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Generation of the Action Potential
Published in Nassir H. Sabah, Neuromuscular Fundamentals, 2020
In contrast to a voltage clamp, a current clamp is a method of intracellular recording involving measurement of the voltage difference across the cellular membrane while injecting a constant positive or negative current (as “square” d.c. pulses) into the cell. Current clamp is used to study the response of a neuron to an inward or outward electric current, which is important, for example, for understanding how the neuron responds to neurotransmitters that act by opening membrane ion channels.
Neuropeptide Receptor-Ion Channel Coupling in the Mammalian Brain
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
The experimental approaches used to identify the effectors linked to a particular neuropeptide in the mammalian brain are the same as those applied in the more widely studied neurotransmitter candidates such as 5-HT, GABA, acetylcholine acting at muscarinic receptors, and adrenergic receptor-effector systems (see reviews1,2,3). Initially electrophysiological recordings are made to ascertain which, if any, ionic currents are affected by the neuropeptide. For this type of experiment intracellular recording, usually from identified neurones in a brain slice preparation, can be undertaken. When discontinuous single-electrode voltage clamp methods are also applied this technique will yield information on the ionic current(s) involved in the neuropeptide response. A more recent introduction to the field of brain slice electrophysiology is the use of whole-cell recording. This variation of the patch clamp technique is also commonly employed for voltage-clamp studies. It offers the advantage of improved voltage-clamp and the possibility to dialyze, at least in part, the cytoplasmic contents of the neurone. This is advantageous when one wishes to introduce suspected intracellular messengers but can be a problem if the initial whole-cell recording protocol leads to loss of neuropeptide response possibly as a result of loss of endogenous messenger.
Electrophysiological Recording of a Gain-of-Function Polycystin-2 Channel with a Two-Electrode Voltage Clamp
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Courtney Ng, Zhifei Wang, Bin Li, Yong Yu
The voltage clamp method allows electrophysiologists to measure the electric current caused by the movement of ions across a cell membrane while holding the Vm at a certain level. When a voltage clamp experiment is done, membrane potential (voltage) will first be measured and then changed (clamped) to the desired value by injecting current. This current is equal, although opposite in direction, to the current passing across the cell membrane through ion channels at the set voltage. Thus, by recording the injected current, we are able to measure the current passing through ion channels. This method was first designed by Cole and Marmont in 1949,35 and was utilized and improved into the two-electrode voltage clamp (TEVC) method by Hodgkin, Huxley, and Katz in 1952 for measuring current from giant squid axons.36 Today, TEVC is a widely used electrophysiological technique for investigating the properties of ion channels and electrogenic transporters expressed in large cells such as Xenopus oocytes. The applications of this technique include characterization of the ion selectivity, activation and desensitization, structural-functional analysis, and screening for agonists or activators and antagonists or inhibitors.
Rituximab Attenuated Lipopolysaccharide-Induced Oxidative Cytotoxicity, Apoptosis, and Inflammation in the Human Retina Cells via Modulating the TRPM2 Signaling Pathways
Published in Ocular Immunology and Inflammation, 2022
Hatice Daldal, Mustafa Nazıroğlu
The whole cell electrophysiology configuration records were obtained in EPC 10 amplifier equipped with Patch-master software (HEKA, Lamprecht, Germany). In the records (n = 4–6), the holding potential (−65 mV) and pipette resistances (3 and 7 MΩ) were kept as described in the previous experiments.4,37 In the records, CaCl2 (1 mM), MgCl2 (1 mM), KCl (5 mM), NaCl (145 mM), D-glucose (10 mM), and HEPES (10 mM) added to the extracellular (patch chamber) solution. For preparing the Na+ free patch chamber solution, we used 150 mM N-methyl-D-glucamine (NMDG+) instead of 145 mM NaCl (pH: 7.4). The cytCa2+ was adjusted to 1 μM instead of 100 nM, when the TRPM2 is gated in the presence of high intracellular cytCa2+.38 The TRPM2 were stimulated in the cells by using the cytosolic ADPR (1 mM in patch pipette), although it was blocked by extracellular ACA (25 μM in patch-chamber). The voltage-clamp results were shown as the current density (pA/pF).
Dieckol is a natural positive allosteric modulator of GABAA-benzodiazepine receptors and enhances inhibitory synaptic activity in cultured neurons
Published in Nutritional Neuroscience, 2021
Sangoh Kwon, Jong Hoon Jung, Suengmok Cho, Kwang-Deog Moon, Jaekwang Lee
Whole-cell recordings were prepared using an Axon 700B patch-clamp amplifier (Molecular Device, Foster City, CA, USA). Membrane currents were sampled and analyzed using a Digidata 1440 interface and a personal computer running Clampex and Clampfit software (Version 10, Molecular Device). In voltage-clamp mode, the membrane potential was held at –60 mV for whole-cell current recording. All electrophysiological experiments were carried out at room temperature (24 ± 2°C). The standard external solution contained (in mM): 150 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 N-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES), and 5.5 glucose (pH 7.3 adjusted with NaOH, 325–330 mOsm/L). The pipette solution for GABAAR response composed of (in mM): 130 CsCl, 2 MgCl2, 2 CaCl2, 10 ethylene glycol tetraacetic acid (EGTA), 2 Mg-ATP, 2 Tris-GTP, 10 HEPES, 10 Glucose (pH 7.2 adjusted with CsOH). To record neuronal activity in current-clamp mode, the pipette solution composed of (in mM): 140 K-gluconate, 7 NaCl, 2 Mg-ATP, 2 Tris-GTP, 10 HEPES, 10 Glucose (pH 7.3 adjusted with KOH).
Curcumin diminishes cisplatin-induced apoptosis and mitochondrial oxidative stress through inhibition of TRPM2 channel signaling pathway in mouse optic nerve
Published in Journal of Receptors and Signal Transduction, 2020
Dilek Özkaya, Mustafa Nazıroğlu
Whole-cell voltage clamp technique records were made from the SH-SY5Y cells using an EPC 10 amplifier and Patchmaster software (HEKA, Lamprecht, Germany). The compositions of standard bath solutions were described in previous studies [25,26]. The TRPM2 channel can be activated by ADPR and H2O2 when there is a high cytosolic Ca2+ concentration in the cells [26]. Consequently, the cytosolic Ca2+ concentration was adjusted to 1 μM instead of 0.1 μM. The holding potentials in the SH-SY5Y cells were kept at −60 mV. In the patch-clamp experiment, the cells were perfused with standard cytosolic solution containing ADPR (1 mM) for stimulation and with extracellular ACA (25 μM) for inhibition. Current-voltage (I–V) relationships were obtained from voltage ramps from −100 to +60 mV applied over 300 ms. The borosilicate pipette resistances were adjusted to about 2–7 MΩ by using a puller (PC-10 Narishige International Limited, London, UK). The maximal current amplitudes (pA) in a single cell were divided by the cell capacitance (pF) and the resulting values were represented as the current density (pA/pF). All patch-clamp analyses were performed daily at room temperature (22 ± 3 °C).