patch clamp method
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2019 ◽  
Vol 20 (7) ◽  
pp. 1713 ◽  
Author(s):  
Vasilii Shteinikov ◽  
Natalia Potapieva ◽  
Valery Gmiro ◽  
Denis Tikhonov

Acid-sensing ion channel 3 (ASIC3) is an important member of the acid-sensing ion channels family, which is widely expressed in the peripheral nervous system and contributes to pain sensation. ASICs are targeted by various drugs and toxins. However, mechanisms and structural determinants of ligands’ action on ASIC3 are not completely understood. In the present work we studied ASIC3 modulation by a series of “hydrophobic monoamines” and their guanidine analogs, which were previously characterized to affect other ASIC channels via multiple mechanisms. Electrophysiological analysis of action via whole-cell patch clamp method was performed using rat ASIC3 expressed in Chinese hamster ovary (CHO) cells. We found that the compounds studied inhibited ASIC3 activation by inducing acidic shift of proton sensitivity and slowed channel desensitization, which was accompanied by a decrease of the equilibrium desensitization level. The total effect of the drugs on the sustained ASIC3-mediated currents was the sum of these opposite effects. It is demonstrated that drugs’ action on activation and desensitization differed in their structural requirements, kinetics of action, and concentration and state dependencies. Taken together, these findings suggest that effects on activation and desensitization are independent and are likely mediated by drugs binding to distinct sites in ASIC3.


Author(s):  
Jingjing Su ◽  
Ting Xu ◽  
Genling Jiang ◽  
Mengru Liang ◽  
Hui Cheng ◽  
...  

Nasopharyngeal carcinoma (NPC) has not been thoroughly studied, and the pathogenesis of NPC is unclear. Scientists have neither discovered effective therapies nor achieved a desirable prognosis. Some studies have found that the regulation of intra- and extracellular ion channels hinges directly on cell apoptosis, and treatment with Gambogenic acid (GNA) brings changes to the volume-sensitive outwardly rectifying chloride (VSOR Cl-) current of CNE-2Z cells recorded by the patch clamp method. Nevertheless, rarely have any researchers probed into the relevance between this variation and the anti-tumor mechanism of GNA. This paper is suggested that GNA activates the VSOR Cl- current on the CNE-2Z cell membrane, and the activation of VSOR Cl- currents by GNA in CNE-2Z cells is blocked by the chloride channel blockers DIDS and DCPIB. GNA induces the down-regulation of GRP78 and up-regulation of ATF4 as well as chop proteins, which is evidence for the induction of CNE-2Z cell apoptosis, and this correlates with ER stress. GNA can activate the VSOR Cl- channel and lead to the occurrence of ER stress, thus inducing the apoptosis of CNE-2Z cells and inhibiting the proliferation of CNE-2Z cells.


2015 ◽  
Vol 93 (4) ◽  
pp. 215-221 ◽  
Author(s):  
Lina Sun ◽  
Yunlong Hou ◽  
Tingting Zhao ◽  
Shanshan Zhou ◽  
Xiaoran Wang ◽  
...  

The phytoestrogen genistein (GST) and magnesium have been independently shown to regulate vascular tone; however, their individual vasodilatory effects are limited. The aim of this study was to examine the combined effects of GST plus magnesium on vascular tone in mesenteric arteries. The effects of pretreatment with GST (0–200 μmol/L), MgCl2 (0–4.8 mmol/L) and GST plus MgCl2 on 10 μmol/L phenylephrine (PE) precontracted mesenteric arteries in rats were assessed by measuring isometric force. BKCa currents were detected by the patch clamp method. GST caused concentration- and partial endothelium-dependent relaxation. Magnesium resulted in dual adjustment of vascular tone. Magnesium-free solution eliminated the vasodilatation of GST in both endothelium-intact and denuded rings. GST (50 μmol/L) plus magnesium (4.8 mmol/L) caused stronger relaxation in both endothelium-intact and denuded rings. Pretreatment with the nitric oxide synthase (NOS) inhibitor l-N-nitroarginine methyl ester (l-NAME, 100 μmol/L) significantly inhibited the effects of GST, high magnesium, and the combination of GST and magnesium. BKCa currents were amplified to a greater extent when GST (50 μmol/L) was combined with 4.8 versus 1.2 mmol/L Mg2+. Our data suggest that GST plus magnesium provides enhanced vasodilatory effects in rat mesenteric arteries compared with that observed when either is used separately, which was related to an eNOS pathway and BKCa current amplification.


2014 ◽  
pp. 771-777
Author(s):  
V. SEPSOVA ◽  
J. KRUSEK ◽  
J. ZDAROVA KARASOVA ◽  
F. ZEMEK ◽  
K. MUSILEK ◽  
...  

Acetylcholinesterase inhibitors (AChEIs) are used in the treatment of myasthenia gravis (MG). We investigated the effects of AChEIs on peripheral nicotinic receptors (nAChR), which play a crucial role in the treatment of MG symptoms. The positive modulation of those receptors by AChE inhibitors could have an added value to the anti-AChE activity and might be useful in the therapy of MG. Furthermore, to estimate the potential drawbacks of the compounds, cytotoxicity has been assessed on various cell lines. The whole-cell mode of the patch-clamp method was employed. The experiments were performed on medulloblastoma/rhabdomyosarcoma cell line TE671 expressing human embryonic muscle-like receptor with subunits α2βγδ. The effect of the compounds on cell viability was measured by standard MTT assay (Sigma Aldrich) on ACHN (renal cell adenocarcinoma), HeLa (immortal cell line derived from a cervical carcinoma), HEPG2 (hepatocellular carcinoma) and BJ (skin fibroblasts) cell lines. No positive modulation by the tested AChE inhibitors was observed. Moreover, the compounds exhibited antagonistic activity on the peripheral nAChR. Standard drugs used in MG treatment were shown to be less potent inhibitors of muscle-type nAChR than the newly synthesized compounds. The new compounds showed very little effect on cell viability, and toxicities were comparable to standards. Newly synthesized AChEIs inhibited peripheral nAChR. Furthermore, the inhibition was higher than that of standards used for the treatment of MG. They could be used for the study of nAChR function, thanks to their high antagonizing potency and fast recovery of receptor activity after their removal. However, since no positive modulation was observed, the new compounds do not seem to be promising candidates for MG treatment, even though their cytotoxic effect was relatively low.


2014 ◽  
Vol 12 (2) ◽  
pp. 54-58
Author(s):  
Anna Alexandrovna Bukinich

The effect of dopamine (DA), its agonists and antagonists on the amplitude of GABA-activated currents of isolated multipolar spinal cord neurons (both motoneurons and interneurons) of larva of the lamprey Lampetra planeri by means of patch-clamp method in the whole cell configuration was studied. (+)-SCH-23390, a D1-DA receptors antagonist was shown to block dopamine effects on GABA-activated currents by 63.0 ± 4.7 % and by 77.1 ± 2.0 %. Effects of (-)quinpirol, a D2-DA receptors agonist, on GABA-activated currents were blocked by means of (+)-SCH-23390 by 78.8 ± 0.4 % and by 85.0 ± 5.7 %. Because of chemoactivated currents are in full accordance with a gradual scale, the results on blocking D1-DA receptors by (+)-SCH-23390 are ideal ones and that is the possible basis to further clinical aprobation of (+)-SCH-23390 for treatment of epilepsy, neurotic reactions and depression.


2012 ◽  
Vol 2012 ◽  
pp. 1-7
Author(s):  
D. Zhang

Cellular and molecular events can be investigated using electrophysiological techniques. In particular, the patch-clamp method provides detailed information. In addition, the patch-clamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. In this paper, recent researches on how acupuncture might modulate electrophysiological responses in the central nervous system (CNS) and affect peripheral structures are reviewed.


Author(s):  
Y Zhao ◽  
S Inayat ◽  
D A Dikin ◽  
J H Singer ◽  
R S Ruoff ◽  
...  

The patch clamp technique permits high-resolution recording of the ionic currents flowing through a cell's plasma membrane. In different configurations, this technique has allowed experimenters to record and manipulate the currents that flow either through single ion channels or those that flow across the whole plasma membrane. Unfortunately, the conventional patch clamp method is laborious, requiring the careful fabrication of electrodes, skillful manipulation of the patch pipette towards a cell, and the clever design of electronics and apparatus to allow low-noise recordings. Advances in microfabrication offer promising technologies for high-throughput patch clamp recordings, particularly suitable for drug screening. This paper provides a review of the advances that have been made in the patch clamp technique over the years and considers where application of nanotechnology might provide significant contributions in the future.


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