RT Journal Article
SR Electronic
T1 High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.11.24.392498
DO 10.1101/2020.11.24.392498
A1 Nina Braun
A1 Søren Friis
A1 Christian Ihling
A1 Andrea Sinz
A1 Jacob Andersen
A1 Stephan A. Pless
YR 2021
UL http://biorxiv.org/content/early/2021/05/07/2020.11.24.392498.abstract
AB Incorporation of non-canonical 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 hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three 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 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.Competing Interest StatementSoren Friis is a full-time employee of Nanion Technologies. The other authors declare no competing interests.