title: Automated Parallel Oocyte Electrophysiology Test station (POETs): a screening platform for identification of ligand-gated ion channel modulators.
abstract: Ligand-gated ion channels (LGICs) play important roles in the regulation of cellular function and signaling and serve as excellent drug targets. However, fast desensitization of most LGICs limits the choice of reliable methods to identify agonists, antagonists, and/or modulators in a high throughput manner. In this study, automated Parallel Oocyte Electrophysiology Test station (POETs) was used to screen a directed compound library against a rapidly desensitizing LGIC and to characterize further the pharmacological properties of the hits. POETs allows up to six two-electrode voltage-clamp experiments to be performed in parallel by automatically loading of the oocytes into flowcells, assessing individual oocyte behavior prior to initiating experiments. Oocytes injected with cRNA were transferred from a chilled 96-well plate into flowcells by the instrument, where they were impaled under software control by two independent electrodes. Expression was tested by measuring current responses to rapid application of agonists. Compounds, prepared in a 96-well format, were tested for effects by coapplication with agonist at a single concentration of 30 microM over 2 s. After compound application, oocytes were washed for a minimum of 30 s, and used repeatedly if the test compounds had no significant effect on the control response. Typical throughput could reach approximately 14 plates/day depending on the protocol. Pilot library screening revealed a hit rate of 0.06%, with active compounds having IC(50) values of 4-40 microM. Hits were also confirmed in native neurons using patch-clamp techniques. We conclude that automated POETs serves as a suitable platform for screening and expedient identification of LGIC modulators.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research Division, Abbott Laboratories, Abbott Park, IL 60064, USA. char-chang.shieh@abbott.com
authors.forename: Char-Chang
authors.initials: CC
authors.lastname: Shieh
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Trumbull
authors.forename: Jay F
authors.initials: JF
authors.lastname: Sarthy
authors.forename: David G
authors.initials: DG
authors.lastname: McKenna
authors.forename: Ashutosh S
authors.initials: AS
authors.lastname: Parihar
authors.forename: Xu-Feng
authors.initials: XF
authors.lastname: Zhang
authors.forename: Connie R
authors.initials: CR
authors.lastname: Faltynek
authors.forename: Murali
authors.initials: M
authors.lastname: Gopalakrishnan
mesh_terms: D000465:Algorithms; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005240:Feasibility Studies; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008839:Microelectrodes; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012371:Robotics / Q000295:instrumentation* / Q000379:methods; D012680:Sensitivity and Specificity; D014584:User-Computer Interface; D014982:Xenopus laevis
chemical_list: D007473:Ion Channels; D008024:Ligands