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
• amiodaron farmakologie   MeSH
• fluorescence MeSH
• imunoprecipitace MeSH
• iontový transport MeSH
• lidé MeSH
• membránové proteiny * MeSH
• Saccharomyces cerevisiae účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• amiodaron MeSH
• membránové proteiny * MeSH

We report proton spin noise spectra of a hyperpolarized solid sample of commonly used "DNP (dynamic nuclear polarization) juice" containing TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine N-oxide) and irradiated by a microwave field at a temperature of 1.2 K in a magnetic field of 6.7 T. The line shapes of the spin noise power spectra are sensitive to the variation of the microwave irradiation frequency and change from dip to bump, when the electron Larmor frequency is crossed, which is shown to be in good accordance with theory by simulations. Small but significant deviations from these predictions are observed, which can be related to spin noise and radiation damping phenomena that have been reported in thermally polarized systems. The non-linear dependence of the spin noise integral on nuclear polarization provides a means to monitor hyperpolarization semi-quantitatively without any perturbation of the spin system by radio frequency irradiation.

• Klíčová slova
• dynamic nuclear polarization, non-linear effects, nuclear magnetic resonance, radiation damping, spin noise,
• Publikační typ
• časopisecké články MeSH

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.

• MeSH
• draslík metabolismus   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály účinky záření   MeSH
• světlo * MeSH
• Trichoderma metabolismus   účinky záření   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík 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.

• Klíčová slova
• GABA-A, GABA-B, HCN2, Hyperpolarization-activated cyclic nucleotide-gated channels, Late IPSP, Optogenetic inhibition, Slow IPSP,
• MeSH
• dopaminergní neurony * metabolismus   MeSH
• draslíkové kanály metabolismus   MeSH
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy * metabolismus   MeSH
• inhibiční postsynaptické potenciály fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nervový útlum * fyziologie   MeSH
• receptory GABA-B * metabolismus   MeSH
• tegmentum mesencephali - area ventralis * metabolismus   MeSH
• úzkost * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslíkové kanály MeSH
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy * MeSH
• receptory GABA-B * 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.

• Klíčová slova
• HCN channels, ZD7288, astrocytes, cortex, focal and global cerebral ischemia, hippocampus,
• MeSH
• AMP cyklický farmakologie   MeSH
• astrocyty účinky léků   metabolismus   MeSH
• gliový fibrilární kyselý protein genetika   metabolismus   MeSH
• ischemie patologie   MeSH
• kationtové kanály řízené cyklickými nukleotidy genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• membránové potenciály účinky léků   fyziologie   MeSH
• modely nemocí na zvířatech MeSH
• mozek cytologie   MeSH
• myši transgenní MeSH
• myši MeSH
• neurony účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• pyrimidiny farmakologie   MeSH
• regulace genové exprese účinky léků   fyziologie   MeSH
• sodík metabolismus   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AMP cyklický MeSH
• gliový fibrilární kyselý protein MeSH
• ICI D2788 MeSH Prohlížeč
• kationtové kanály řízené cyklickými nukleotidy MeSH
• proteiny nervové tkáně MeSH
• pyrimidiny MeSH
• sodík 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
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádory farmakoterapie   MeSH
• nervové kmenové buňky metabolismus   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy 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
• buněčná membrána metabolismus   MeSH
• draslík metabolismus   MeSH
• draslíkové kanály genetika   metabolismus   MeSH
• kationty metabolismus   MeSH
• membránové potenciály * MeSH
• Na(+)-H(+) antiport metabolismus   MeSH
• proteiny přenášející kationty metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• draslíkové kanály MeSH
• ENA1 protein, S cerevisiae MeSH Prohlížeč
• kationty MeSH
• Na(+)-H(+) antiport MeSH
• NHA1 protein, S cerevisiae MeSH Prohlížeč
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• sodíko-draslíková ATPasa MeSH
• TOK1 protein, S cerevisiae MeSH Prohlížeč

The aim of this study was to determine the relative contribution of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in histamine-induced relaxation of isolated pulmonary artery from normotensive and hypertensive rats. The hypertension was induced by oral administration of NO synthase inhibitor N(G)-nitro-L-arginine methylester (L-NAME, 50 mg/kg/day) to normotensive rats for 8 weeks. In phenylephrine-precontracted arterial rings the histamine-induced relaxation was significantly reduced in L-NAME-treated rats compared to the controls. Indomethacin (cyclooxygenase inhibitor) and glibenclamide (ATP-sensitive K+-channel blocker) did not inhibit the relaxation response in either control or hypertensive rats. On the other hand, tetraethylammonium (TEA), a K+-channel blocker with a broad specificity, significantly reduced histamine-induced relaxation in the pulmonary artery from both groups examined. The TEA-resistant relaxation was completely abolished by additional administration of L-NAME to the incubation medium. The results indicate that histamine-induced relaxation of the pulmonary artery in both normotensive and hypertensive rats is mediated mainly by nitric oxide, whereas EDHF seems to play a minor role.

• MeSH
• acetylcholin farmakologie   MeSH
• arteria pulmonalis patofyziologie   MeSH
• blokátory draslíkových kanálů MeSH
• draslíkové kanály fyziologie   MeSH
• histamin farmakologie   MeSH
• hypertenze chemicky indukované   patofyziologie   MeSH
• indomethacin farmakologie   MeSH
• inhibitory cyklooxygenasy farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• NG-nitroargininmethylester farmakologie   MeSH
• oxid dusnatý fyziologie   MeSH
• potkani Wistar MeSH
• prostaglandiny fyziologie   MeSH
• relaxace svalu účinky léků   MeSH
• svaly hladké cévní patofyziologie   MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholin MeSH
• blokátory draslíkových kanálů MeSH
• draslíkové kanály MeSH
• histamin MeSH
• indomethacin MeSH
• inhibitory cyklooxygenasy MeSH
• inhibitory enzymů MeSH
• NG-nitroargininmethylester MeSH
• oxid dusnatý MeSH
• prostaglandiny MeSH
• synthasa oxidu dusnatého 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 regulace účinky léků   fyziologie   MeSH
• draslíkové kanály biosyntéza   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy antagonisté a inhibitory   biosyntéza   MeSH
• krátkodobá paměť účinky léků   fyziologie   MeSH
• lokomoce účinky léků   fyziologie   MeSH
• methamfetamin aplikace a dávkování   toxicita   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• náhodné rozdělení MeSH
• prefrontální mozková kůra účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslíkové kanály MeSH
• Hcn1 protein, mouse MeSH Prohlížeč
• hyperpolarizační iontové kanály řízené cyklickými nukleotidy MeSH
• methamfetamin MeSH

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

• Klíčová slova
• 3D NMR, BEST-HNCO, Direct 13C detection, Dissolution-dynamic nuclear polarization (D-DNP), Hyperpolarization, Non-uniform sampling,
• MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• osteopontin chemie   MeSH
• ubikvitin chemie   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• osteopontin MeSH
• SPP1 protein, human MeSH Prohlížeč
• ubikvitin MeSH
• vnitřně neuspořádané proteiny MeSH
• voda MeSH