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Single channel patch clamp

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https://www.readbyqxmd.com/read/28202726/two-tonoplast-mate-proteins-function-as-turgor-regulating-chloride-channels-in-arabidopsis
#1
Haiwen Zhang, Fu-Geng Zhao, Ren-Jie Tang, Yuexuan Yu, Jiali Song, Yuan Wang, Legong Li, Sheng Luan
The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel...
February 15, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28197715/vacuolar-ion-channels-in-the-liverwort-marchantia-polymorpha-influence-of-ion-channel-inhibitors
#2
Mateusz Koselski, Kazimierz Trebacz, Halina Dziubinska
Potassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwort Marchantia polymorpha were characterized in respect to calcium dependence, selectivity, and pharmacology. The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents-predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages...
February 14, 2017: Planta
https://www.readbyqxmd.com/read/28197552/altered-channel-conductance-states-and-gating-of-gabaa-receptors-by-a-pore-mutation-linked-to-dravet-syndrome
#3
Ciria C Hernandez, Weijing Kong, Ningning Hu, Yujia Zhang, Wangzhen Shen, Laurel Jackson, Xiaoyan Liu, Yuwu Jiang, Robert L Macdonald
We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABAA receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABAA receptors using structural modeling...
January 2017: ENeuro
https://www.readbyqxmd.com/read/28192183/measuring-kinetics-and-potency-of-herg-block-for-cipa
#4
Monique J Windley, Najah Abi-Gerges, Bernard Fermini, Jules C Hancox, Jamie I Vandenberg, Adam P Hill
INTRODUCTION: The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol...
February 10, 2017: Journal of Pharmacological and Toxicological Methods
https://www.readbyqxmd.com/read/28183797/ca-2-binding-protein-2-inhibits-ca-2-channel-inactivation-in-mouse-inner-hair-cells
#5
Maria Magdalena Picher, Anna Gehrt, Sandra Meese, Aleksandra Ivanovic, Friederike Predoehl, SangYong Jung, Isabelle Schrauwen, Alberto Giulio Dragonetti, Roberto Colombo, Guy Van Camp, Nicola Strenzke, Tobias Moser
Ca(2+)-binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca(2+) channels of type 1.3 (CaV1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2(LacZ/LacZ) ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs)...
February 9, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28174298/the-startle-disease-mutation-e103k-impairs-activation-of-human-homomeric-%C3%AE-1-glycine-receptors-by-disrupting-an-intersubunit-salt-bridge-across-the-agonist-binding-site
#6
Fatemah Safar, Elliot Hurdiss, Marios Erotocritou, Timo Greiner, Remigijus Lape, Mark W Irvine, Guangyu Fang, David Jane, Rilei Yu, Marc A Dämgen, Philip C Biggin, Lucia G Sivilotti
Glycine receptors (GlyR) belong to the pentameric ligand gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface...
February 7, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28131627/down-regulation-of-kir2-6-channel-by-c-termini-mutation-d252n-and-its-association-with-the-susceptibility-to-thyrotoxic-periodic-paralysis
#7
Rolf Matias Paninka, Estevão Carlos-Lima, Susan C Lindsey, Ilda S Kunii, Magnus R Dias-da-Silva, Manoel Arcisio-Miranda
Inward rectifying potassium - Kir - channels drive the resting potential to potassium reversal potential and, when disrupted, might be related to muscular diseases. Recently, Thyrotoxic Periodic Paralysis (TPP) has emerged as a channelopathy related to mutations in KCNJ18 gene, which encodes Kir2.6 channel. TPP is a neuromuscular disorder characterized by a triad of muscle weakness, hypokalemia, and thyrotoxicosis, the latter being essential for the crisis. Direct sequencing revealed two heterozygous mutations - D252N and R386C - in two TPP patients...
January 25, 2017: Neuroscience
https://www.readbyqxmd.com/read/28119170/existence-of-atp-sensitive-potassium-currents-on-human-periodontal-ligament-cells
#8
Phuong Thi Huyen Tran, Young Hee Lee, Janardhan Prasad Bhattarai, Soo Joung Park, Ho-Keun Yi, Seong Kyu Han
OBJECTIVE: Potassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis...
January 17, 2017: Archives of Oral Biology
https://www.readbyqxmd.com/read/28114421/immediate-effects-of-repetitive-magnetic-stimulation-on-single-cortical-pyramidal-neurons
#9
Jineta Banerjee, Mary E Sorrell, Pablo A Celnik, Galit Pelled
Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons...
2017: PloS One
https://www.readbyqxmd.com/read/28075010/stimulatory-actions-of-a-novel-thiourea-derivative-on-large-conductance-calcium-activated-potassium-channels
#10
Sheng-Nan Wu, Jyh-Haur Chern, Santai Shen, Hwei-Hisen Chen, Ying-Ting Hsu, Chih-Chin Lee, Ming-Huan Chan, Ming-Chi Lai, Feng-Shiun Shie
AIM: In this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels. METHODS: The effects of compound #326 on Ca(2+) -activated K(+) channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration. RESULTS: In pituitary GH3 cells, compound #326 increased the amplitude of Ca(2+) -activated K(+) currents (IK(Ca) ) with an EC50 value of 11...
January 11, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/28052704/conflicting-effects-by-antibodies-against-connexin36-during-the-action-of-intracellular-cyclic-amp-onto-electrical-synapses-of-retinal-ganglion-cells
#11
Soh Hidaka
Alpha-type retinal ganglion cells (alpha cells) of the same class in mammalian retina are connected by gap junctions. Electrical synapses between alpha cells were examined using combined techniques of dual patch-clamp recordings, intracellular labeling and electron microscopy in the albino rat retina. In simultaneous dual whole-cell recordings from pairs of neighboring alpha cells, bidirectional electrical synapses with symmetrical junction conductance were observed in pairs with cells of the same morphological type...
January 4, 2017: Journal of Integrative Neuroscience
https://www.readbyqxmd.com/read/28039402/high-and-low-conductance-nmda-receptors-are-present-in-layer-4-spiny-stellate-and-layer-2-3-pyramidal-neurons-of-mouse-barrel-cortex
#12
Christian Scheppach
N-Methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate in the mammalian brain and are important in synaptic function and plasticity, but are also found in extrasynaptic locations and influence neuronal excitability. There are different NMDA receptor subtypes which differ in their single-channel conductance. Recently, synaptic plasticity has been studied in the mouse barrel cortex, the primary sensory cortex for input from the animal's whiskers. Pharmacological data imply the presence of low-conductance NMDA receptors in spiny stellate neurons of cortical layer 4, but of high-conductance NMDA receptors in pyramidal neurons of layer 2/3...
December 2016: Physiological Reports
https://www.readbyqxmd.com/read/27989687/role-of-protein-kinase-c-in-metabolic-regulation-of-the-cardiac-na-channel
#13
Man Liu, Guangbin Shi, Kai-Chien Yang, Lianzhi Gu, Anumantha G Kanthasamy, Vellareddy Anantharam, Samuel C Dudley
BACKGROUND: The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activates protein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), and down-regulates the cardiac Na(+) channel (NaV1.5). OBJECTIVE: The purpose of this study was to determine how NADH signals down-regulation of NaV1.5. METHODS: Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoROS with MitoSOX Red...
December 15, 2016: Heart Rhythm: the Official Journal of the Heart Rhythm Society
https://www.readbyqxmd.com/read/27899943/effects-of-allitridum-on-the-transient-outward-potassium-current-in-rats-with-heart-failure
#14
Xiao-Jing Zhao, Kun Lin, Yu Zhang, Bin Xu, Li Liu, Yi-Cheng Fu, Xi Chen, Zhong-Qi Cai, Zhi-Juan Wu, Yun Huang, Yang Li
OBJECTIVE: To study the effect of allitridum on the transient outward potassium current (Ito) of ventricular myocytes in heart failure (HF). METHODS: The dual enzymatic method was used to separate single ventricular myocytes from Sprague Dawley rats. Patch-clamping was used to record Ito and analyze the effect of allitridum on the current. RESULTS: The Ito current had a significant decrease in the HF group, compared with the control group...
September 2016: Journal of Geriatric Cardiology: JGC
https://www.readbyqxmd.com/read/27859335/extracellular-protons-enable-activation-of-the-calcium-dependent-chloride-channel-tmem16a-ano1
#15
Silvia Cruz-Rangel, José J De Jesus-Pérez, Iván A Aréchiga-Figueroa, Aldo A Rodríguez-Menchaca, Patricia Pérez-Cornejo, H Criss Hartzell, Jorge Arreola
TMEM16A (ANO1), the pore forming subunit of a Ca(2+) -dependent Cl(-) channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca(2+) . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H(+) ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch clamp recordings. We found that increasing the [H(+) ]o from 10(-10) to 10(-5...
November 17, 2016: Journal of Physiology
https://www.readbyqxmd.com/read/27856251/local-calcium-signalling-is-mediated-by-mechanosensitive-ion-channels-in-mesenchymal-stem-cells
#16
Vladislav I Chubinskiy-Nadezhdin, Valeria Y Vasileva, Natalia A Pugovkina, Irina O Vassilieva, Elena A Morachevskaya, Nikolay N Nikolsky, Yuri A Negulyaev
Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs)...
January 22, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27827370/ethanol-sensitive-pacemaker-neurons-in-the-mouse-external-globus-pallidus
#17
Karina P Abrahao, Jessica H Chancey, C Savio Chan, David M Lovinger
Although ethanol is one of the most widely used drugs, we still lack a full understanding of which neuronal subtypes are affected by this drug. Pacemaker neurons exert powerful control over brain circuit function, but little is known about ethanol effects on these types of neurons. Neurons in the external globus pallidus (GPe) generate pacemaker activity that controls basal ganglia, circuitry associated with habitual and compulsive drug use. We performed patch-clamp recordings from GPe neurons and found that bath application of ethanol dose-dependently decreased the firing rate of low-frequency GPe neurons, but did not alter the firing of high-frequency neurons...
December 14, 2016: Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology
https://www.readbyqxmd.com/read/27823931/two-p2x1-receptor-transcripts-able-to-form-functional-channels-are-present-in-most-human-monocytes
#18
Cintya López-López, Josue Jaramillo-Polanco, Diana P Portales-Pérez, Karen S Gómez-Coronado, Jessica G Rodríguez-Meléndez, Juan D Cortés-García, Rosa Espinosa-Luna, Luis M Montaño, Carlos Barajas-López
To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics...
December 15, 2016: European Journal of Pharmacology
https://www.readbyqxmd.com/read/27805790/interplay-among-distinct-ca-2-conductances-drives-ca-2-sparks-spontaneous-transient-outward-currents-in-rat-cerebral-arteries
#19
Ahmed M Hashad, Neil Mazumdar, Monica Romero, Anders Nygren, Kamran Bigdely-Shamloo, Osama F Harraz, Jose L Puglisi, Edward J Vigmond, Sean M Wilson, Donald G Welsh
KEY POINTS: Distinct Ca(2+) channels work in a coordinated manner to grade Ca(2+) spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca(2+) channel to Ca(2+) spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na(+) /Ca(2+) exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca(2+) sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction...
February 15, 2017: Journal of Physiology
https://www.readbyqxmd.com/read/27802315/exosomal-gapdh-from-proximal-tubule-cells-regulate-enac-activity
#20
Kishore Kumar Jella, Ling Yu, Qiang Yue, Daniel Friedman, Billie J Duke, Abdel A Alli
Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells...
2016: PloS One
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