• MeSH
• Potassium metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials radiation effects   MeSH
• Light MeSH
• Trichoderma metabolism   radiation effects   MeSH

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

• MeSH
• Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Neoplasms drug therapy   MeSH
• Neural Stem Cells metabolism   MeSH
• Cell Proliferation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

During GABAergic synaptic transmission, G protein-coupled GABAB receptors (GBRs) activate K+ channels that prolong the duration of inhibitory postsynaptic potentials (IPSPs). We now show that KCTD16, an auxiliary GBR subunit, anchors hyperpolarization-activated cyclic nucleotide-gated (HCN) channels containing HCN2/HCN3 subunits to GBRs. In dopamine neurons of the VTA (DAVTA neurons), this interaction facilitates activation of HCN channels via hyperpolarization during IPSPs, counteracting the GBR-mediated late phase of these IPSPs. Consequently, disruption of the GBR/HCN complex in KCTD16-/- mice leads to prolonged optogenetic inhibition of DAVTA neuron firing. KCTD16-/- mice exhibit increased anxiety-like behavior in response to stress - a behavior replicated by CRISPR/Cas9-mediated KCTD16 ablation in DAVTA neurons or by intra-VTA infusion of an HCN antagonist in wild-type mice. Our findings support that the retention of HCN channels at GABAergic synapses by GBRs in DAVTA neurons provides a negative feedback mechanism that restricts IPSP duration and mitigates the development of anxiety.

• MeSH
• Dopaminergic Neurons * metabolism   MeSH
• Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels * metabolism   genetics   MeSH
• Inhibitory Postsynaptic Potentials physiology   drug effects   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Receptors, GABA-B * metabolism   MeSH
• Ventral Tegmental Area * metabolism   MeSH
• Anxiety * metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca(2+) that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential. We show that low concentrations of amiodarone (0.1-2 microm) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 microm), hyperpolarization is transient and is followed by depolarization, coincident with influx of Ca(2+) and H(+) and loss in cell viability. Proton and alkali metal cation transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Diminishment of membrane potential by glucose removal or addition of salts or in pma1, tok1Delta, ena1-4Delta, or nha1Delta mutants protected against drug toxicity, suggesting that initial hyperpolarization was important in the mechanism of antifungal activity. Furthermore, we show that the link between membrane hyperpolarization and drug toxicity is pH-dependent. We propose the existence of pH- and hyperpolarization-activated Ca(2+) channels in yeast, similar to those described in plant root hair and pollen tubes that are critical for cell elongation and growth. Our findings illustrate how membrane-active compounds can be effective microbicidals and may pave the way to developing membrane-selective agents.

• MeSH
• Amiodarone pharmacology   MeSH
• Financing, Organized MeSH
• Fluorescence MeSH
• Immunoprecipitation MeSH
• Ion Transport MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Saccharomyces cerevisiae physiology   drug effects   MeSH
• Check Tag
• Humans MeSH

The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation (HCN) channels play a potential role in the neurological basis underlying drug addiction. However, little is known about the role of HCN channels in methamphetamine (METH) abuse. In the present study, we examined the changes in working memory functions of METH re-exposed mice through Morris water maze test, and investigated the protein expression of HCN1 channels and potential mechanisms underlying the modulation of HCN channels by Western blotting analysis. Mice were injected with METH (1 mg/kg, i.p.) once per day for 6 consecutive days. After 5 days without METH, mice were re-exposed to METH at the same concentration. We found that METH re-exposure caused an enhancement of working memory, and a decrease in the HCN1 channels protein expression in both hippocampus and prefrontal cortex. The phosphorylated extracellular regulated protein kinase 1/2 (p-ERK1/2), an important regulator of HCN channels, was also obviously reduced in hippocampus and prefrontal cortex of mice with METH re-exposure. Meanwhile, acute METH exposure did not affect the working memory function and the protein expressions of HCN1 channels and p-ERK1/2. Overall, our data firstly showed the aberrant protein expression of HCN1 channels in METH re-exposed mice with enhanced working memory, which was probably related to the down-regulation of p-ERK1/2 protein expression.

• MeSH
• Down-Regulation drug effects   physiology   MeSH
• Potassium Channels biosynthesis   MeSH
• Hippocampus drug effects   metabolism   MeSH
• Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels antagonists & inhibitors   biosynthesis   MeSH
• Memory, Short-Term drug effects   physiology   MeSH
• Locomotion drug effects   physiology   MeSH
• Methamphetamine administration & dosage   toxicity   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Random Allocation MeSH
• Prefrontal Cortex drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: During heart development, it has been hypothesized that apoptosis of atrioventricular canal myocardium and replacement by fibrous tissue derived from the epicardium are imperative to develop a mature atrioventricular conduction. To test this, apoptosis was blocked using an established caspase inhibitor and epicardial growth was delayed using the experimental epicardial inhibition model, both in chick embryonic hearts. RESULTS: Chicken embryonic hearts were either treated with the peptide caspase inhibitor zVAD-fmk by intrapericardial injection in ovo (ED4) or underwent epicardial inhibition (ED2.5). Spontaneously beating embryonic hearts isolated (ED7-ED8) were then stained with voltage-sensitive dye Di-4-ANEPPS and imaged at 0.5-1 kHz. Apoptotic cells were quantified (ED5-ED7) by whole-mount LysoTracker Red and anti-active caspase 3 staining. zVAD-treated hearts showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from the right atrioventricular junction, a pattern never observed in control hearts. zVAD-treated hearts showed decreased numbers of apoptotic cells in the atrioventricular canal myocardium at ED7. Hearts with delayed epicardial outgrowth showed also increased immature activation patterns at ED7.5 and ED8.5. However, the ventricular activation always originated from the left atrioventricular junction. Histological examination showed no changes in apoptosis rates, but a diminished presence of atrioventricular sulcus tissue compared with controls. CONCLUSIONS: Apoptosis in the atrioventricular canal myocardium and controlled replacement of this myocardium by epicardially derived HCN4-/Trop1- sulcus tissue are essential determinants of mature ventricular activation pattern. Disruption can lead to persistence of accessory atrioventricular connections, forming a morphological substrate for ventricular pre-excitation. Developmental Dynamics 247:1033-1042, 2018. © 2018 Wiley Periodicals, Inc.

• MeSH
• Epithelial Cell Adhesion Molecule MeSH
• Apoptosis * MeSH
• Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MeSH
• Chick Embryo MeSH
• Pericardium * MeSH
• Pre-Excitation Syndromes etiology   MeSH
• Heart Conduction System physiopathology   MeSH
• Ventricular Remodeling * MeSH
• Atrial Remodeling * MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

• MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Child MeSH
• Adult MeSH
• Potassium Channels genetics   MeSH
• Electric Stimulation MeSH
• Epilepsy, Generalized genetics   MeSH
• Genetic Association Studies MeSH
• Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels genetics   MeSH
• Infant MeSH
• Spasms, Infantile genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Membrane Potentials genetics   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Models, Molecular MeSH
• Mutation genetics   MeSH
• Mutagenesis, Site-Directed methods   MeSH
• Child, Preschool MeSH
• Aged MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Astrocytes respond to ischemic brain injury by proliferation, the increased expression of intermediate filaments and hypertrophy, which results in glial scar formation. In addition, they alter the expression of ion channels, receptors and transporters that maintain ionic/neurotransmitter homeostasis. Here, we aimed to demonstrate the expression of Hcn1-4 genes encoding hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following focal cerebral ischemia (FCI) or global cerebral ischemia (GCI) and to characterize their functional properties. A permanent occlusion of the middle cerebral artery (MCAo) was employed to induce FCI in adult GFAP/EGFP mice, while GCI was induced by transient bilateral common carotid artery occlusion combined with hypoxia in adult rats. Using FACS, we isolated astrocytes from non-injured or ischemic brains and performed gene expression profiling using single-cell RT-qPCR. We showed that 2 weeks after ischemia reactive astrocytes express high levels of Hcn1-4 transcripts, while immunohistochemical analyses confirmed the presence of HCN1-3 channels in reactive astrocytes 5 weeks after ischemia. Electrophysiological recordings revealed that post-ischemic astrocytes are significantly depolarized, and compared with astrocytes from non-injured brains, they display large hyperpolarization-activated inward currents, the density of which increased 2-3-fold in response to ischemia. Their activation was facilitated by cAMP and their amplitudes were decreased by ZD7288 or low extracellular Na(+) concentration, suggesting that they may belong to the family of HCN channels. Collectively, our results demonstrate that regardless of the type of ischemic injury, reactive astrocytes express HCN channels, which could therefore be an important therapeutic target in poststroke therapy.

• MeSH
• Cyclic AMP pharmacology   MeSH
• Astrocytes drug effects   metabolism   MeSH
• Glial Fibrillary Acidic Protein genetics   metabolism   MeSH
• Ischemia pathology   MeSH
• Cyclic Nucleotide-Gated Cation Channels genetics   metabolism   MeSH
• Rats MeSH
• Membrane Potentials drug effects   physiology   MeSH
• Disease Models, Animal MeSH
• Brain cytology   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Neurons drug effects   metabolism   MeSH
• Rats, Wistar MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Pyrimidines pharmacology   MeSH
• Gene Expression Regulation drug effects   physiology   MeSH
• Sodium metabolism   MeSH
• In Vitro Techniques MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.

• MeSH
• Cell Membrane metabolism   MeSH
• Potassium metabolism   MeSH
• Potassium Channels genetics   metabolism   MeSH
• Cations metabolism   MeSH
• Membrane Potentials MeSH
• Sodium-Hydrogen Exchangers metabolism   MeSH
• Cation Transport Proteins metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   physiology   MeSH
• Sodium-Potassium-Exchanging ATPase metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Endothelium, Vascular MeSH
• Endothelium-Dependent Relaxing Factors MeSH
• Endothelins * MeSH
• Blood Flow Velocity MeSH
• Muscle, Smooth, Vascular MeSH
• Vasodilator Agents MeSH
• Publication type
• Congress MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• angiologie
• NML Publication type
• kolektivní monografie