High Throughput Validation Of Non Canonical Amino Acid Incorporation Into Acid Sensing Ion Channel 1a

Human acid-sensing ion channel 1a (ASIC1a) is a trimeric proton-sensitive cation channel expressed in the central nervous system, where it plays a crucial role in the initiation of neuropathic pain and acid-induced ischemia after stroke. In order to target these conditions therapeutically, the molecular links between ASIC1a activity and its surrounding cellular network have to be identified. We are thus aiming to investigate protein-protein and protein-peptide interactions of ASIC1a with a combination of patch-clamp electrophysiology and UV-induced cross-linking. This will allow mapping of both intra- and extracellular interaction sites. For the purpose of cross-linking, the non-canonical amino acids (ncAAs) AzF, Bpa and Se-AbK are site-specifically incorporated at selected positions in ASIC1a via the nonsense suppression method. Here we introduce a workflow to functionally assess site-specific incorporation of ncAAs into a library of 103 ASIC1a variants. After removing endogenous ASIC1a from HEK293 cells using CRISPR-Cas9, we transiently transfect the test constructs into these knock-out cells. Following transfection, cells are submitted to fluorescence-activated cell sorting, after which we perform high-throughput patch-clamp recordings on a SyncroPatch 384PE. Incorporation of ncAAs is tolerated throughout all channel domains, with highest success rates for AzF (61%) and lowest for Se-AbK (44%). Bpa is preferably incorporated at positions of aromatic origin, while no comparable trend is observed for the other ncAAs. Interestingly, replacement of residues around the acidic pocket produces channels with significantly reduced proton affinity. In conclusion, this approach allows rapid identification of suitable positions for cross-linking experiments, as well as efficient biophysical characterization of ncAA-containing ASIC1a variants.