ArticlePLoS biology2021
High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
Article in PLoS biology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Molecular mechanisms and hotspots of pH sensing in ASIC1a revealed by computational and functional analysis.Communications biology · 2025Article
- Noncanonical Amino Acid Tools and Their Application to Membrane Protein Studies.Chemical reviews · 2024Review
- Engineering Pyrrolysine Systems for Genetic Code Expansion and Reprogramming.Chemical reviews · 2024Review
- Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions.Biophysical journal · 2024Article
- Structural basis for excitatory neuropeptide signaling.Nature structural & molecular biology · 2024Article
- Photomodulation of the ASIC1a acidic pocket destabilizes the open state.Protein science : a publication of the Protein Society · 2023Article
- Genetically encoded crosslinkers to address protein-protein interactions.Protein science : a publication of the Protein Society · 2023Review
- Article
- Metabolic and electrolyte abnormalities as risk factors in drug-induced long QT syndrome.Biophysical reviews · 2022Review
- Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms.Frontiers in molecular neuroscience · 2022Article
- The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs.The Journal of general physiology · 2021Article
- Molecular Investigation of Chicken Acid-Sensing Ion Channel 1 β11-12 Linker Isomerization and Channel Kinetics.Frontiers in cellular neuroscience · 2021Article
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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