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2021 - Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms

icon pl   Patchliner and   icon sp96  SyncroPatch 384i (a predecessor model of SyncroPatch 384) Biophysical Society Meeting 2021  logo pdf   (2 MB)

Abstract:

Acid-sensing ion channels (ASICs) are proton-gated ion channels which are highly sensitive to extracellular acidosis and are permeable to cations, predominantly Na+. To date, six different ASIC subunits (1a, 1b, 2a, 2b, 3 and 4) encoded by four genes have been identified. Three subunits assemble to form homomeric or heteromeric channels which are expressed throughout the CNS and PNS. Excitatory ASIC channels have a proposed role in nociception and pain, and other neurological diseases such as ischaemia, inflammation and learning and memory. Ligand-gated ion channels with rapid activation and desensitization present specific challenges on automated patch clamp (APC) systems due to low channel expression, the need for rapid application and wash-off of ligands, and loss of responsiveness due to short- and longterm desensitization. In this study we used ASIC1a channels expressed in CHO cells as an exemplar rapid ligand-gated ion channel to develop reliable screening assays on medium (Patchliner) and high throughput (SyncroPatch 384i) APC devices. ASIC1a-mediated current responses were reproducible and could be repetitively activated with similar peak amplitudes at least 7 times in the same cell using techniques which minimize agonist exposure time. ASIC1a was activated by decreasing pH and blocked by benzamil and amiloride. Finally, we demonstrate successful implementation of the traditional input resistance/conductance-tracking technique to measure ligand-gated current activation on an APC platform.

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