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

PLoS Biol. 2021 Sep 7;19(9):e3001321. doi: 10.1371/journal.pbio.3001321. eCollection 2021 Sep.


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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Sensing Ion Channels / chemistry*
  • Acid Sensing Ion Channels / genetics
  • Acid Sensing Ion Channels / pharmacology*
  • Amino Acids / chemistry*
  • HEK293 Cells
  • Humans
  • Peptides / chemistry
  • Spider Venoms / chemistry
  • Transfection


  • Acid Sensing Ion Channels
  • Amino Acids
  • PcTX1 protein, Psalmopoeus cambridgei
  • Peptides
  • Spider Venoms

Grants and funding

We acknowledge the Lundbeck Foundation (R139-2012-12390 to SAP and R218-2016-1490 to NB), the Boehringer Ingelheim Fond (to NB) and the Deutsche Forschungsgemeinschaft (#391498659 to AS) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.