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Single channel patch clamp

Floriana Fruscione, Pierluigi Valente, Bruno Sterlini, Alessandra Romei, Simona Baldassari, Manuela Fadda, Cosimo Prestigio, Giorgia Giansante, Jacopo Sartorelli, Pia Rossi, Alicia Rubio, Antonio Gambardella, Thierry Nieus, Vania Broccoli, Anna Fassio, Pietro Baldelli, Anna Corradi, Federico Zara, Fabio Benfenati
Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c...
March 15, 2018: Brain: a Journal of Neurology
Guang-Zhe Huang, Mutsuo Taniguchi, Ye-Bo Zhou, Jing-Ji Zhang, Fumino Okutani, Yoshihiro Murata, Masahiro Yamaguchi, Hideto Kaba
The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB...
April 2018: Learning & Memory
Peng Yu, Lili Hu, Jinyan Xie, Sisi Chen, Lin Huang, Zixuan Xu, Xiao Liu, Qiongqiong Zhou, Ping Yuan, Xia Yan, Jiejin Jin, Yang Shen, Wengen Zhu, Linghua Fu, Qi Chen, Jianhua Yu, Jianxin Hu, Qing Cao, Rong Wan, Kui Hong
BACKGROUND: Cardiovascular complications are major causes of mortality and morbidity in diabetic patients. The mechanisms underlying the progression of diabetic heart (DH) to ventricular arrhythmias are unclear. O-linked GlcNAcylation (O-GlcNAc) is a reversible post-translational modification for the regulation of diverse cellular processes. The purpose of this study was to assess whether the cardiac voltage-gated sodium channel (Nav1.5) is subjected to O-linked GlcNAcylation (O-GlcNAc), which plays an essential role in DH-induced arrhythmias...
February 27, 2018: International Journal of Cardiology
Mickaël F El Hachmane, Charlotta S Olofsson
Ca2+ impacts a large array of cellular processes in every known cell type. In the white adipocyte, Ca2+ is involved in regulation of metabolic processes such as lipolysis, glucose uptake and hormone secretion. Although the importance of Ca2+ in control of white adipocyte function is clear, knowledge is still lacking regarding the control of dynamic Ca2+ alterations within adipocytes and mechanisms inducing intracellular Ca2+ changes remain elusive. Own work has recently demonstrated the existence of store-operated Ca2+ entry (SOCE) in lipid filled adipocytes...
March 7, 2018: Biochemical and Biophysical Research Communications
Rajeev Yadav, H Peter Lu
The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording...
March 8, 2018: Physical Chemistry Chemical Physics: PCCP
Katharina T Schrapers, Gerhard Sponder, Franziska Liebe, Hendrik Liebe, Friederike Stumpff
Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging...
2018: PloS One
Jing-Na Ren, Ben-Long Rao, Hong-Xin Ma, Mao-Mao Sha, Yi Kuang, Zheng-Xin Xu
To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC₅₀ of the drug was in the range of 10-30 μmol•L⁻¹, and the drug concentration of 1-3 μmol•L⁻¹ for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current (Ito) of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied...
December 2017: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
Monika Plescher, Gerald Seifert, Jan Niklas Hansen, Peter Bedner, Christian Steinhäuser, Annett Halle
Microglia, the central nervous system resident innate immune cells, cluster around Aβ plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aβ plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aβ plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes...
March 1, 2018: Glia
Yingfei Xiong, Sasa Teng, Lianghong Zheng, Suhua Sun, Jie Li, Ning Guo, Mingli Li, Li Wang, Feipeng Zhu, Changhe Wang, Zhiren Rao, Zhuan Zhou
Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether/how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. In contrast, two recent papers report vesicular ATP release using more direct assays. In the present work, by using patch-clamped "ATP-sniffer-cells", we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes...
February 28, 2018: Journal of Physiology
Bailin Liu, Yanping Liu, Ruixiu Shi, Xueqin Feng, Xiang Li, Wenna Zhang, Jue Wu, Na Li, Xiuwen Zhou, Miao Sun, Zhice Xu
BACKGROUND/AIMS: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. METHODS: Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR...
February 21, 2018: Cellular Physiology and Biochemistry
Man-Song Li, Elizabeth A Cowley, Yassine El Hiani, Paul Linsdell
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals". Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electro-diffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown...
February 23, 2018: Journal of Biological Chemistry
Shunsuke Ohura, Haruyuki Kamiya
Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the functional significance, possible activity-dependent modulation of axonal spikes has not been studied extensively, partly due to inaccessibility of the small structures of axons for electrophysiological recordings. In this study, we tested the possibility of use-dependent changes in axonal spikes at the hippocampal mossy fibers, where direct recordings from the axon terminals are readily feasible...
January 2018: ENeuro
Guangyan Wu, Lin Li, Bo Chen, Chuan Chen, Duqiang Luo, Bingjun He
A new meroterpenoid, named acetoxydehydroaustin A (1) and the known meroterpenoid austin (2) were isolated from the plant pathogenic fungus Verticillium albo-atrum. Their structures were established based on general spectroscopic techniques and the relative configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. We first investigated and identified their significant electrophysiological effects on the gating kinetics of voltage-gated sodium channels in central neurons acutely dissociated from Helicoverpa armigera using whole-cell patch clamp technique...
January 2018: Pesticide Biochemistry and Physiology
Chunli Wang, Ying Chen, Bixia Zheng, Mengshu Zhu, Jia Fan, Juejin Wang, Zhanjun Jia, Songming Huang, Aihua Zhang
Inactivated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patient's genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2bp from the 5' splice donor site in exon 15, further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother...
February 14, 2018: American Journal of Physiology. Renal Physiology
Nicholas C Vierra, Matthew T Dickerson, Kelli L Jordan, Prasanna K Dadi, Ketaki A Kadare, Molly K Altman, Sarah C Milian, David A Jacobson
OBJECTIVE: Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in β-cells, K+ flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR) from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca2+ handling and somatostatin secretion...
January 31, 2018: Molecular Metabolism
Hugo Poulin, Pascal Gosselin-Badaroudine, Savine Vicart, Karima Habbout, Damien Sternberg, Serena Giuliano, Bertrand Fontaine, Saïd Bendahhou, Sophie Nicole, Mohamed Chahine
Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP)...
February 1, 2018: Scientific Reports
Guohui Zhang, Jianmin Cui
The Xenopus oocyte expression system is ideal for electrophysiological characterization of voltage-dependent and ligand-dependent ion channels because of its relatively low background of endogenous channels and the large size of the cell. Here, we present a protocol to study voltage- and ligand-dependent activation of ion channels expressed in Xenopus oocytes using patch-clamp techniques designed to control both the membrane voltage and the intracellular solution. In this protocol, the large conductance voltage- and Ca2+-activated K+ (BK) channel is studied as an example...
January 30, 2018: Cold Spring Harbor Protocols
Dongze Zhang, Huiyin Tu, Liang Cao, Hong Zheng, Robert L Muelleman, Michael C Wadman, Yu-Long Li
BACKGROUND: Attenuated cardiac vagal activity is associated with ventricular arrhythmogenesis and related mortality in patients with chronic heart failure. Our recent study has shown that expression of N-type Ca2+ channel α-subunits (Cav2.2-α) and N-type Ca2+ currents are reduced in intracardiac ganglion neurons from rats with chronic heart failure. Rat intracardiac ganglia are divided into the atrioventricular ganglion (AVG) and sinoatrial ganglion. Ventricular myocardium receives projection of neuronal terminals only from the AVG...
January 15, 2018: Journal of the American Heart Association
Xingxing Liu, Haibo Song, Jun Yang, Cheng Zhou, Yi Kang, Linghui Yang, Jin Liu, Wensheng Zhang
OBJECTIVE: (R)-2-methoxyethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate hydrochloride (ET-26 HCl) is a novel etomidate analogue. The purpose of this study was to characterize whether ET-26 HCl could retain the superior myocardial performance of etomidate in vivo and in vitro. METHODS: In vivo, the influence of ET-26 HCl and etomidate on the cardiac function of dogs was confirmed using echocardiography and electrocardiogram. In vitro, a Langendorff preparation was used to examine direct myocardial performance in isolated rat hearts, and a whole-cell patch-clamp technique was used to study effects on the human ether-a-go-go-related gene (hERG) channel...
2018: PloS One
Jia Liu, Hermann Bihler, Carlos M Farinha, Nikhil T Awatade, Ana M Romão, Dayna Mercadante, Yi Cheng, Isaac Musisi, Walailak Jantarajit, Yiting Wang, Zhiwei Cai, Margarida D Amaral, Martin Mense, David N Sheppard
BACKGROUND AND PURPOSE: Rescue of F508del-cystic fibrosis transmembrane conductance regulator (CFTR), the most common cystic fibrosis (CF) mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004 (from WO2010/068863), a small molecule designed to independently correct protein processing and channel gating defects. EXPERIMENTAL APPROACH: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques...
January 10, 2018: British Journal of Pharmacology
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