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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
Recently, the endolysosomal planar patch clamp method was successfully applied to characterize endolysosomal two-pore channels (TPC) and endolysosomal members of the transient receptor potential superfamily of non-selective cation channels, i.e. mucolipins (TRPML) on intact endolysosomes, isolated, e.g., from fibroblasts that endogenously express the channel or HEK293 cell lines that stably express the channel (Schieder et al., 2010b; Chen et al., 2014; Grimm et al., 2014; Ruas et al., 2015). Solid-matrix planar glass chips were used in the Port-a-Patch system (Nanion, Munich) which represents a planar patch system, where isolated vesicles are attached to a small aperture (<1 μm in diameter) in a microstructured planar borosilicate chip. This glass chip with small aperture allows even small and native endolysosomes to be analyzed. A significant advantage of this approach is the combination of an automated patch clamp device with a pressure control system, which also provides a low-noise and zero-vibration environment. The purification of endolysosomes is the most crucial step of this method. The solid base electrophysiology allows floating cells and vesicles to be patched by an automatic pressure control system, which is essential to form a high-resistance seal (gigaseal) and to establish the whole-endolysosome recording configuration. However, purification requires multiple ultra-centrifugation steps which are relatively time-consuming. The other drawbacks are (1) no vision control by a microscope during measurements; (2) limited choices of solution compositions: high concentrations of luminal Ca2+ and cytosolic F− are needed; (3) purification requires large amounts of cells (>2 × 107) which can be a challenge for certain types of primary cells; and (4) no inside-out or outside-out patches can be established. Nevertheless, the application of the solid base endolysosomal patch clamp approach is advantageous when cells are growing only in suspension and not on glass coverslips, an essential prerequisite for the manual endolysosomal patch clamp approach. In addition, the possible bias towards patch clamping very large vesicles is less of a concern in the solid base endolysosomal patch clamp approach, which does not discriminate between smaller and larger vesicles, as long as they are larger than the aperture of the chip.
New methodological approaches to atrial fibrillation drug discovery
Published in Expert Opinion on Drug Discovery, 2021
Initial screening of molecules with a desired effect on individual ion channels can be achieved in a high-throughput manner using systems such as fluorescence-based screening technologies and automated patch clamp with commercially available validated cell lines that express target ion channels. [99] To further study the drug effect on actual cardiomyocytes, it is necessary to sacrifice animals to harvest cardiomyocytes. During drug development, numerous candidate compounds may need to be tested, hence it may be necessary to sacrifice many animals. This raises ethical concerns and consumes a large amount of resources. There have been attempts to immortalize murine atrial cardiomyocytes to produce cell lines in order to produce an alternative source of cardiomyocytes. These have included transfecting neonatal rat cardiomyocytes with a lentiviral vector carrying doxycycline-controlled expression of simian virus 40 large T antigen to control cell proliferation versus maturation. [100]
Automated patch clamp in drug discovery: major breakthroughs and innovation in the last decade
Published in Expert Opinion on Drug Discovery, 2021
Alison Obergrussberger, Søren Friis, Andrea Brüggemann, Niels Fertig
Patch-clamp electrophysiology remains an important technique in studying ion channels; indeed, it is still considered the gold standard since it was first described by Neher and Sakmann in the 1970s [1]. Ion channels are integral membrane proteins which allow ion current flow across the cell membrane. They are involved in almost all physiological processes, and their malfunction underlies many disease states, making them important pharmacological targets. Conventional patch clamp is a very information-rich technique, but it requires skilled personnel to perform experiments, and typically, only one experiment can be performed at a time. In the late 1990s and early 2000s, the field of ion-channel research was revolutionized by the development of the automated patch-clamp (APC) technique. The most successful approach involved replacing the patch-clamp pipette with a planar substrate (for review, see [2]), making the experiments easier to perform and offering the option for recording multiple cells in parallel. In the last two decades, much has changed in the field of ion-channel drug discovery and APC, with increased throughput and enhanced simplicity. We summarize the main changes in the last decade and attempt to look into the future of what’s to come.
An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery
Published in Expert Opinion on Drug Discovery, 2018
Alison Obergrussberger, Tom A. Goetze, Nina Brinkwirth, Nadine Becker, Søren Friis, Markus Rapedius, Claudia Haarmann, Ilka Rinke-Weiß, Sonja Stölzle-Feix, Andrea Brüggemann, Michael George, Niels Fertig
The ability to record ion channels at physiological temperature is an important feature for automated patch clamp systems. The Patchliner, IonFlux, IonFlux Mercury, and QPatch all offer temperature control at physiological temperature. Indeed, using the IonFlux [20,21] and Patchliner [5], the temperature-dependent effects of different compounds, for example, erythromycin could be demonstrated on the hERG channel. The Patchliner has also been used to show temperature-dependent effects of allosteric modulators on the nAChα7R [22]. The allosteric potentiation of the nAChα7R by PNU120596 was reduced at physiological temperature compared with room temperature [22] as previously reported using manual patch clamp [23]. In addition to recordings at physiological temperature, brief temperature pulses using the Patchliner, that is, heating the temperature of the external solution inside the pipette and rapidly applying this to cells expressing TRPV1 or TRPV3, have been used to study heat activated channels [5,24]. It has been proposed that potential pain therapeutics exert their hyperthermic side effects by blocking the proton-induced activation of TRPV1 [25]. Therefore, the ability to test inhibition of compounds on heat, capsaicin, and proton-activated TRPV1 responses separately may be a huge advantage in the search for safe pain therapeutics targeting TRPV1.