scholarly journals Isolating Nuclei from Cultured Cells for Patch-Clamp Electrophysiology of Intracellular Ca 2+ Channels

2013 ◽  
Vol 2013 (9) ◽  
pp. pdb.prot073056 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Horia Vais ◽  
King-Ho Cheung ◽  
J. Kevin Foskett
Keyword(s):  
Patch Clamp ◽  
Cultured Cells ◽  
Intracellular Ca ◽  
Patch Clamp Electrophysiology

scholarly journals Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1716
Author(s):  
Kun Tong ◽  
Ruotian Zhang ◽  
Fengzhi Ren ◽  
Tao Zhang ◽  
Junlin He ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Formalin Test ◽  
Sodium Ion ◽  
Inhibitory Potency ◽  
Voltage Gated ◽  
Patch Clamp Electrophysiology ◽  
Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

Na+/Ca2+ exchanger isoform 1 takes part to the Ca2+-related prosurvival pathway of SOD1 in primary motor neurons exposed to beta-methylamino-L-alanine

2021 ◽  
Author(s):  
Agnese Secondo ◽  
Tiziana Petrozziello ◽  
Francesca Boscia ◽  
Valentina Tedeschi ◽  
Anna Pannaccione ◽  
...  
Keyword(s):  
Motor Neurons ◽  
P2x7 Receptor ◽  
Nuclear Translocation ◽  
Neuroprotective Effect ◽  
Integrated Approach ◽  
Double Labeling ◽  
Reverse Mode ◽  
Intracellular Ca ◽  
Patch Clamp Electrophysiology ◽  
Enriched Cultures

Abstract Background: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), causing the amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca2+ homeostasis. By activating Akt/ERK1/2 pathway, the Cu,Zn-superoxide dismutase (SOD1) and its non-metallated form, ApoSOD1, prevent endoplasmic reticulum (ER) stress-induced cell death in motor neurons exposed to LBMAA. This occurs through the rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in part flowing from the extracellular compartment and in part released from ER. However, the molecular components of this mechanism remain uncharacterized.Methods: By an integrated approach consisting on the use of siRNA strategy, Western blotting, confocal double labeling immunofluorescence, patch-clamp electrophysiology, and Fura 2- /SBFI-single-cell imaging, we explored in rat motor neuron-enriched cultures the involvement of plasma membrane Na+/Ca2+ exchanger (NCX) and the purinergic P2X7 receptor as well as of the intracellular cADP-ribose (cADPR) pathway in the rapid and neuroprotective mechanism of SOD1.Results: we showed that SOD1-induced [Ca2+]i rise was prevented by the pan inhibitor of NCX CB-DMB but not by A430879, a P2X7 receptor specific antagonist, or by 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose. The same occurred for the ApoSOD1. Confocal double labeling immunofluorescence showed a huge expression of plasmalemmal NCX1 and intracellular NCX3 isoforms. Furthermore, we identified NCX1 reverse mode as the main mechanism responsible for the neuroprotective ER Ca2+ refilling elicited by SOD1 and ApoSOD1. Furthermore, SOD1 and ApoSOD1 promoted translocation of active Akt in some nuclei of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CNPYB2 protected motor neurons from L-BMAA-induced cell death.Conclusion: collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons.

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology

2000 ◽  
Vol 278 (5) ◽  
pp. C1055-C1061 ◽  
Author(s):  
Tim Robinson ◽  
Lars Thomsen ◽  
Jan D. Huizinga
Keyword(s):  
Patch Clamp ◽  
Data Acquisition ◽  
Front Panel ◽  
Analysis Program ◽  
Digital To Analog Converter ◽  
Analog Converter ◽  
Data Acquisition Card ◽  
System Requirements ◽  
User Need ◽  
Patch Clamp Electrophysiology

An acquisition and analysis program, “LabPatch,” has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/ .

scholarly journals An assessment of KIR channel function in human cerebral arteries

2019 ◽  
Vol 316 (4) ◽  
pp. H794-H800 ◽  
Author(s):  
Maria Sancho ◽  
Yuan Gao ◽  
Bjorn O. Hald ◽  
Hao Yin ◽  
Melfort Boulton ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Patch Clamp ◽  
Cerebral Arteries ◽  
Channel Expression ◽  
Expression Activity ◽  
Patch Clamp Electrophysiology ◽  
Resting Tone ◽  
The Impact ◽  
Arterial Tone

In the rodent cerebral circulation, inward rectifying K+ (KIR) channels set resting tone and the distance over which electrical phenomena spread along the arterial wall. The present study sought to translate these observations into human cerebral arteries obtained from resected brain tissue. Computational modeling and a conduction assay first defined the impact of KIR channels on electrical communication; patch-clamp electrophysiology, quantitative PCR, and immunohistochemistry then characterized KIR2.x channel expression/activity. In keeping with rodent observations, computer modeling highlighted that KIR blockade should constrict cerebral arteries and attenuate electrical communication if functionally expressed. Surprisingly, Ba2+ (a KIR channel inhibitor) had no effect on human cerebral arterial tone or intercellular conduction. In alignment with these observations, immunohistochemistry and patch-clamp electrophysiology revealed minimal KIR channel expression/activity in both smooth muscle and endothelial cells. This absence may be reflective of chronic stress as dysphormic neurons, leukocyte infiltrate, and glial fibrillary acidic protein expression was notable in the epileptic cortex. In closing, KIR2.x channel expression is limited in human cerebral arteries from patients with epilepsy and thus has little impact on resting tone or the spread of vasomotor responses. NEW & NOTEWORTHY KIR2.x channels are expressed in rodent cerebral arterial smooth muscle and endothelial cells. As they are critical to setting membrane potential and the distance signals conduct, we sought to translate this work into humans. Surprisingly, KIR2.x channel activity/expression was limited in human cerebral arteries, a paucity tied to chronic brain stress in the epileptic cortex. Without substantive expression, KIR2.x channels were unable to govern arterial tone or conduction.

scholarly journals Identification of a New Pyk2 Target Protein with Arf-GAP Activity

1999 ◽  
Vol 19 (3) ◽  
pp. 2338-2350 ◽  
Author(s):  
J. Andreev ◽  
J.-P. Simon ◽  
D. D. Sabatini ◽  
J. Kam ◽  
G. Plowman ◽  
...  
Keyword(s):  
Protein Tyrosine Kinase ◽  
Cultured Cells ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Stable Complex ◽  
Pleckstrin Homology Domain ◽  
Intracellular Ca ◽  
Constitutive Secretion ◽  
Weak Activity

ABSTRACT Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal α-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

scholarly journals Interferon γ Gene Expression in Sensory Neurons: Evidence for Autocrine Gene Regulation

1997 ◽  
Vol 186 (12) ◽  
pp. 2023-2031 ◽  
Author(s):  
Harald Neumann ◽  
Hannes Schmidt ◽  
Elke Wilharm ◽  
Lüder Behrens ◽  
Hartmut Wekerle
Keyword(s):  
Patch Clamp ◽  
Sensory Neurons ◽  
Nuclear Translocation ◽  
Interferon Γ ◽  
Dorsal Root Ganglion Neurons ◽  
Neuronal Membrane ◽  
Cultured Neurons ◽  
Ifn Γ ◽  
Patch Clamp Electrophysiology ◽  
Ganglion Neurons

We explored expression and possible function of interferon-γ (IFN-γ) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-γ protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-γ and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-γ receptor. Locally produced IFN-γ acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-γ–dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-γ. Our findings indicate a role of IFN-γ in autocrine regulation of sensory neurons.

scholarly journals 793Validation of cardiac optogenetics against patch clamp electrophysiology in human cardiomyocyte drug efficacy and safety studies

EP Europace ◽  
2017 ◽  
Vol 19 (suppl_3) ◽  
pp. iii141-iii141
Author(s):  
S. Bjork ◽  
E. Ojala ◽  
T. Nordstrom ◽  
E. Kankuri ◽  
E. Mervaala
Keyword(s):  
Patch Clamp ◽  
Drug Efficacy ◽  
Efficacy And Safety ◽  
Safety Studies ◽  
Patch Clamp Electrophysiology

Malate-Regulated Channels Permeable to Anions in Vacuoles of Arabidopsis thaliana

1995 ◽  
Vol 22 (1) ◽  
pp. 115 ◽  
Author(s):  
R Cerana ◽  
L Giromini ◽  
R Colombo
Keyword(s):  
Arabidopsis Thaliana ◽  
Patch Clamp ◽  
High Resistance ◽  
Cultured Cells ◽  
Reversal Potential ◽  
Anion Channels ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Inward Currents ◽  
Cytoplasmic Side

Anion channels in isolated vacuoles of Arabidopsis thaliana cultured cells were studied by means of the patch clamp technique in the whole-vacuole configuration. In symmetrical 100 mM KCl, a high resistance of the membrane at positive potentials inside the vacuole was observed. In symmetrical 100 mM K2-malate positive potentials inside the vacuole elicited slowly developing inward currents, due to the opening of channels, which, according to measurements of reversal potential, are selective for malate. The activation potential of the channels shifted as a function of the cytoplasmic malate concentration, but it was always such that the channels opened only to mediate malate influx into the vacuole. The channels were also permeable to succinate, fumarate and, to a lesser extent, oxaloacetate. In vacuoles preincubated with cytoplas- mic malate, inward currents were also elicited in the presence of KCl or KNO3 at the cytoplasmic side of the tonoplast. Malate channels were different from the cation slow vacuolar-type channels with regard to their sensitivity to changes in the cytoplasmic concentrations of Ca2+ and ATP, and in temperature between 10 and 20�C.

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