Cyclodextrins Exert a Ligand-like Current Inhibitory Effect on the KV1.3 Ion Channel Independent of Membrane Cholesterol Extraction

Cyclodextrins (CDs) are cyclic oligosaccharides capable of forming water-soluble complexes with a variety of otherwise poorly soluble molecules including cholesterol and different drugs. Consistently, CDs are widely used in research and clinical practice to deplete cholesterol from cellular membranes or to increase solubility and bioavailability of different pharmaceuticals at local concentrations in the millimolar range. Effects of CDs exerted on cellular functions are generally thought to originate from reductions in cholesterol levels. Potential direct, ligand-like CD effects are largely neglected in spite of several recent studies reporting direct interaction between CDs and proteins including AMP-activated protein kinase, β-amyloid peptides, and α-synuclein. In this study, by using patch-clamp technique, time-resolved quantitation of cholesterol levels and biophysical parameters and applying cholesterol-extracting and non-cholesterol-extracting CDs at 1 and 5 mM concentrations, we provide evidence for a previously unexplored ligand-like, cholesterol-independent current inhibitory effect of CDs on KV1.3, a prototypical voltage-gated potassium channel with pathophysiological relevance in various autoimmune and neurodegenerative disorders. Our findings propose that potential direct CD effects on KV channels should be taken into consideration when interpreting functional consequences of CD treatments in both research and clinical practice. Furthermore, current-blocking effects of CDs on KV channels at therapeutically relevant concentrations might contribute to additional beneficial or adverse effects during their therapeutic applications.

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Modulating Tumor Cell Functions by Tunable Nanopatterned Ligand Presentation

Nanomaterials ◽

10.3390/nano10020212 ◽

2020 ◽

Vol 10 (2) ◽

pp. 212

Author(s):

Katharina Amschler ◽

Michael P. Schön

Keyword(s):

Extracellular Matrix ◽

Tumor Cell ◽

Cell Fate ◽

Epithelial Mesenchymal Transition ◽

Model Systems ◽

Cellular Functions ◽

Biophysical Parameters ◽

Ligand Density ◽

Mesenchymal Transition ◽

Cell Functions

Cancer comprises a large group of complex diseases which arise from the misrouted interplay of mutated cells with other cells and the extracellular matrix. The extracellular matrix is a highly dynamic structure providing biochemical and biophysical cues that regulate tumor cell behavior. While the relevance of biochemical signals has been appreciated, the complex input of biophysical properties like the variation of ligand density and distribution is a relatively new field in cancer research. Nanotechnology has become a very promising tool to mimic the physiological dimension of biophysical signals and their positive (i.e., growth-promoting) and negative (i.e., anti-tumoral or cytotoxic) effects on cellular functions. Here, we review tumor-associated cellular functions such as proliferation, epithelial-mesenchymal transition (EMT), invasion, and phenotype switch that are regulated by biophysical parameters such as ligand density or substrate elasticity. We also address the question of how such factors exert inhibitory or even toxic effects upon tumor cells. We describe three principles of nanostructured model systems based on block copolymer nanolithography, electron beam lithography, and DNA origami that have contributed to our understanding of how biophysical signals direct cancer cell fate.

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Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

Journal of Bacteriology ◽

10.1128/jb.01670-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2496-2504 ◽

Cited By ~ 25

Author(s):

Po-Chi Soo ◽

Yu-Tze Horng ◽

Jun-Rong Wei ◽

Jwu-Ching Shu ◽

Chia-Chen Lu ◽

...

Keyword(s):

Serratia Marcescens ◽

Binding Site ◽

Promoter Region ◽

Transcriptional Start Site ◽

Direct Interaction ◽

Underlying Mechanism ◽

Inhibitory Effect ◽

Start Codon ◽

Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

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Thapsigargin inhibits voltage-activated calcium channels in adrenal glomerulosa cells

Biochemical Journal ◽

10.1042/bj2960309 ◽

1993 ◽

Vol 296 (2) ◽

pp. 309-312 ◽

Cited By ~ 40

Author(s):

M F Rossier ◽

C P Python ◽

M M Burnay ◽

W Schlegel ◽

M B Vallotton ◽

...

Keyword(s):

Cell Types ◽

Inhibitory Effect ◽

Zona Glomerulosa ◽

High Threshold ◽

Patch Clamp Technique ◽

Dual Effect ◽

Blocking Voltage ◽

Current Voltage ◽

Glomerulosa Cells ◽

Ca2 Channels

Thapsigargin, an inhibitor of the microsomal Ca2+ pumps, has been extensively used to study the intracellular Ca2+ pool participating in the generation of the agonist-induced Ca2+ signal in various cell types. A dual effect of this agent was observed in bovine adrenal zona glomerulosa cells. At nanomolar concentrations, thapsigargin stimulated a sustained Ca2+ influx, probably resulting from Ca(2+)-store depletion. In contrast, when added at micromolar concentrations, thapsigargin prevented the rise in cytosolic free Ca2+ concentration ([Ca2+]c) induced by K+. This inhibitory effect of thapsigargin on voltage-activated Ca2+ channels was confirmed by measuring Ba2+ currents by the patch-clamp technique. Both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels were affected by micromolar concentrations of thapsigargin. Analysis of the current-voltage relationship for T-type channels revealed that thapsigargin did not modify the sensitivity of these channels to the voltage, but decreased the maximal current flowing through the channels. In conclusion, thapsigargin appears to exert a dual effect on adrenal glomerulosa cells. At lower concentrations, this agent induces a sustained Ca2+ entry, whereas at higher concentrations it decreases [Ca2+]c by blocking voltage-activated Ca2+ channels.

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A novel role for WAVE1 in controlling actin network growth rate and architecture

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1477 ◽

2015 ◽

Vol 26 (3) ◽

pp. 495-505 ◽

Cited By ~ 9

Author(s):

Meredith O. Sweeney ◽

Agnieszka Collins ◽

Shae B. Padrick ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Specific Activity ◽

Inhibitory Effect ◽

Actin Network ◽

Inhibitory Effects ◽

Cellular Functions ◽

Network Growth ◽

Assembly Kinetics ◽

Filament Networks ◽

First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

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An interdomain bridge influences RNA binding of the human La protein

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.003995 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1529-1540 ◽

Cited By ~ 4

Author(s):

Stefano A. Marrella ◽

Kerene A. Brown ◽

Farnaz Mansouri-Noori ◽

Jennifer Porat ◽

Derek J. Wilson ◽

...

Keyword(s):

Rna Binding ◽

Rna Polymerase Iii ◽

Conformational Dynamics ◽

Direct Interaction ◽

Rna Recognition Motif ◽

Binding Modes ◽

Nuclear Retention ◽

Time Resolved ◽

La Protein ◽

C Terminus

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3′OH–tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3′OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen–deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein–protein interdomain interaction in La proteins.

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Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis

Molecules ◽

10.3390/molecules23112884 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2884 ◽

Cited By ~ 4

Author(s):

Eun-Jung In ◽

Yuno Lee ◽

Sushruta Koppula ◽

Tae-Yeon Kim ◽

Jun-Hyuk Han ◽

...

Keyword(s):

Cell Death ◽

Md Simulation ◽

Ischemia Reperfusion Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Direct Interaction ◽

Inhibitory Effect ◽

Therapeutic Implications ◽

Pathological Conditions ◽

Tnf Α

Necroptosis, or caspase-independent programmed cell death, is known to be involved in various pathological conditions, such as ischemia/reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. Although several inhibitors of necroptosis have been identified, none of them are currently in clinical use. In the present study, we identified a new compound, 4-({[5-(4-aminophenyl)-4-ethyl-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-N-(1,3-thiazol-2-yl) benzamide (NTB451), with significant inhibitory activity on the necroptosis induced by various triggers, such as tumor necrosis factor-α (TNF-α) and toll-like receptor (TLR) agonists. Mechanistic studies revealed that NTB451 inhibited phosphorylation and oligomerization of mixed lineage kinase domain like (MLKL), and this activity was linked to its inhibitory effect on the formation of the receptor interacting serine/threonine-protein kinase 1 (RIPK1)-RIPK3 complex. Small interfering RNA (siRNA)-mediated RIPK1 knockdown, drug affinity responsive target stability assay, and molecular dynamics (MD) simulation study illustrated that RIPK1 is a specific target of NTB451. Moreover, MD simulation showed a direct interaction of NTB451 and RIPK1. Further experiments to ensure that the inhibitory effect of NTB451 was restricted to necroptosis and NTB451 had no effect on nuclear factor-κB (NF-κB) activation or apoptotic cell death upon triggering with TNF-α were also performed. Considering the data obtained, our study confirmed the potential of NTB451 as a new necroptosis inhibitor, suggesting its therapeutic implications for pathological conditions induced by necroptotic cell death.

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The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

Physiological Research ◽

10.33549/physiolres.931155 ◽

2008 ◽

pp. 55-62

Author(s):

HY Xu ◽

X Huang ◽

M Yang ◽

J-B Sun ◽

L-H Piao ◽

...

Keyword(s):

Guinea Pig ◽

Phosphodiesterase Inhibitor ◽

Potassium Currents ◽

Inhibitory Effect ◽

Delayed Rectifier ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Whole Cell Patch Clamp ◽

Dependent Protein ◽

Gastric Myocytes

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.

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Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS) for on- and off-line analysis of atmospheric gas and aerosol species

10.5194/amt-2016-132 ◽

2016 ◽

Cited By ~ 1

Author(s):

Jordan E. Krechmer ◽

Michael Groessl ◽

Xuan Zhang ◽

Heikki Junninen ◽

Paola Massoli ◽

...

Keyword(s):

Mass Spectrometry ◽

Ion Mobility Spectrometry ◽

Ion Mobility ◽

Measurement Techniques ◽

Molecular Ions ◽

Molecular Structures ◽

Water Soluble ◽

Oxidation Products ◽

Time Resolved ◽

Organic Species

Abstract. Measurement techniques that provide molecular-level information are needed to elucidate the multi-phase processes that produce secondary organic aerosol (SOA) species in the atmosphere. Here we demonstrate the application of ion mobility spectrometry-mass spectrometry (IMS-MS) to the simultaneous characterization of the elemental composition and molecular structures of organic species in the gas and particulate phases. Molecular ions of gas-phase organic species are measured online with IMS-MS after ionization with a custom build nitrate chemical ionization (CI) source. This CI-IMS-MS technique is used to obtain time-resolved measurements (5 min) of highly oxidized organic molecules during the 2013 Southern Oxidant and Aerosol Study (SOAS) ambient field campaign in the forested SE US. The ambient IMS-MS signals are consistent with laboratory IMS-MS spectra obtained from single-component carboxylic acids and multicomponent mixtures of isoprene and monoterpene oxidation products. Mass-mobility correlations in the 2-dimensional IMS-MS space provide a means of identifying ions with similar molecular structures within complex mass spectra and are used to separate and identify monoterpene oxidation products in the ambient data that are produced from different chemical pathways. Water-soluble organic carbon (WSOC) constituents of fine aerosol particles that are not resolvable with standard analytical separation methods, such as liquid chromatography (LC), are shown to be separable with IMS-MS coupled to an electrospray ionization (ESI) source. The capability to use ion mobility to differentiate between isomers is demonstrated for organosulfates derived from the reactive uptake of isomers of isoprene epoxydiols (IEPOX) onto wet acidic sulfate aerosol. Controlled fragmentation of precursor ions by collisional dissociation (CID) in the transfer region between the IMS and the MS is used to validate MS peak assignments, elucidate structures of oligomers, and confirm the presence of the organosulfate functional group.

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Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.4.l475 ◽

1998 ◽

Vol 274 (4) ◽

pp. L475-L484 ◽

Cited By ~ 49

Author(s):

Lucky Jain ◽

Xi-Juan Chen ◽

Lou Ann Brown ◽

Douglas C. Eaton

Keyword(s):

Nitric Oxide ◽

Protein Kinase ◽

Single Channel ◽

Inhibitory Effect ◽

Cation Channel ◽

Alveolar Type ◽

Apical Surface ◽

Type Ii ◽

Patch Clamp Technique ◽

Open Probability

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

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Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.1.c248 ◽

2001 ◽

Vol 281 (1) ◽

pp. C248-C256 ◽

Cited By ~ 21

Author(s):

Dominique Trouet ◽

Iris Carton ◽

Diane Hermans ◽

Guy Droogmans ◽

Bernd Nilius ◽

...

Keyword(s):

Tyrosine Kinase ◽

Inhibitory Effect ◽

Kinase Domain ◽

Wild Type ◽

Patch Clamp Technique ◽

Src Tyrosine Kinase ◽

Src Homology ◽

Cpae Cells ◽

Src Homology Domains ◽

Homology Domains

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I Cl,swell, the swelling-induced Cl−current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I Cl,swell. However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I Cl,swell in CPAE cells. Kinase activity was dispensable for the inhibition of I Cl,swell, since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.

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