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Single channel patch clamp

Nan Zhang, Shengchang Yang, Chang Wang, Jianghua Zhang, Lifang Huo, Yiru Cheng, Chuan Wang, Zhanfeng Jia, Leiming Ren, Lin Kang, Wei Zhang
Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by β-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca(2+) homeostasis, the underlying mechanisms have not been fully explored...
October 12, 2016: Neuropharmacology
Stephen P Brazier, Vsevolod Telezhkin, Paul J Kemp
Proteomic studies have suggested a biochemical interaction between α subunit of the large conductance, voltage- and Ca(2+)-activated potassium channel (BKCaα), and annexin A5 (ANXA5), which we verify here by coimmunoprecipitation and double labelling immunocytochemistry. The observation that annexin is flipped to the outer membrane leaflet of the plasma membrane during apoptosis, together with the knowledge that the intracellular C-terminal of BKCaα contains both Ca(2+)-binding and a putative annexin-binding motif, prompted us to investigate the functional consequences of this protein partnership to cell death...
2016: Oxidative Medicine and Cellular Longevity
I Kolb, W A Stoy, E B Rousseau, O A Moody, A Jenkins, C R Forest
Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute...
October 11, 2016: Scientific Reports
Donatella Contini, Steven D Price, Jonathan J Art
Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we use dual patch-clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulate membrane potentials and extend the range of information transfer. High-fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance...
September 16, 2016: Journal of Physiology
Y H Liu, Y P Wang, Y Wang, K T Ma, J Q Si, L Li
OBJECTIVE: The purpose of this paper was to study the electrophysiological properties and the type of potassium channels on cell membrane in the stria vascularis pericytes in cochlear of guinea pig. METHODS: Firstly examined the expression of the stria vascularis pericytes by desmin, a marker of pericytes, in cochlear of guinea pig with immunofluorescent method. Using whole-cell patch clamp recording techniques to observe electrophysiological properties in the cochlear pericytes in stria vascularis of guinea pig...
August 7, 2016: Zhonghua Er Bi Yan Hou Tou Jing Wai Ke za Zhi, Chinese Journal of Otorhinolaryngology Head and Neck Surgery
Kiril L Hristov, Shankar P Parajuli, Aaron Provence, Georgi V Petkov
In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability...
September 7, 2016: American Journal of Physiology. Renal Physiology
Jose L Sanchez-Alonso, Anamika Bhargava, Thomas O'Hara, Alexey V Glukhov, Sophie Schobesberger, Navneet Bhogal, Markus B Sikkel, Catherine Mansfield, Yuri E Korchev, Alexander R Lyon, Prakash P Punjabi, Viacheslav O Nikolaev, Natalia A Trayanova, Julia Gorelik
RATIONALE: Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. OBJECTIVE: To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF. METHODS AND RESULTS: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes...
September 30, 2016: Circulation Research
Jeffery A Boychuk, Jordan S Farrell, Laura A Palmer, Anna C Singleton, Quentin J Pittman, G Campbell Teskey
The mechanisms by which distinct movements of a forelimb are generated from the same area of motor cortex have remained elusive. Here we examined a role for HCN channels, given their ability to alter synaptic integration, in the expression of forelimb movement responses during intracortical microstimulation (ICMS) and movements of the forelimb on a skilled reaching task. We used short-duration high resolution ICMS to evoke forelimb movements following pharmacological (ZD7288), experimental (electrically induced cortical seizures) or genetic approaches that we confirmed with whole-cell patch clamp to substantially reduce Ih current...
August 29, 2016: Journal of Physiology
Shijun Yan, Fang Du, Long Wu, Zhihua Zhang, Changjia Zhong, Qing Yu, Yongfu Wang, Lih-Fen Lue, Douglas G Walker, Justin T Douglas, Shirley Shidu Yan
Mitochondrial abnormalities are well-known to cause cognitive decline. However, the underlying molecular basis of mitochondria-associated neuronal and synaptic dysfunction in the diabetic brain remains unclear. Here, using a mitochondrial single channel patch clamp and cyclophilin D (CypD)-deficient mice (Ppif (-/-)) with streptozotocin (STZ)-induced diabetes, we observed an increase in the probability of Ca(2+)-induced mitochondrial permeability transition pore (mPTP) opening in brain mitochondria of diabetic mice, which was further confirmed by mitochondrial swelling and cytochrome c release induced by Ca(2+) overload...
August 23, 2016: Diabetes
S Rezania, S Kammerer, C Li, B Steinecker-Frohnwieser, A Gorischek, T T J DeVaney, S Verheyen, C A Passegger, N Ghaffari Tabrizi-Wizsy, H Hackl, D Platzer, A H Zarnani, E Malle, S W Jahn, T Bauernhofer, W Schreibmayer
BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced...
2016: BMC Cancer
Masahiro Ishii, Hirofumi Hashimoto, Jun-Ichi Ohkubo, Toyoaki Ohbuchi, Takeshi Saito, Takashi Maruyama, Mitsuhiro Yoshimura, Yukiyo Yamamoto, Koichi Kusuhara, Yoichi Ueta
Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons...
September 6, 2016: Neuroscience Letters
Tengis S Pavlov, Vladislav Levchenko, Daria V Ilatovskaya, Carol Moreno, Alexander Staruschenko
OBJECTIVE: The Dahl salt-sensitive rat is a well-established model of salt-sensitive hypertension. The goal of this study was to assess the expression and activity of renal sodium channels and transporters in the renin-deficient salt-sensitive rat. METHODS: Renin knockout (Ren(-/-)) rats created on the salt-sensitive rat background were used to investigate the role of renin in the regulation of ion transport in salt-sensitive hypertension. Western blotting and patch-clamp analyses were utilized to assess the expression level and activity of Na(+) transporters...
July 2016: Journal of the Renin-angiotensin-aldosterone System: JRAAS
Sergey Yechikov, Raul Copaciu, Jessica M Gluck, Wenbin Deng, Nipavan Chiamvimonvat, James W Chan, Deborah K Lieu
Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles...
July 19, 2016: Stem Cells
Ye-Tao Li, Xin-Ling Du
Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated...
June 2016: Journal of Huazhong University of Science and Technology. Medical Sciences
Giedrius Kanaporis, Lothar A Blatter
Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca(2+)-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization...
November 2016: Channels
Chang Liu, Changan Xie, Khade Grant, Zhuocheng Su, Weihua Gao, Qinglian Liu, Lei Zhou
Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide-regulated (HCN) channels...
July 2016: Journal of General Physiology
M Q Sun, L L Qian, S P Dang, Y Wu, X Tang, Y Ji, X Y Wang, D Y Xia, W Wang, Q Chai, T Lu, R X Wang
OBJECTIVE: To investigate the mechanisms of docosahexaenoic acids (DHA) on activating large conductance calcium-activated potassium channels (BK channels) in normal rat coronary smooth muscle cells. METHODS: Normal coronary smooth muscle cells were isolated by enzyme digestion from Sprague-Dawley rats. BK currents were recorded by patch clamp in whole cell and single channel configurations, respectively. The effects of DHA on cytosolic calcium concentrations were examined by recording the changes of fluorescence intensity ratios...
June 24, 2016: Zhonghua Xin Xue Guan Bing za Zhi
Frank J P Kühn, Cornelia Kühn, Mathis Winking, Daniel C Hoffmann, Andreas Lückhoff
The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of hTRPM2. The TRPM2 orthologue from Nematostella vectensis (nv) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca2+-imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells...
2016: PloS One
Héctor Solís-Chagoyán, Edgar Flores-Soto, Jorge Reyes-García, Marcela Valdés-Tovar, Eduardo Calixto, Luis M Montaño, Gloria Benítez-King
In adulthood, differentiation of precursor cells into neurons continues in several brain structures as well as in the olfactory neuroepithelium. Isolated precursors allow the study of the neurodevelopmental process in vitro. The aim of this work was to determine whether the expression of functional Voltage-Activated Ca(2+) Channels (VACC) is dependent on the neurodevelopmental stage in neuronal cells obtained from the human olfactory epithelium of a single healthy donor. The presence of channel-forming proteins in Olfactory Sensory Neurons (OSN) was demonstrated by immunofluorescent labeling, and VACC functioning was assessed by microfluorometry and the patch-clamp technique...
2016: International Journal of Molecular Sciences
Linda G Zachariassen, Ljudmila Katchan, Anna G Jensen, Darryl S Pickering, Andrew J R Plested, Anders S Kristensen
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells...
July 5, 2016: Proceedings of the National Academy of Sciences of the United States of America
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