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

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.

Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

“…These experiments indicate that PcTx1 interacts with F350L* and introduces conformational changes despite the lack of functional consequences at these concentrations. This is in line with earlier results that suggested PcTx1 to still bind to F352L hASIC1a as pre-incubation with PcTx1 prevented the modulatory effects of BigDyn ( Sherwood and Askwith, 2009 ) and PcTx1 could still be crosslinked with the mutant channel ( Braun et al, 2021 ). Similarly, experiments from ASIC1a/2a concatemers of different subunit stoichiometries suggested that PcTx1 also binds to ASIC1a-2a interfaces ( Joeres et al, 2016 ) despite the fact that ASIC2a subunits contain a leucine at the position corresponding to 350 in mASIC1a.…”

Molecular Investigation of Chicken Acid-Sensing Ion Channel 1 β11-12 Linker Isomerization and Channel Kinetics

“…Mutations in the thumb domain influence desensitization kinetics ( Krauson et al, 2013 ; Krauson and Carattino, 2016 ; Braun et al, 2021 ) as does simple extracellular anion substitution ( Kusama et al, 2010 , 2013 ). How can these data be reconciled with a model where linker flipping is the crucial feature governing ASIC desensitization?…”

Metabolic and electrolyte abnormalities as risk factors in drug-induced long QT syndrome

“…As a result, investigations of the effects of variation in divalent ion concentrations in the physiological range are limited in these systems. This issue is particularly salient in automated high-throughput patch-clamp systems, where calcium fluoride seal enhancers are critical in establishing high-quality seals (Braun et al 2021 ), meaning thorough investigation of the effects of divalent ions on drug block of hERG at large scale remains technically difficult. In addition to this practical challenge, there is also the issue of what is physiologically or clinically relevant.…”