The current chemical biology tool box for studying ion channels

Abstract

Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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

“…Over the past 2 decades, this method has greatly facilitated protein modification and functionalization beyond the confines of the genetic code [1]. Ion channels have proven highly suited to ncAA incorporation, as evidenced by the success in introducing photocrosslinking, photoswitchable, or fluorescent ncAAs into numerous members of this large and diverse protein family [2][3][4]. Among the ncAA subclasses, photocrosslinkers have proven particularly versatile, as they allow for the trapping of ion channels in certain conformational states [5][6][7][8], capturing of protein-protein interactions [9][10][11][12], and covalent linking of receptor-ligand complexes to delineate ligand binding sites [13][14][15][16][17].…”

“…Typically, incorporation of ncAAs is achieved by repurposing a stop codon to encode for an ncAA supplied by an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair. But the incorporation efficiency can be variable, and unspecific incorporation of naturally occurring amino acids can result in inhomogeneous protein populations [2]. Verification of site-specific ncAA incorporation can therefore be laborious and time consuming, especially in combination with detailed functional characterization.…”

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

“…Over the past two decades, this method has greatly facilitated protein modification and functionalization beyond the confines of the genetic code (Chin, 2017). Ion channels have proven highly suited to ncAA incorporation, as evidenced by the success in introducing photocrosslinking, photoswitchable or fluorescent ncAAs into numerous members of this large and diverse protein family (Klippenstein et al ., 2018; Paoletti et al ., 2019; Braun et al ., 2020). Among the ncAA subclasses, photocrosslinkers have proven particularly versatile, as they allow for the trapping of ion channels in certain conformational states (Klippenstein et al ., 2014; Zhu et al ., 2014; Poulsen et al ., 2019; Rook et al ., 2020b), capturing of protein-protein interactions (Martin et al ., 2016; Murray et al ., 2016; Tian & Ye, 2016; Westhoff et al ., 2017) and covalent linking of receptor-ligand complexes to delineate ligand binding sites (Coin et al ., 2013; Rannversson et al ., 2016; Reiners et al ., 2018; Borg et al ., 2020; Bottke et al ., 2020).…”

“…Typically, incorporation of ncAAs is achieved by repurposing a stop codon to encode for a ncAA supplied by an orthogonal tRNA/aminoacyl tRNA synthetase (aaRS) pair. But the incorporation efficiency can be variable and unspecific incorporation of naturally occurring amino acids can result in inhomogeneous protein populations (Braun et al ., 2020). Verification of site-specific ncAA incorporation can therefore be laborious and time-consuming, especially in combination with detailed functional characterization.…”

“…Over the past two decades, this method has greatly facilitated protein modification and functionalization beyond the confines of the genetic code [1]. Ion channels have proven highly suited to ncAA incorporation, as evidenced by the success in introducing photocrosslinking, photoswitchable or fluorescent ncAAs into numerous members of this large and diverse protein family [2–4]. Among the ncAA subclasses, photocrosslinkers have proven particularly versatile, as they allow for the trapping of ion channels in certain conformational states [5–8], capturing of protein-protein interactions [9–12] and covalent linking of receptor-ligand complexes to delineate ligand binding sites [13–17].…”