High Throughput Electrophysiology with Xenopus Oocytes

Abstract

Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis.Until recently, cellular electrophysiology was performed manually, one cell at a time by a single operator. However, methods of high-throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high-throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications.

α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

“…Since expression of nAChRs in Xenopus oocytes have several limitations, including the possible formation of different subunit stoichiometries, and a current contribution from calcium-dependent chloride channels [32,33], we also investigated the function of α7 versus α7β2 nAChRs expressed in the mammalian cell line HEK293. In HEK293 cells, epibatidine exhibited similar peak response currents in α7 and α7β2 nAChR expressing cells, which differs from the 5 fold difference seen in Xenopus oocytes, whereas α7β2 nAChRs displayed a reduced current response to compound B compared to α7 nAChR homomers.…”

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

“…The ease with which oocytes can be injected and the high efficiency of cRNA expression may facilitate the study of heteromeric channels formed between mutant versions of MscS (or between MscS and its five E. coli homologues; Booth et al, 2007) when coinjected, and will allow for the incorporation of unnatural amino acids (Nowak et al, 1998;Torrice et al, 2009). It may also be possible to make use of high-throughput technologies to quickly analyze MscS variants or the effect of drugs on MscS function (Dunlop et al, 2008;Papke and Smith-Maxwell, 2009).…”

Patch-Clamp Recordings of Action Potentials From Human Atrial Myocytes: Optimization Through Dynamic Clamp

“…Various in vitro (Papke and Smith-Maxwell, 2009;Ebert et al, 2012;Devalla et al, 2015;Hu et al, 2018) and in vivo animal models (Olgin and Verheule, 2002;Finet et al, 2009;Nishida et al, 2010), including AF models, are available for atrial drug testing, all with their individual strengths and limitations. While these models provide an excellent starting point for atrial and AFrelated drug studies, assessments of action potentials (APs) of native human atrial cardiomyocytes remain essential due to distinct inter-species differences in ion channel expression profiles (Varró et al, 2021), and because drugs frequently act through AP shortening or prolonging effects, which may be frequency dependent (Peyronnet and Ravens, 2019).…”