A dual voltage clamp technique to study gap junction hemichannels in astrocytes cultured from neonatal rodent spinal cords

Astrocytes express surface channels involved in purinergic signaling, and among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) mediate ATP release that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, i.e., open and close, under physiological and pathological conditions, single channel conductance of Px1 HCs is not well defined. Here, we developed a dual voltage clamp technique in HeLa cells overexpressing human Px1-YFP, and then applied this system to rodent spinal astrocytes. Single channels were recorded in cell attached patches and evoked with ramp cycles of 2 s duration and -/+ 80-100 mV amplitude or rectangular pulses through another pipette in whole cell clamp. Conductance of Px1 HC openings recorded during ramp stimuli ranged 25-110 pS. Based on their single channel conductances, Px1 HCs could be distinguished from Cx43 HCs and P2X7 receptors (P2X7Rs) in spinal astrocytes during dual voltage clamp experiments. Furthermore, we found that single channel activity of Cx43 HCs and P2X7Rs was increased, and that of Px1 HCs was decreased, in spinal astrocytes treated for 7h with FGF-1, a growth factor implicated in neurodevelopment, repair and inflammation.

Ginsenoside Rg3 Alleviates Antithyroid Cancer Drug Vandetanib-Induced QT Interval Prolongation

Inhibition of human ether-a-go-go-related gene (hERG) potassium channel is responsible for acquired long QT syndromes, which leads to life-threatening cardiac arrhythmia. A multikinase inhibitor, vandetanib, prolongs the progression-free survival time in advanced medullary thyroid cancer. However, vandetanib has been reported to induce significant QT interval prolongation, which limits its clinical application. Some studies have showed that ginsenoside Rg3 decelerated hERG K(+) channel tail current deactivation. Therefore, in this study, we aim to confirm whether ginsenoside Rg3 targeting hERG K(+) channel could be used to reverse the vandetanib-induced QT interval prolongation. Electrocardiogram (ECG) and monophasic action potential (MAP) were recorded using electrophysiology signal sampling and analysis system in Langendorff-perfused rabbit hearts. The current clamp mode of the patch-clamp technique was used to record transmembrane action potential. The whole-cell patch-clamp technique was used to record the hERG K+ current. In Langendorff-perfused hearts, vandetanib prolonged the QT interval in a concentration-dependent manner with an IC50 of 1.96 μmol/L. In human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), vandetanib significantly prolonged the action potential duration at 50%, 70%, and 90% repolarization (APD50, APD70, and APD90). In stable transfected human hERG gene HEK293 cells, vandetanib caused concentrate-dependent inhibition in the step and tail currents of hERG. As expected, ginsenoside Rg3 relieved vandetanib-induced QT interval prolongation in Langendorff-perfused heart and reversed vandetanib-induced APD prolongation in hiPSC-CMs. Furthermore, ginsenoside Rg3 alleviated vandetanib-induced hERG current inhibition and accelerated the process of the channel activation. Ginsenoside Rg3 may be a promising cardioprotective agent against vandetanib-induced QT interval prolongation through targeting hERG channel. These novel findings highlight the therapeutic potential of ginsenoside to prevent vandetanib-induced cardiac arrhythmia.

Nanotechnology: new opportunities for the development of patch‐clamps

The patch-clamp technique is one of the best approaches to investigate neural excitability. Impressive improvements towards the automation of the patch-clamp technique have been made, but obvious limitations and hurdles still exist, such as parallelization, volume displacement in vivo, and long-term recording. Nanotechnologies have provided opportunities to overcome these hurdles by applying electrical devices on the nanoscale. Electrodes based on nanowires, nanotubes, and nanoscale field-effect transistors (FETs) are confirmed to be robust and less invasive tools for intracellular electrophysiological recording. Research on the interface between the nanoelectrode and cell membrane aims to reduce the seal conductance and further improve the recording quality. Many novel recording approaches advance the parallelization, and precision with reduced invasiveness, thus improving the overall intracellular recording system. The combination of nanotechnology and the present intracellular recording framework is a revolutionary and promising orientation, potentially becoming the next generation electrophysiological recording technique and replacing the conventional patch-clamp technique. Here, this paper reviews the recent advances in intracellular electrophysiological recording techniques using nanotechnology, focusing on the design of noninvasive and greatly parallelized recording systems based on nanoelectronics.

Electrophysiology study using patch clamp technique in sensory neurons

The electrophysiological and pharmacological study involving sensory and autonomic neurons enables the development of new effective agents in the treatment of neuropathic disorders, since they enable the elucidation of the mechanisms underlying the malfunction of the nervous system. In this context, the patch clamp technique increased the study of cells, providing a high-resolution method at the molecular level for observing the flow of ions through ion channels characteristic of excitable cells [1], such as the neurons. When using different protocols with combinations of intracellular and extracellular solutions with specific pharmacological agents, this technique allows different unit and/or macroscopic records of active and passive electrical variables of cellular activity [2] that it favored the Nobel Prize in physiology or medicine to Erwin Neher and Bert Sakmann in 1991. Although the whole cell mode is the most used configuration in health-related researches, little is known in health courses. To apply this technique to neurons, it is commonly necessary to dissociate neurossomas. Figure 01 shows sensory neurossome of the dorsal root ganglion (GRD) of rats from the bioterium of the State University of Ceará (CEUA process number 10339956-9). The process of isolating neurossomas from the intact ganglion consists of two phases: 1) Collagenase (1mg / ml for 75 min) and Trypsin + EDTA (0.25% and 0.025%, respectively, for 12 minutes); 2) Mechanical dispersion with 3 Pasteur glass pipettes with decreasing diameter (2.5 mm, 1 mm and 0.5 mm, respectively). Then, the neurossomas were plated on coverslips previously treated with poly-D-lysine maintained in supplemented DMEM and incubated at 37 °C and 5% CO2 [3]. The figure shows a neurossoma 24h after plating. This cell has approximately 25 µM in diameter, which it plays role nociception function [4]. Furthermore, the nucleus is not centralized, the cell does not have neurites. As for the micropipette, capillaries were used for micro-hematocrit without heparin (75 mm length, 1 mm inner diameter and 1.5 mm outer diameter) for making with tip resistance range from 1 and 3 MΩ after filling with the solution to compose intracellular medium [5]. In this technique, a microelectrode was micrometrically move toward until it lightly touched the plasma membrane. Then, a continuous negative pressure was applied to increase the contact of the glass with the membrane, stabilizing the seal (interaction between membrane and glass) and increasing it until its resistance reaches the order of 109 ohm (GΩ). Then, more suction was applied to cause the cell surface under the microelectrode to rupture, thus providing access to the interior of the cell, allowing excellent control of the cell membrane potential and, consequently, high-fidelity records of ionic currents that flow through ion channels present in the plasma membrane of neurossomas.

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.