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

被引:13
作者
Braun, Nina [1 ]
Friis, Soren [2 ]
Ihling, Christian [3 ]
Sinz, Andrea [3 ]
Andersen, Jacob [1 ,4 ]
Pless, Stephan A. [1 ]
机构
[1] Univ Copenhagen, Dept Drug Design & Pharmacol, Copenhagen, Denmark
[2] Nanion Technol GmbH, Munich, Germany
[3] Martin Luther Univ Halle Wittenberg, Inst Pharm, Dept Pharmaceut Chem & Bioanalyt, Ctr Struct Mass Spectrometry, Halle, Saale, Germany
[4] H Lundbeck & Co AS, Lundbeck Res, Valby, Denmark
关键词
PHOTO-CROSS-LINKING; DYNORPHIN PEPTIDE; AMINO-ACIDS; SITE; INHIBITION; VENOM; NEUROPROTECTION; DESENSITIZATION; EFFICIENT; DYNAMICS;
D O I
10.1371/journal.pbio.3001321
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页数:30
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共 84 条
[1]   Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system [J].
Ahmed, Raju ;
Spikings, Emma ;
Zhou, Shaobo ;
Thompsett, Andrew ;
Zhang, Tiantian .
JOURNAL OF IMMUNOLOGICAL METHODS, 2014, 406 :143-147
[2]   Probing Protein-Protein Interactions with a Genetically Encoded Photo-crosslinking Amino Acid [J].
Ai, Hui-wang ;
Shen, Weijun ;
Sagi, Amit ;
Chen, Peng R. ;
Schultz, Peter G. .
CHEMBIOCHEM, 2011, 12 (12) :1854-1857
[3]   Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes [J].
Baconguis, Isabelle ;
Gouaux, Eric .
NATURE, 2012, 489 (7416) :400-U86
[4]   Venom toxins in the exploration of molecular, physiological and pathophysiological functions of acid-sensing ion channels [J].
Baron, Anne ;
Diochot, Sylvie ;
Salinas, Miguel ;
Deval, Emmanuel ;
Noel, Jacques ;
Lingueglia, Eric .
TOXICON, 2013, 75 :187-204
[5]  
Boddum K, 2021, METHODS MOL BIOL, V2188, P311, DOI 10.1007/978-1-0716-0818-0_16
[6]   Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers [J].
Boettke, Thore ;
Ernicke, Stefan ;
Serfling, Robert ;
Ihling, Christian ;
Burda, Edyta ;
Gurevich, Vsevolod V. ;
Sinz, Andrea ;
Coin, Irene .
EMBO REPORTS, 2020, 21 (11)
[7]   A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain [J].
Bohlen, Christopher J. ;
Chesler, Alexander T. ;
Sharif-Naeini, Reza ;
Medzihradszky, Katalin F. ;
Zhou, Sharleen ;
King, David ;
Sanchez, Elda E. ;
Burlingame, Alma L. ;
Basbaum, Allan I. ;
Julius, David .
NATURE, 2011, 479 (7373) :410-U167
[8]   Mechanism and site of action of big dynorphin on ASIC1a [J].
Borg, Christian B. ;
Braun, Nina ;
Heusser, Stephanie A. ;
Bay, Yasmin ;
Weis, Daniel ;
Galleano, Iacopo ;
Lund, Camilla ;
Tian, Weihua ;
Haugaard-Kedstrom, Linda M. ;
Bennett, Eric P. ;
Lynagh, Timothy ;
Stromgaard, Kristian ;
Andersen, Jacob ;
Pless, Stephan A. .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2020, 117 (13) :7447-7454
[9]   The current chemical biology tool box for studying ion channels [J].
Braun, N. ;
Sheikh, Z. P. ;
Pless, S. A. .
JOURNAL OF PHYSIOLOGY-LONDON, 2020, 598 (20) :4455-4471
[10]   Compounds commonly used in equine medicine inhibits the voltage-gated potassium channel Kv11.1 [J].
Calloe, Kirstine ;
Rognant, Salome ;
Friis, Soren ;
Shaughnessy, Catherine ;
Klaerke, Dan A. ;
Trachsel, Dagmar .
RESEARCH IN VETERINARY SCIENCE, 2019, 123 :239-246