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

View ORCID ProfileNina Braun, Søren Friis, Christian Ihling, View ORCID ProfileAndrea Sinz, View ORCID ProfileJacob Andersen, View ORCID ProfileStephan A. Pless
doi: https://doi.org/10.1101/2020.11.24.392498
Nina Braun
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Søren Friis
2Nanion Technologies GmbH, Munich, Germany
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Christian Ihling
3Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany
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Andrea Sinz
3Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany
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Jacob Andersen
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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Stephan A. Pless
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
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  • For correspondence: stephan.pless@sund.ku.dk
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Abstract

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 Statement

Soren Friis is a full-time employee of Nanion Technologies. The other authors declare no competing interests.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 07, 2021.
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High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology
Nina Braun, Søren Friis, Christian Ihling, Andrea Sinz, Jacob Andersen, Stephan A. Pless
bioRxiv 2020.11.24.392498; doi: https://doi.org/10.1101/2020.11.24.392498
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High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology
Nina Braun, Søren Friis, Christian Ihling, Andrea Sinz, Jacob Andersen, Stephan A. Pless
bioRxiv 2020.11.24.392498; doi: https://doi.org/10.1101/2020.11.24.392498

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