Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

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Clinical Factors Associated with Time-Specific Distribution of 18F-Fluorodeoxyglucose in Large-Vessel Vasculitis

Scientific Reports ◽

10.1038/s41598-019-51800-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Joel S. Rosenblum ◽

Kaitlin A. Quinn ◽

Casey A. Rimland ◽

Nehal N. Mehta ◽

Mark A. Ahlman ◽

...

Keyword(s):

Fdg Pet ◽

Blood Pool ◽

Large Vessel Vasculitis ◽

Time Dependent ◽

Large Vessel ◽

Dependent Manner ◽

Clinical Factors ◽

Factors Associated ◽

Fdg Uptake ◽

Specific Distribution

Abstract 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can detect vascular inflammation in large-vessel vasculitis (LVV). Clinical factors that influence distribution of FDG into the arterial wall and other tissues have not been characterized in LVV. Understanding these factors will inform analytic strategies to quantify vascular PET activity. Patients with LVV (n = 69) underwent 141 paired FDG-PET imaging studies at one and two hours per a delayed image acquisition protocol. Arterial uptake was quantified as standardized uptake values (SUVMax). SUVMean values were obtained for background tissues (blood pool, liver, spleen). Target-to-background ratios (TBRs) were calculated for each background tissue. Mixed model multivariable linear regression was used to identify time-dependent associations between FDG uptake and selected clinical features. Clinical factors associated with FDG distribution differed in a tissue- and time-dependent manner. Age, body mass index, and C-reactive protein were significantly associated with arterial FDG uptake at both time points. Clearance factors (e.g. glomerular filtration rate) were significantly associated with FDG uptake in background tissues at one hour but were weakly or not associated at two hours. TBRs using liver or blood pool at two hours were most strongly associated with vasculitis-related factors. These findings inform standardization of FDG-PET protocols and analytic approaches in LVV.

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Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1992.262.4.r712 ◽

1992 ◽

Vol 262 (4) ◽

pp. R712-R715 ◽

Cited By ~ 11

Author(s):

M. A. Perez-Pinzon ◽

M. Rosenthal ◽

T. J. Sick ◽

P. L. Lutz ◽

J. Pablo ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Tissue Oxygenation ◽

Mammalian Brain ◽

Survival Strategy ◽

Voltage Gated ◽

Channel Density ◽

Spike Threshold ◽

Channel Arrest ◽

Key Factor

In contrast to mammalian brain, which exhibits rapid degeneration during anoxia, the brains of certain species of turtles show an extraordinary capacity to survive prolonged anoxia. The decrease in energy expenditure shown by the anoxic turtle brain is likely to be a key factor for anoxic survival. The "channel arrest" hypothesis proposes that ion channels, which regulate brain electrical activity in normoxia, may be altered during anoxia in the turtle brain as a mechanism to spare energy. Goals of present research were to test this hypothesis and to determine whether down-regulation of sodium channels is a possible explanation for spike threshold shifts seen during anoxia in isolated turtle cerebellum. We report here that anoxia induced a significant (42%) decline in voltage-gated sodium channel density as determined by studies of the binding of a sodium channel ligand, [3H]brevetoxin. This study demonstrates that sodium channel densities in brain may be regulated by tissue oxygenation or by physiological events associated with anoxia. Moreover, it also suggests that downregulation of sodium channels may be a basis for changes in action potential thresholds, the electrical depression and energy conservation that provide the unique anoxic tolerance of turtle brain.

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The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽

10.1182/blood.v120.21.5120.5120 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5120-5120

Author(s):

Hatice Demet Kiper ◽

Burcin Tezcanli Kaymaz ◽

Ozlem Purclutepe ◽

Ceyda Tunakan Dalgic ◽

Nur Selvi ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Drug Exposure ◽

Conflicts Of Interest ◽

Time Dependent ◽

Blue Dye ◽

Dependent Manner ◽

Down Regulation ◽

Expression Levels ◽

Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

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Abstract 14602: Physical and Biophysical Coupling of the Cardiac Sodium Channel Involves 14-3-3

Circulation ◽

10.1161/circ.132.suppl_3.14602 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Jerome C Clatot ◽

Malcolm Hoshi ◽

Haiyan Liu ◽

Xiaoping Wan ◽

Krekwit Shinlapawittayatorn ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Dominant Negative ◽

Hek293 Cells ◽

Alpha Subunit ◽

Gene Encoding ◽

Cardiac Sodium Channel ◽

Channel Assembly ◽

Cardiac Sodium Channels ◽

Biophysical Interactions

Introduction: Mutations in SCN5A, the gene encoding for the cardiac sodium channel, produce alterations of the cardiac action potential that lead to life-threatening arrhythmias such as Long QT Syndrome (LQT3) and Brugada Syndrome (BrS). The conventional wisdom that sodium channels exist in complexes containing a single alpha-subunit has been challenged by the existence of dominant-negative (DN) mutations in BrS and the presence of polymorphisms that can restore trafficking and gating deficiencies of mutant channels in LQT and BrS. In fact, we have previously demonstrated that SCN5A subunits can interact with each other. Here we hypothesized that the physical and biophysical interactions between SCN5A alpha-subunits involve the partner protein 14-3-3, known to form dimers. Methods: SCN5A DN-BrS mutants and LQT3 gating deficient mutants were expressed in HEK293 cells and in commercially available iPS-derived cardiomyocytes, iCells©, in presence or absence of 14-3-3 inhibition. Resulting currents were measured using patch-clamp. Results: In order to investigate if the DN-effect seen by some BrS mutants is due to interaction of the sodium channel with the protein 14-3-3 which in turn would be involved in the alpha-alpha interaction, we expressed two different BrS DN-mutants in HEK293 cells with and without difopein, a specific 14-3-3 inhibitor. The presence of difopein abolished the DN-effect of both mutants. The DN-effect was also abolished when we mutated the putative 14-3-3 binding site on SCN5A and expressed the DN-mutants either in HEK293 cells or in iCells©. Inhibition of 14-3-3 also impaired the biophysical coupling observed in presence of SCN5A gating deficient mutants that affect either activation or inactivation of not only the mutants but also of the wild-type channel. Conclusions: Our results suggest that binding of 14-3-3 to the cardiac sodium channel alpha-subunit is involved in the alpha-alpha interaction and biophysical coupling of the channel. This study not only shifts paradigms in regards to sodium channel assembly and structure, but also puts forward the idea that physical and biophysical uncoupling of cardiac sodium channels could be a new therapy target for cardiac arrhythmias caused by SCN5A mutations.

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Interaction of n-alkylguanidines with the sodium channels of squid axon membrane.

The Journal of General Physiology ◽

10.1085/jgp.76.3.315 ◽

1980 ◽

Vol 76 (3) ◽

pp. 315-335 ◽

Cited By ~ 20

Author(s):

G E Kirsch ◽

J Z Yeh ◽

J M Farley ◽

T Narahashi

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Sodium Current ◽

Time Dependent ◽

Squid Axon ◽

Axon Membrane ◽

Inactivation Mechanism ◽

Hydrophobic Binding ◽

Sodium Inactivation ◽

Guanidino Group

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.

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Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels

British Journal of Pharmacology ◽

10.1038/sj.bjp.0703960 ◽

2001 ◽

Vol 132 (7) ◽

pp. 1455-1466 ◽

Cited By ~ 13

Author(s):

Seiji Shiraishi ◽

Izumi Shibuya ◽

Yasuhito Uezono ◽

Hiroki Yokoo ◽

Yumiko Toyohira ◽

...

Keyword(s):

Cell Surface ◽

Sodium Channel ◽

Sodium Channels ◽

Mrna Levels ◽

Cytoplasmic Calcium ◽

Down Regulation ◽

Subunit Mrna

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Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA

Marine Drugs ◽

10.3390/md17090510 ◽

2019 ◽

Vol 17 (9) ◽

pp. 510 ◽

Cited By ~ 2

Author(s):

Rocio K. Finol-Urdaneta ◽

Jeffrey R. McArthur ◽

Vyacheslav S. Korkosh ◽

Sun Huang ◽

Denis McMaster ◽

...

Keyword(s):

Skeletal Muscle ◽

Sodium Channel ◽

Sodium Channels ◽

Cell Voltage ◽

Hill Coefficient ◽

Dependent Manner ◽

Binding Modes ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Conducting Pathway

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10−12 to 10−5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

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Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

The Journal of General Physiology ◽

10.1085/jgp.94.5.813 ◽

1989 ◽

Vol 94 (5) ◽

pp. 813-831 ◽

Cited By ~ 7

Author(s):

D S Duch ◽

E Recio-Pinto ◽

C Frenkel ◽

S R Levinson ◽

B W Urban

Keyword(s):

Sodium Channel ◽

Lipid Bilayers ◽

Sodium Channels ◽

Single Channel ◽

Reversal Potential ◽

Channel Structure ◽

Dependent Manner ◽

Channel Conductance ◽

Single Channel Conductance ◽

Protein Subunit

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

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Simulated Microgravity Increases the Permeability of HUVEC Monolayer through Up-Regulation of Rap1GAP and Decreased Rap2 Activation

International Journal of Molecular Sciences ◽

10.3390/ijms23020630 ◽

2022 ◽

Vol 23 (2) ◽

pp. 630

Author(s):

Shuliang Shi ◽

Jing Li ◽

Erzhuo Li ◽

Wenqi Guo ◽

Yao He ◽

...

Keyword(s):

Endothelial Cells ◽

Simulated Microgravity ◽

Microgravity Condition ◽

Adherens Junction ◽

Time Dependent ◽

Culture Vessel ◽

Dependent Manner ◽

Down Regulation ◽

Protein Levels ◽

Huvec Cells

Space microgravity condition has great physiological influence on astronauts’ health. The interaction of endothelial cells, which control vascular permeability and immune responses, is sensitive to mechanical stress. However, whether microgravity has significant effects on the physiological function of the endothelium has not been investigated. In order to address such a question, a clinostat-based culture model with a HUVEC monolayer being inside the culture vessel under the simulated microgravity (SMG) was established. The transmittance of FITC-tagged dextran was used to estimate the change of integrity of the adherens junction of the HUVEC monolayer. Firstly, we found that the permeability of the HUVEC monolayer was largely increased after SMG treatment. To elucidate the mechanism of the increased permeability of the HUVEC monolayer under SMG, the levels of total expression and activated protein levels of Rap1 and Rap2 in HUVEC cells, which regulate the adherens junction of endothelial cells, were detected by WB and GST pull-down after SMG. As the activation of both Rap1 and Rap2 was significantly decreased under SMG, the expression of Rap1GEF1 (C3G) and Rap1GAP in HUVECs, which regulate the activation of them, was further determined. The results indicate that both C3G and Rap1GAP showed a time-dependent increase with the expression of Rap1GAP being dominant at 48 h after SMG. The down-regulation of the expression of junctional proteins, VE-cadherin and β-catenin, in HUVEC cells was also confirmed by WB and immunofluorescence after SMG. To clarify whether up-regulation of Rap1GAP is necessary for the increased permeability of the HUVEC monolayer after SMG, the expression of Rap1GAP was knocked down by Rap1GAP-shRNA, and the change of permeability of the HUVEC monolayer was detected. The results indicate that knock-down of Rap1GAP reduced SMG-induced leaking of the HUVEC monolayer in a time-dependent manner. In total, our results indicate that the Rap1GAP-Rap signal axis was necessary for the increased permeability of the HUVEC monolayer along with the down-regulation of junctional molecules including VE-cadherin and β-catenin.

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