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The Electrical Properties of Cells
Published in Richard C. Niemtzow, Transmembrane Potentials and Characteristics of Immune and Tumor Cell, 2020
In point of fact, a membrane is not actually necessary in order to observe a potential between two solutions of differing ion concentrations in contact with one another. If a solution of 0.01 M NaCl is carefully layered on top of a solution of 0.1 M NaCl, a potential can be recorded between the two solutions. The potential arises because of the difference in mobility of the Na+ ion and the Cl- ion. Since the rate of movement of Cl- in solution is greater than that of Na+, anions will move across the solution boundary from higher concentration to lower somewhat faster than cations, thus producing a negative potential in the solution of lower concentration. The formula which describes the magnitude of this liquid junction potential is
Endolysosomal Patch Clamping
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Cheng-Chang Chen, Christian Grimm, Christian Wahl-Schott, Martin Biel
Ion selectivity refers to the ability of a channel to permeate specific ions through cell membranes, which is fundamental for multiple important biological functions. Using specific solutions (e.g., bi-ionic solutions) and the equations shown in the Appendix, endolysosomal ion channel selectivity can be determined. The relative permeability to different cations can be calculated from the measured reversal potential using the Goldman–Hodgkin–Katz equation and solutions containing two or more cations (Appendix). To determine the relative permeability of TPC2 for monovalent cations relative to Na+, bi-ionic solutions and equation III.1 in the Appendix were used (Chao et al., 2017). Before measuring the reversal potentials, all reversal potentials should be corrected for liquid junction potentials according to the solutions used in the experiment. The reversal potentials were obtained with a voltage-ramp protocol (−100 mV to +100 mV, 500 ms), using bath solutions containing 160 mM (pH 7.2, HEPES 5 mM) of the respective cations (Na+, Li+, K+, Rb+ and Cs+), and a pipette solution containing 160 mM NaCl (pH 7.2, HEPES 5 mM). The recordings were started with symmetric Na+ solutions (160 mM NaCl in both the pipette and bath), and then the bath solution was exchanged by the respective monovalent cation solution. The results indicate that the selectivity of wild-type TPC2 is comparable to the Eisenman selectivity sequence X: Na+ > Li+ > K+ > Rb+ > Cs+. To estimate the pore diameter of TPC2, the relative permeability ratios of cations relative to Na+ can be plotted against the size of the cations (referring to Stoke’s diameter, e.g., the ionic radii of Li+, K+, Rb+ and Cs+ are 0.6, 1.33, 1.48 and 1.69 Å respectively). Fitting the following equation to the plot will then yield an estimation of the pore diameter:
Superparamagnetic iron oxide nanoparticles (SPIONs) modulate hERG ion channel activity
Published in Nanotoxicology, 2019
Roberta Gualdani, Andrea Guerrini, Elvira Fantechi, Francesco Tadini-Buoninsegni, Maria Rosa Moncelli, Claudio Sangregorio
Electrophysiological recordings were performed in whole-cell patch-clamp configuration. The extracellular solution had the following composition: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4 with NaOH. The pipette contained: 130 mM KCl, 10 mM EGTA, 1 mM MgCl2, 2 mM Mg-ATP, 10 mM HEPES, pH 7.20 with KOH. Solutions were applied to the cell via a gravity-fed perfusion system coupled with a Fast Exchange Open Diamond Bath chamber (VC-6 Six Channel Valve Controller, Warner Instruments, Hamden, CT), in order to allow very fast solution exchange. Currents were sampled at 20 kHz and digitally filtered at 2.9 kHz. The currents were not corrected for the liquid junction potential. Series resistance was electronically compensated. Patch-clamp recordings of cell cultures were carried out at room temperature 48 h after transfection. Patch-clamp electrodes were pulled from Sutter capillary glass (Novato, San Francisco, CA, USA) on a Flaming/Brown type puller (Sutter P-87), and fire polished to 3–4 MΩ resistance, using a microforge (Narishige, Tokyo, Japan). For recordings a Multiclamp 700B amplifier (Molecular Devices Inc., Sunnyvale, CA, USA) and Digidata 1440 data acquisition board (Molecular Devices Inc., Sunnydale, CA, USA) with pCLAMP 10 software (Molecular Devices Inc., Sunnyvale, CA, USA) were used. Data analysis was performed using Origin version 8.0 (OriginLab Corporation, Northampton, MA, USA).
Calcium-dependent, non-apoptotic, large plasma membrane bleb formation in physiologically stimulated mast cells and basophils
Published in Journal of Extracellular Vesicles, 2019
C. Jansen, C Tobita, E. U. Umemoto, J. Starkus, N. M. Rysavy, L. M. N. Shimoda, C. Sung, A.J. Stokes, H Turner
Patch-clamp experiments were performed in the tight-seal whole-cell configuration at 21–25°C. Current and membrane capacitance recordings were captured with EPC-10 amplifier (HEKA, Lambrecht, Germany). RBL-2H3 cells were grown on glass coverslips and bathed in the external Ringer solution. External Ringer solution (in mM): 140 NaCl, 2.8 KCl, 1 CaCl2, 2 MgCl2 and 10 NaHEPES. Internal solution in the pipette contained the following (in mM): 120 Cs-glutamate, 8 NaCl, 1 MgCl2, 8.5 CaCl2, 10 Cs-BAPTA and 10 CsHEPES, which resulted in 1.2 µM buffered internal calcium. The internal solution-filled patch pipettes had a resistance between 2 and 4 MΩ. Following break-in, voltage ramps of 50 ms duration from −100 mV – +100 mV were delivered to the cells with a holding potential of 0 mV at a rate of 0.5 Hz over the period of the recording. All voltages were corrected for a liquid junction potential of 10 mV. Currents were filtered at 2.9 kHz and digitized at 100 µs intervals. Capacitance measurements specifically employed the OnCell_Cm protocol in PATCHMASTER on the HEKA EPC-10 amplifier.
n-3 Polyunsaturated fatty acid supplementation restored impaired memory and GABAergic synaptic efficacy in the hippocampus of stressed rats
Published in Nutritional Neuroscience, 2018
Miguel Ángel Pérez, Valentín Peñaloza-Sancho, Juan Ahumada, Marco Fuenzalida, Alexies Dagnino-Subiabre
Whole-cell recordings were made from the soma of CA1 pyramidal neurons with patch pipettes (4–8 MΩ) filled with an internal solution that contained (in mM): 100 Cs-Gluconate, 10 HEPES, 10 EGTA, 4 Na2-ATP, 10 TEA-Cl, and 1 MgCl2–6H2O, buffered to pH 7.2–7.3 with CsOH. Recordings were made in voltage-clamp modes using an EPC-7 patch-clamp amplifier (HEKA Instruments, Massachusetts, MA USA). In voltage-clamp experiments, the Vh was adjusted to –65 or 0 mV to record excitatory post-synaptic currents (EPSCs) or IPSCs, respectively. Series resistance in the voltage-clamp configuration was compensated to ∼70% and neurons were accepted only when seal resistance was >1 GΩ and series resistance (7–14 MΩ) did not change by more than 10% during the experiment. The liquid junction potential was measured (∼6 mV) but was not corrected. Voltage-clamp data were low-pass filtered at 3.0 kHz and sampled at rates between 6.0 and 10.0 kHz using an A/D converter (ITC-16, InstruTech, Massachusetts, MA USA) and stored with Pulse FIT software (HEKA Instruments, Massachusetts, MA USA). The Pulse Fit program was used to generate stimulus timing signals and transmembrane current pulses. The recording analysis was made off-line with the pClamp software (Clamp-fit, Molecular Devices Corporation, Chicago, IL, USA). EPSCs and IPSCs were evoked with a concentric bipolar electrode (60 µm diameter, tip separation ∼100 µm (FHC Inc., Maine, ME, USA), placed at the stratum radiatum and close to recorded pyramidal neurons (∼100 µm). An average of EPSC (n = 10, 6 rats) and IPSC (n = 10, 6 rats) were obtained under voltage clamp by repeated stimulation at 0.3 Hz. Chemicals were purchased from Sigma-Aldrich Chemistry (Santiago, Chile), and Tocris (Bioscience, Pittsburgh, PA, USA). To establish the pre- or post-synaptic locus expression of these synaptic changes, we analyzed the paired-pulse ratio (PPR), which was quantified by (R2/R1)*100, where R1 and R2 are the amplitude of the first and second IPSC or EPSC, respectively. To determine whether chronic stress simultaneously affects glutamatergic and GABAergic pyramidal neuron synapses (PNs), we voltage-clamped CA1 PNs at the reversal potential for evoked EPSCs or IPSCs, respectively. Values of the reversal potential of EPSCs and IPSCs were estimated from current–voltage relationships of EPSCs (0.3 ± 0.5 mV; n = 10) and IPSCs (−64.2 ± 2.3 mV; n = 10), respectively. In some experiments, excitatory or inhibitory synaptic transmissions were isolated after blocking GABAA receptors with picrotoxin (10 µM) or NMDA and AMPA receptors with D-AP5 (50 µM) and CNQX (20 µM).