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

['Nina Braun', 'Department Of Drug Design', 'Pharmacology', 'University Of Copenhagen', 'Copenhagen', 'Søren Friis', 'Nanion Technologies Gmbh', 'Munich', 'Christian Ihling', 'Department Of Pharmaceutical Chemistry']

Date: 2021-09

Development of an APC screen to validate ncAA incorporation into hASIC1a

In order to efficiently assess functional incorporation of ncAAs into hASIC1a, we developed an APC screen to record proton-gated channel activation (Fig 1). To this end, we co-transfected 103 different hASIC1a variants containing individual TAG stop codons throughout the protein together with the suppressor tRNA/ncAA-RS pair for either AzF, Bpa, or Se-AbK and a GFP reporter carrying a TAG at Y40 (for Bpa and Se-AbK) or Y151 (for AzF, as we observed a higher degree of unspecific incorporation in the Y40TAG variant with AzF) into custom-made ASIC1a-KO HEK 293T cells [17,42–44]. The corresponding ncAA was supplied in the cell culture medium 6 hours after transfection or omitted from the experiment in incorporation control samples. To increase cell viability and uptake efficiency, we synthesized the methylester derivates of AzF and Bpa [8,45]. This allowed us to supplement the cell media with 50- and 100-fold lower ncAA concentration compared to previous studies, respectively [7,13].

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TIFF original image Download: Fig 1. Schematic illustration of the workflow to assess ncAA incorporation into hASIC1a. HEK 293T ASIC1a-KO cells are transfected with hASIC1a containing a TAG stop codon at the site of interest, a coevolved suppressor tRNA/ncAA-RS pair, and a TAG-containing GFP reporter. ncAA is supplied in the cell culture medium. Moreover, 48 hours after transfection, cells are sorted for green fluorescence on a FACS BD Aria I, and those showing fluorescence are subjected to APC on a SyncroPatch 384PE to measure proton concentration response curves. APC, automated patch clamp; FACS, fluorescence-activated cell sorting; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; WT, wild type. https://doi.org/10.1371/journal.pbio.3001321.g001

After 48 hours, cells grown in the presence of ncAA were sorted for green fluorescence to enrich the population of transfected cells, which were then submitted to APC to record proton-gated currents. Using GFP fluorescence as a proxy, we determined a transfection efficiency of 62.9 ± 9.5% for hASIC1a wild type (WT) and an average of 11.2 ± 5% for the ncAA variants (S1 Fig, S1 Table). Without the FACS step, the latter rate would translate into less than 10% of the APC wells being occupied by transfected cells, precluding efficient APC experiments. By contrast, the cell sorting improves occupation to around 46% of wells with successful patch also displaying proton-gated currents (62% for AzF, 29% for Bpa, and 48% for Se-AbK) and is therefore an indispensable element for the use of transiently transfected cells in APC (S1 Fig).

The 384-well system of the SyncroPatch 384PE allows for parallel concentration response curve measurements on 24 different samples, enabling us to test 11 different channel variants with corresponding incorporation controls (cells grown in the absence of ncAA), as well as hASIC1a WT and untransfected cells in less than 1 hour, with up to 16 replicates per sample. Specifically, we embarked to functionally interrogate 103 positions throughout the hASIC1a sequence: 38 positions in the N-terminal domain (S2 Fig), 24 positions in the TMD and interface region (S3 Fig), 29 positions in the carboxyl-terminal domain (S4 Fig), and 12 positions around the acidic pocket (Fig 3, S8 Fig). The current traces in Fig 2A show typical pH-induced inward currents of hASIC1a WT with a pH 50 of 6.64 ± 0.12 (n = 182), in line with previous studies [46,47], as well as a variant with lower proton sensitivity containing AzF in the acidic pocket (T236AzF, pH 50 6.17 ± 0.14, n = 10). Interestingly, the incorporation of Bpa, AzF, and Se-AbK at position W46 did not result in proton-gated currents (Fig 2A, S3 Fig), despite a previous report showing functional incorporation of a bulky ncAA at this conserved Trp in the M1 helix [48]. We analyzed all variants for mean peak current size and pH 50 to compare incorporation efficiency and proton sensitivity, respectively (S2–S4 and S8 Figs, S1 Table). Furthermore, we routinely assessed the extent of tachyphylaxis [49], and variants displaying >20% current decrease after reaching the peak current are indicated in Fig 3 and S2–S8 Figs as well as S1 Table.

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TIFF original image Download: Fig 2. Incorporation of ncAA crosslinkers is tolerated in all domains of hASIC1a and produces functional channel variants. (A) Representative current traces for pH response curves of hASIC1a WT, T236AzF, and W46Bpa recorded on the SyncroPatch 384PE, pH response curve in bottom right corner (WT pH 50 6.64 ± 0.12, n = 182; T236AzF pH 50 6.17 ± 0.14, n = 10). (B–D) Snake plot representations indicating specific, unspecific, and unsuccessful incorporation (no current) for AzF (B), Bpa (C), and Se-AbK (D). Specific incorporation (circles with darker shade) is defined as pH-dependent peak currents >1 nA observed in cells grown in the presence, but not in the absence of ncAA, whereas unspecific incorporation (circles with lighter shade) indicates that currents were observed both in the presence and absence of ncAA. Positions indicated by gray circles did not yield functional channel variants when replaced by an ncAA (no current), while those colored in white were not tested. The gray area highlights positions distal of L465. (E) Relative incorporation rates of AzF (red), Bpa (blue), and Se-AbK (green) at 80 different hASIC1a positions. Exchanged amino acids are grouped for original side chain properties and position within the channel, respectively. Relative incorporation rates were calculated by dividing the number of positions successfully replaced with an ncAA by the total number of positions at which incorporation was attempted. Positions distal of L465 were excluded from the analysis (highlighted in gray in B–D), as more distal deletions result in truncated, but functional channels (see S4–S6 Figs). The underlying data have been deposited at zenodo.org (https://doi.org/10.5281/zenodo.4906985; files 02–08 and 15–28). AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; C-term <L465, carboxyl terminus up to and including L465; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; ICD, intracellular domain; ncAA, noncanonical amino acid; N-term, N-terminus; Se-AbK, (R)-2-Amino-3-{2-[2-(3-methyl-3H-diazirin-3-yl)-ethoxycarbonylamino]-ethylselanyl}-propionic acid; TM, transmembrane; TMD, transmembrane domain; WT, wild type. https://doi.org/10.1371/journal.pbio.3001321.g002

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TIFF original image Download: Fig 3. Incorporation of ncAA photocrosslinkers into the acidic pocket results in channel variants with lowered proton sensitivity and accelerated current decay. (A) Snake plot of hASIC1a with the assessed positions highlighted in blue. (B) Representative current traces of variants T239Bpa and D357AzF as recorded on the SyncroPatch 384PE, with arrows indicating the time of the proton application. Dashed lines indicate WT current in response to pH 6.0 application. Bar graph shows mean t½ ± SD of current decay. (C) Incorporation of AzF (red), Bpa (blue), and Se-AbK (green) at 8 positions around the acidic pocket results in lowered proton sensitivity for several variants. Dot plots comparing pH 50 (left) and peak current sizes (right); bars indicate mean ± SD, (#) indicates >20% tachyphylaxis (see also S8 Fig and S1 Table), and (*) denotes significant difference between t½ of current decay, p < 0.05; (***): p < 0.001; Mann–Whitney test (see also S3 Table). The underlying data have been deposited at zenodo.org (https://doi.org/10.5281/zenodo.4906985; files 09, 10, and 33). AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; ICD, intracellular domain; ncAA, noncanonical amino acid; SD, standard deviation; Se-AbK, (R)-2-Amino-3-{2-[2-(3-methyl-3H-diazirin-3-yl)-ethoxycarbonylamino]-ethylselanyl}-propionic acid; TMD, transmembrane domain; WT, wild type. https://doi.org/10.1371/journal.pbio.3001321.g003

To provide a comprehensive overview, we mapped incorporation patterns for the 3 photocrosslinkers onto snake plots schematically depicting an ASIC1a subunit (Fig 2B–2D). We defined specific incorporation (circles with dark color shade) as proton-gated currents of >1 nA observed in the presence of ncAA and minimal (<500 pA) proton-gated currents in the absence of ncAA. If currents >500 pA were observed in the absence of ncAA, incorporation was considered unspecific (circles with lighter color shade), while positions labeled in gray did not yield substantial currents in either condition (<1 nA). However, we cannot exclude the possibility of underestimating the degree of unspecific incorporation, as enriching transfected cells grown in the absence of ncAA by FACS was not feasible due to the low number of cells displaying GFP fluorescence (2.2 ± 1.7%). On the other hand, by defining incorporation as not successful for currents <1 nA, we are aware that we may have potentially excluded variants in which specific ncAA incorporation resulted in reduced open probability or lower conductance.

As is apparent from the snake plots, we observed robust incorporation in the N-terminus, around the acidic pocket, and in the proximal carboxyl terminus. Indeed, among the 80 positions tested up to and including L465, AzF resulted in functional channel variants in 61% of cases, compared to 50% for Bpa and 44% for Se-AbK (Fig 2E).

By contrast, all 3 crosslinkers showed mostly unspecific incorporation distal of L465, with WT-like current phenotypes from position 467 onward (S4 and S5A–S5C Figs). This led us to hypothesize that channel constructs truncated in this region are functional. To investigate this further, we inserted an additional TGA stop codon for several variants, confirmed channel truncation by comparing molecular weight on a western blot and measured concentration response curves in APC and two-electrode voltage clamp (TEVC) (S5D and S5E Fig). We found that channels truncated after H463 or K464 yielded no current in either APC or TEVC, but truncation after L465 produced a variant with strong tachyphylaxis in HEK 293T cells (S5D Fig), and truncation after C466 or R467 resulted in channels with WT-like proton sensitivity in both APC and TEVC. We conclude that the carboxyl terminus distal of position 465 is not essential for proton-gated channel activity and that it is not possible to differentiate between currents originating from truncated and full-length protein to evaluate ncAA incorporation. We therefore added a carboxyl-terminal 1D4-tag to the hASIC1a construct to selectively purify full-length protein and compare the amounts in cells grown in the presence or absence of ncAA. This strategy confirms efficient incorporation in the distal carboxyl terminus (S6A Fig). Additionally, liquid chromatography/tandem mass spectrometry data revealed that Bpa can be specifically incorporated at positions distal of L465 (A480, S6B Fig).

For the 80 positions up to and including L465, we evaluated incorporation efficiency by comparing how many positions could be functionally replaced by each of the ncAA photocrosslinkers, based on the nature of the side chain occupying the position in the native channel and the position within the protein overall. We did not find evidence for pronounced global trends, but, for instance, Bpa incorporation was tolerated best at originally aromatic side chains (79%), while replacement of basic residues was least successful (27%) (Fig 2E). The 3 tested prolines could not be exchanged for any of the ncAAs. Interestingly, and in contrast to our expectations, Se-AbK incorporation only produced functional variants in 33% of cases when replacing structurally similar Lys and Arg side chains, while success rates were higher at polar and acidic side chains (58% and 54%, respectively). AzF incorporation rates were similar throughout all protein domains, whereas Bpa was better tolerated in the transmembrane regions and less in the N-terminal and carboxyl terminal and Se-AbK incorporation in the M2 helix and carboxyl terminus was negligible (Fig 2E). Overall, incorporating the 3 photocrosslinkers produced functional variants in all protein domains, albeit with varying success rates.

Together, we show that combining FACS with APC affords the time-efficient functional characterization of over 300 hASIC1a variants and provides a versatile platform to assess successful ncAA incorporation throughout all protein domains.

To evaluate if the established APC screen can also serve as a platform for other ion channels, we applied it to selected TAG variants of the rat P2X2 and rat GluA2 receptors. Specifically, we compared currents upon exposure to 2 different concentrations of ATP or glutamate, respectively (S7 Fig, S2 Table [5,7,50]). Incorporation of AzF into position K296 of the rP2X2 receptor is unspecific, whereas that of Bpa is efficient and specific (S7A Fig). For GluA2, incorporation patterns at Y533 and S729 are identical to those observed in previous studies using manual patch clamp (S7B Fig [5,7]). Incorporation of AzF at Y533 is tolerated with currents of 1.21 ± 0.96 nA (n = 30, compared to 600 ± 100 pA (n = 15) reported by Poulsen and colleagues), while incorporation of Bpa does not produce a functional channel. At position S729, we observe small currents of 390 ± 330 pA for AzF (n = 17) and 280 ± 240 pA for Bpa (n = 16, compared to 470 ± 50 pA reported by Klippenstein and colleagues for S729Bpa). Importantly, as GluA2 gating is fast compared to the perfusion speed of the SyncroPatch 384PE and Klippenstein and colleagues report increased desensitization rates for S729 variants, we preincubated cells with 100 μM cyclothiazide to slow desensitization and increase the likelihood of resolving the GluA2 peak current [51]. While our GluA2 and P2X2 data show that target-specific optimization of the ligand application protocols is required, they illustrate that our APC screening approach can be applied to a variety of different ion channels and yields results comparable to those obtained with conventional ncAA incorporation protocols.

[END]

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