High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function these variants be studied great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional pharmacological investigations ncAA-containing human acid-sensing channel 1a (hASIC1a) in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions assess resulting 309 automated patch clamp (APC). demonstrate that efficient versatile, it amenable assessing even complex modulation by peptides. The data show acidic pocket major determinant for current decay, live-cell provides insight hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, provide evidence applied other P2X2 GluA2 receptors. therefore anticipate enable future APC-based studies channels