scholarly journals Hydrophobic Amines and Their Guanidine Analogues Modulate Activation and Desensitization of ASIC3

2019 ◽  
Vol 20 (7) ◽  
pp. 1713 ◽  
Author(s):  
Vasilii Shteinikov ◽  
Natalia Potapieva ◽  
Valery Gmiro ◽  
Denis Tikhonov
Keyword(s):  
Nervous System ◽  
Patch Clamp ◽  
Chinese Hamster ◽  
Pain Sensation ◽  
Structural Determinants ◽  
Structural Requirements ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
Kinetics Of ◽  
Patch Clamp Method

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.

scholarly journals Patch Clamp: A Powerful Technique for Studying the Mechanism of Acupuncture

2012 ◽  
Vol 2012 ◽  
pp. 1-7
Author(s):  
D. Zhang
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Patch Clamp ◽  
Powerful Method ◽  
Patch Clamp Technique ◽  
Powerful Technique ◽  
Molecular Events ◽  
Electrophysiological Responses ◽  
The Central Nervous System ◽  
Patch Clamp Method

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.

scholarly journals Neutrophil Secretion Induced by an Intracellular Ca2+Rise and Followed by Whole-Cell Patch-Clamp Recordings Occurs Without any Selective Mobilization of Different Granule Populations

2006 ◽  
Vol 2006 ◽  
pp. 1-7 ◽  
Author(s):  
Daniel Granfeldt ◽  
Olle Harbecke ◽  
Åse Björstad ◽  
Anna Karlsson ◽  
Claes Dahlgren
Keyword(s):  
Patch Clamp ◽  
Human Neutrophils ◽  
Lag Phase ◽  
Measuring Time ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
High Concentrations ◽  
Kinetics Of

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (△Cm). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca2+. A sustained secretion following a short lag phase was induced by high concentrations of Ca2+(100μM and higher). A stable plateau was reached after 5–7 minutes at△Cmvalues corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca2+concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca2+-sensitivity.

scholarly journals Q&A: using Patch-seq to profile single cells

BMC Biology ◽  
2017 ◽  
Vol 15 (1) ◽  
Author(s):  
Cathryn R. Cadwell ◽  
Rickard Sandberg ◽  
Xiaolong Jiang ◽  
Andreas S. Tolias
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Patch Clamp ◽  
Single Cell ◽  
Single Cells ◽  
Cell Types ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Whole Cell Patch Clamp ◽  
Single Cell Rna Sequencing

Abstract Individual neurons vary widely in terms of their gene expression, morphology, and electrophysiological properties. While many techniques exist to study single-cell variability along one or two of these dimensions, very few techniques can assess all three features for a single cell. We recently developed Patch-seq, which combines whole-cell patch clamp recording with single-cell RNA-sequencing and immunohistochemistry to comprehensively profile the transcriptomic, morphologic, and physiologic features of individual neurons. Patch-seq can be broadly applied to characterize cell types in complex tissues such as the nervous system, and to study the transcriptional signatures underlying the multidimensional phenotypes of single cells.

scholarly journals The Effect of Hydrophobic Monoamines on Acid-Sensing Ion Channels ASIC1B

Acta Naturae ◽  
2015 ◽  
Vol 7 (2) ◽  
pp. 95-101 ◽  
Author(s):  
E. I. Nagaeva ◽  
N. N. Potapieva ◽  
D. B. Tikhonov
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Recombinant Receptors ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
The Hill ◽  
Cooperative Activation

Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 M concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.

scholarly journals Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition

2013 ◽  
Vol 304 (1) ◽  
pp. C89-C101 ◽  
Author(s):  
Nobuyoshi Kusama ◽  
Mamta Gautam ◽  
Anne Marie S. Harding ◽  
Peter M. Snyder ◽  
Christopher J. Benson
Keyword(s):  
Ion Channels ◽  
Ph Dependence ◽  
Extracellular Fluid ◽  
Dorsal Root Ganglion Neurons ◽  
Subunit Composition ◽  
Fluid Composition ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
Ganglion Neurons ◽  
Kinetics Of

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl−-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl−-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl− is a novel mechanism to sense shifts in extracellular fluid composition.

Expression Clone of TMEM16A as a Calcium-Activated Chloride Channels in CHO Cells

2013 ◽  
Vol 709 ◽  
pp. 832-835
Author(s):  
Feng Hao ◽  
Yi Ju Hou ◽  
Chen Zhao ◽  
Li Zhang ◽  
Zhi Qiang Tong ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cho Cells ◽  
Chloride Channels ◽  
Chinese Hamster ◽  
Green Fluorescence Protein ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

There is compelling evidence that TMEM16A fuctions as calcium-activated chloride channels (CaCCS), which was discovered by three independent labs in 2008 after Calcium-activated chloride channel current was first recorded in the 1980s. CaCCs are involved in many physiological processes, including transepithelial fluid secretion, smooth muscle contraction , sensory signal transduction and others. CaCCs are considers as potential drug therapy of hypertension, secretoy diarrheas, neuropathic pain, asthma, cystic fibrosis and certain tumors. In our previous study, TMEM16A with green fluorescence protein (GFP) fusion protein were subcloned into pcDNA3.1/Zeo. In this study, TMEM16A transient transfection conditon of Chinese hamster ovary (CHO) cells were optimized through liposome transfection and CHO cells expressing TMEM16A were got by stable transfection in which the classical calcium-activated chloride channels current was recorded by whole cell patch clamp technique. By a comparison between the results in this study and the results in previous study, both CHO and FRT cells are suitable for TMEM16A-pcDNA3.1 expression through liposome transfection and currents were recorded in both FRT and CHO cells by whole cell patch clamp technique, but our results indicated different purposes should require different cell lines and methods. These results were beneficial for the delving into study of TMEM16A-CaCCs by patch clamp technique which is the gold standard for real-time investigation of ion channels and their effectors.

scholarly journals Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype

2011 ◽  
Vol 301 (4) ◽  
pp. G644-G655 ◽  
Author(s):  
Subhash Kulkarni ◽  
Bende Zou ◽  
Jesse Hanson ◽  
Maria-Adelaide Micci ◽  
Gunjan Tiwari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nervous System ◽  
Patch Clamp ◽  
Neural Stem Cells ◽  
Functional Characterization ◽  
Enteric Neurons ◽  
Separate Experiment ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na+ currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca2+-dependent K+ channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

scholarly journals Structural Requirements for Differential Sensitivity of KCNQ K+ Channels to Modulation by Ca2+/Calmodulin

2005 ◽  
Vol 16 (8) ◽  
pp. 3538-3551 ◽  
Author(s):  
Nikita Gamper ◽  
Yang Li ◽  
Mark S. Shapiro
Keyword(s):  
Patch Clamp ◽  
Sympathetic Neurons ◽  
Chinese Hamster ◽  
Differential Sensitivity ◽  
Carboxy Terminus ◽  
Structural Requirements ◽  
K Channels ◽  
M Current ◽  
Amino Terminal ◽  
Carboxy Terminal

Calmodulin modulation of ion channels has emerged as a prominent theme in biology. The sensitivity of KCNQ1–5 K+ channels to modulation by Ca2+/calmodulin (CaM) was studied using patch-clamp, Ca2+ imaging, and biochemical and pharmacological approaches. Coexpression of CaM in Chinese hamster ovary (CHO) cells strongly reduced currents of KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3. In simultaneous current recording/Ca2+ imaging experiments, CaM conferred Ca2+ sensitivity to KCNQ4 and KCNQ5, but not to KCNQ1, KCNQ3, or KCNQ1/KCNE1 channels. A chimera constructed from the carboxy terminus of KCNQ4 and the rest KCNQ1 displayed Ca2+ sensitivity similar to KCNQ4. Chimeras constructed from different lengths of the KCNQ4 carboxy terminal and the rest KCNQ3 localized a region that confers sensitivity to Ca2+/CaM. Lobe-specific mutations of CaM revealed that its amino-terminal lobe mediates the Ca2+ sensitivity of the KCNQ/CaM complex. The site of CaM action within the channel carboxy terminus overlaps with that of the KCNQ opener N-ethylmaleimide (NEM). We found that CaM overexpression reduced NEM augmentation of KCNQ2, KCNQ4, and KCNQ5, and NEM pretreatment reduced Ca2+/CaM-mediated suppression of M current in sympathetic neurons by bradykinin. We propose that two functionally distinct types of carboxy termini underlie the observed differences among this channel family.

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