Biomaterial Database

Studying Nicotinic Acetylcholine Receptors Using the IonFlux™ Microfluidic-Based Automated Patch-Clamp System with Continuous Perfusion and Fast Solution Exchange. 2020

Ali Yehia, and Haiyang Wei

Fluxion Biosciences, Alameda, California.

Automated patch-clamp (APC) systems have become indispensable tools of drug-discovery programs by allowing high-throughput electrophysiology-based screening of ion channel compounds. The recent development and introduction of microfluidics-based APC systems have made it possible to study the interactions of ligand-gated ion channels with pharmacological reagents, such as agonists, antagonists, or positive allosteric modulators (PAMs), with reliable pharmacological results comparable to those of the gold-standard manual patch-clamp technique while maintaining high-throughput capacity. Many ligand-gated ion channels exhibit rapid desensitization upon repetitive introduction of ligands; this loss of channel activity in the absence of pharmacological interaction poses a challenge for developing accurate, precise, and robust assays with high success rate, low run-down, and reliable pharmacological results. Here we present procedures to study nicotinic acetylcholine receptors (nAChRs) with the IonFlux™, an automated patch-clamp system with continuous flow and precise fluidic exchange; these procedures can also be generalized to the study of other ligand-gated ion channels. We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand-gated ion channels, generating data that closely mimic those from manual patch-clamp conditions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring agonist concentration-dependent response Basic Protocol 2: Measuring antagonist concentration-dependent response Basic Protocol 3: Measuring positive allosteric modulator (PAM) concentration-dependent response Support Protocol 1: Basic IonFlux system operation Support Protocol 2: Plate care and filling Support Protocol 3: Plate preparation for water rinsing Support Protocol 4: Water rinsing of plates Support Protocol 5: Plate priming Support Protocol 6: General assay Support Protocol 7: Editing the compound addition sequence (compound list) Support Protocol 8: Creating compound list for agonist concentration-dependent response Support Protocol 9: Creating compound list for antagonist or PAM concentration-dependent response Support Protocol 10: Defining the different compounds used or compound list Support Protocol 11: Maintenance Support Protocol 12: Data analysis Support Protocol 13: Cell culture.

UI	MeSH Term	Description	Entries
D008024	Ligands	A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)	Ligand
D008564	Membrane Potentials	The voltage differences across a membrane. For cellular membranes they are computed by subtracting the voltage measured outside the membrane from the voltage measured inside the membrane. They result from differences of inside versus outside concentration of potassium, sodium, chloride, and other ions across cells' or ORGANELLES membranes. For excitable cells, the resting membrane potentials range between -30 and -100 millivolts. Physical, chemical, or electrical stimuli can make a membrane potential more negative (hyperpolarization), or less negative (depolarization).	Resting Potentials,Transmembrane Potentials,Delta Psi,Resting Membrane Potential,Transmembrane Electrical Potential Difference,Transmembrane Potential Difference,Difference, Transmembrane Potential,Differences, Transmembrane Potential,Membrane Potential,Membrane Potential, Resting,Membrane Potentials, Resting,Potential Difference, Transmembrane,Potential Differences, Transmembrane,Potential, Membrane,Potential, Resting,Potential, Transmembrane,Potentials, Membrane,Potentials, Resting,Potentials, Transmembrane,Resting Membrane Potentials,Resting Potential,Transmembrane Potential,Transmembrane Potential Differences
D010477	Perfusion	Treatment process involving the injection of fluid into an organ or tissue.	Perfusions
D003412	Cricetulus	A genus of the family Muridae consisting of eleven species. C. migratorius, the grey or Armenian hamster, and C. griseus, the Chinese hamster, are the two species used in biomedical research.	Hamsters, Armenian,Hamsters, Chinese,Hamsters, Grey,Armenian Hamster,Armenian Hamsters,Chinese Hamster,Chinese Hamsters,Grey Hamster,Grey Hamsters,Hamster, Armenian,Hamster, Chinese,Hamster, Grey
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000494	Allosteric Regulation	The modification of the reactivity of ENZYMES by the binding of effectors to sites (ALLOSTERIC SITES) on the enzymes other than the substrate BINDING SITES.	Regulation, Allosteric,Allosteric Regulations,Regulations, Allosteric
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D016466	CHO Cells	CELL LINE derived from the ovary of the Chinese hamster, Cricetulus griseus (CRICETULUS). The species is a favorite for cytogenetic studies because of its small chromosome number. The cell line has provided model systems for the study of genetic alterations in cultured mammalian cells.	CHO Cell,Cell, CHO,Cells, CHO
D044085	Microfluidics	The study of fluid channels and chambers of tiny dimensions of tens to hundreds of micrometers and volumes of nanoliters or picoliters. This is of interest in biological MICROCIRCULATION and used in MICROCHEMISTRY and INVESTIGATIVE TECHNIQUES.	Microfluidic
D055724	Electrophysiological Phenomena	The electrical properties, characteristics of living organisms, and the processes of organisms or their parts that are involved in generating and responding to electricity.	Electrophysiological Concepts,Electrophysiological Processes,Electrophysiologic Concepts,Electrophysiologic Phenomena,Electrophysiological Phenomenon,Electrophysiological Process,Concept, Electrophysiologic,Concept, Electrophysiological,Concepts, Electrophysiologic,Concepts, Electrophysiological,Electrophysiologic Concept,Electrophysiological Concept,Phenomena, Electrophysiologic,Phenomena, Electrophysiological,Phenomenon, Electrophysiological,Process, Electrophysiological,Processes, Electrophysiological