initiation, restore microvasculature function and promote a healthy brain environment to limit disease progression in AD patients.

Functions of potassium channels blocked by low micromolar 4-aminopyridine in the crayfish nervous system.

4-aminopyridine (4-AP) is a potassium channel blocker that has been used to treat patients with multiple sclerosis and Lambert-Eaton disease. The concentration of this drug in the blood of patients was estimated to be in low or submicromolar range. Animal studies have shown that 4-AP at such low concentration selectively blocks a subset of channels in Kv1 or Kv3 families. The crayfish opener neuromuscular junction and ventral superficial flexor (VSF) preparations were used to examine functions of K+ channels blocked by low concentrations of 4-AP. At opener motor axons, intracellular recordings show that 4-AP could increase action potential (AP) amplitude, duration, and after-depolarization (ADP) at 10 μM. As 4-AP concentration was increased, in twofold steps, AP amplitude did not increase further up to 5mM. AP duration and ADP increased significantly mainly in two concentration ranges, 10-50 μM and 1-5mM. The effects of 50 μM 4-AP on the VSF were less consistent than that observed at the opener motor axons. 4-AP did not change AP amplitude of motor axons recorded with an extracellular electrode and change in AP repolarizing potential was observed in ∼25% of the axons. EPSP recorded simultaneously with AP showed an increase in amplitude with 4-AP treatment only in 30% of the axon-EPSP pairs. 4-AP also increased firing frequencies of ∼50% of axons. In four animals, 4-AP "awakened" the firing of APs from an axon that was silent before the drug. The mixture of positive and negative 4-AP effects summarized above was observed in the same VSF preparations in all cases (n=8). We propose that there is a significant diversity in the density 4-AP-sensitive potassium channels among motor axons of the VSF. Functional significance in the differences of 4-AP sensitivity of the two motor systems is discussed.

Discovery of Novel Activators of Large-Conductance Calcium-Activated Potassium Channels for the Treatment of Cerebellar Ataxia.

Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative spinocerebellar ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and are therefore poor templates for future drug development. We implemented an automated patch clamp platform for high-throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channel activators that were then retested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that exhibited a more potent half-maximal activation of BK current (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on small-conductance calcium-activated potassium (SK)1-3 channels but interestingly was a potent blocker of the T-type calcium channel, Cav3.1 (IC50 = 1.05 μM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. SIGNIFICANCE STATEMENT: Motor impairment associated with altered Purkinje cell spiking due to dysregulation of large-conductance calcium-activated potassium (BK) channel expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug development for the treatment of these disabling disorders.

An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.