title: Sub-millisecond ligand probing of cell receptors with multiple solution exchange.
abstract: The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10-15 s. This protocol, which has been validated in our recent studies and takes 20-60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.
authors.affiliation: UCL Institute of Neurology, University College London, London, UK. s.sylantyev@ucl.ac.uk
authors.forename: Sergiy
authors.initials: S
authors.lastname: Sylantyev
authors.forename: Dmitri A
authors.initials: DA
authors.lastname: Rusakov
mesh_terms: D002138:Calibration; D004867:Equipment Design; D018756:GABA Antagonists / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D055616:Microtechnology / Q000295:instrumentation*; D008967:Molecular Biology / Q000379:methods*; D018408:Patch-Clamp Techniques / Q000295:instrumentation; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D011963:Receptors, GABA-A / Q000032:analysis / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D018756:GABA Antagonists; D008024:Ligands; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate