(a, b) Electrophysiological recordings of ELIC activated by the agonist GABA and in the presence of increasing concentrations of PAM-Nb (a, green) or NAM-Nb (b, red). The curve represents a fit to …
Electrophysiological recordings of ELIC with PAM-Nb and NAM-Nb.
(a) An example control experiment with two consecutive applications of a series of GABA concentrations (5–40 mM). ELIC is prone to desensitization, but this effect can be minimized by excluding GABA …
ELIC concentration-activation curves in the presence of PAM-Nb and NAM-Nb.
The white mesh shows a simulated annealing omit map calculated in PHENIX and contoured at a sigma level of 1.0 around a 12 Å sphere zoom of the ELIC-Nb interface. In the omit calculation an omit box …
The white mesh shows a simulated annealing omit map calculated in PHENIX and contoured at a sigma level of 1.0 around a 12 Å sphere zoom of the ELIC-Nb interface. In the omit calculation, an omit …
(a) A detailed view of the interaction between PAM-Nb (green) and a single ELIC subunit (blue). The binding site for PAM-Nb overlaps with a known allosteric binding site, in a related pLGIC, for a …
Analysis of pore radius profiles.
(a–b) The cartoon shows that the binding site of the PAM-Nb (green) in ELIC (blue) (a) is homologous to the CU2017 (yellow spheres) site in α7-AChBP (cyan) (b). For clarity, only residues at the …
List of amino acid interactions in the PAM-Nb bound ELIC structure versus CU2017 bound alpha7-AChBP structure.
(a) The cartoon shows that the binding site of the NAM-Nb (red) is adjacent to the flurazepam site (yellow spheres) in the vestibule of ELIC (orange). For clarity, only residues at the receptor site …
List of amino acid interactions in the NAM-Nb bound ELIC structure versus flurazepam bound ELIC structure.
(a) Pore radius profiles extending into lateral fenestrations (green, magenta, blue) at subunit interfaces or the extracellular vestibule entrance (amber) in the NAM-Nb bound structure. (b) Pore …
(a–c) Yellow ribbon representation of a single subunit ligand binding domain. Part of the vestibule site (shown in blue cartoon), called the Ω-loop, adopts three distinct conformations in different …
The vestibule site is depicted in the ‘putty’ cartoon presentation. Yellow-red regions indicate example structures with higher average B-factors per residue compared to cyan-blue regions. B-factors …
(a) Example traces of agonist-evoked channel responses of the L151C 5-HT3AR mutant before and after modification with MTSEA-biotin show potentiation after cysteine modification. (b) Location of …
Electrophysiological recordings of 5-HT3R mutants before and after treatment with MTSEA-biotin.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene | Erwinia ligand-gated ion channel (ELIC) | Synthetic gene from Genscript | UniProt P0C7B7 | |
Gene | Human 5-hydroxytryptamine receptor 3A (5-HT3A R) | John Peters laboratory (Belelli et al., 1995) | GenBank NM_000869 | |
Cell line | C43 E. coli strain | Lucigen | #60446 | |
Cell line | WK6 E. coli strain | Zell and Fritz, 1987 | ||
Cell line | TG1 E. coli strain | Lucigen | #60502 | |
Antibody | PAM-Nb | Nanobody obtained by immunizing an adult llama glama with recombinant ELIC protein. PAM-Nb was used at 1–30 μM concentration in electrophysiology experiments. In protein crystals the PAM-Nb concentration is > 1 mM. | ||
Antibody | NAM-Nb | Nanobody obtained by immunizing an adult llama glama with recombinant ELIC protein. NAM-Nb was used at 0.03–3 μM concentration in electrophysiology experiments. In protein crystals the NAM-Nb concentration is > 1 mM. | ||
Recombinant DNA reagent | pGEM-HE | Liman et al. (1992) | ||
Recombinant DNA reagent | pMESy4 | GenBank KF415192 | ||
Sequence-based reagent | P111C oligonucleotide forward and reverse | 5’-caccaagttgtccatcTGcacggacagcatctgg-3’ 5’-ccagatgctgtccgtgCAgatggacaacttggtg- 3’ | ||
Sequence-based reagent | T112C oligonucleotide forward and reverse | 5’-caagttgtccatccccTGCgacagcatctgggtcc-3’ 5’-ggacccagatgctgtcGCAggggatggacaacttg-3’ | ||
Sequence-based reagent | N147C oligonucleotide forward and reverse | 5’-caaggcgaagttcagTGctacaagccccttcagg-3’ 5’-cctgaaggggcttgtagCActgaacttcgccttg-3’ | ||
Sequence-based reagent | K149C oligonucleotide forward and reverse | 5’-ggcgaagttcagaactacTGCccccttcaggtggtga-3’ 5’- tcaccacctgaaggggGCAgtagttctgaacttcgcc-3’ | ||
Sequence-based reagent | L151C oligonucleotide forward and reverse | 5’- gttcagaactacaagcccTGtcaggtggtgactgc-3’ 5’-gcagtcaccacctgaCAgggcttgtagttctgaac-3’ | ||
Sequence-based reagent | Y86C oligonucleotide forward and reverse | 5’- ctggtaccggcagtGctggactgatgag-3’ 5’-ctcatcagtccagCactgccggtaccag-3’ | ||
Sequence-based reagent | F125C oligonucleotide forward and reverse | 5’-ggacattctcatcaatgagtGcgtggatgtggg-3’ 5’-cccacatccacgCactcattgatgagaatgtcc-3’ | ||
Sequence-based reagent | Primer for nanobody library generation CALL001 | Pardon et al. (2014) | 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ | |
Sequence-based reagent | Primer for nanobody library generation CALL002 | Pardon et al. (2014) | 5′-GGT ACGTGCTGTTGAACTGTTCC-3′ | |
Sequence-based reagent | Primer for nanobody library generation VHH-Back | Pardon et al. (2014) | 5′-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3′(PstI) | |
Sequence-based reagent | Primer for nanobody library generation VHH-For | Pardon et al. (2014) | 5′-CTAGTGCGGCCGCTGG AGACGGTGACCTGGGT-3′(Eco91I) | |
Sequence-based reagent | Primer for nanobody library analysis MP57 | Pardon et al. (2014) | 5′-TTATGCTTCCGGCTC GTATG-3′ | |
Peptide, recombinant protein | Primer for nanobody library analysis GIII | Pardon et al. (2014) | 5′-CCACAGACAGCCCTCATAG-3′ | |
Commercial assay or kit | mMessage mMachine T7 Transcription kit | ThermoFisher Scientific | #AM1344 | |
Commercial assay or kit | Ni Sepharose 6 Fast Flow resin | GE Healthcare | #17531802 | |
Commercial assay or kit | Superdex 75 10/300 GL column | GE Healthcare | # 17-5174-01 | |
Commercial assay or kit | Superdex 200 Increase 10/300 GL | GE Healthcare | # 28990944 | |
Commercial assay or kit | QuikChange Site-Directed Mutagenesis Kit | Agilent | # 200518 | |
Chemical compound, drug | 5-HT creatinine sulphate | Sigma-Aldrich | #S2805 | |
Chemical compound, drug | MTSEA-biotin | Biotium | #90064 | |
Chemical compound, drug | GABA | Sigma-Aldrich | #A2129 | |
Chemical compound, drug | E. coli total lipid extract | Avanti Polar Lipids | #100500P | |
Chemical compound, drug | Anagrade n-undecyl-β-D-maltoside (UDM) | Anatrace | #U300 | |
Software, algorithm | GraphPad Prism Software v4.03 | RRID:SCR_002798 | ||
Software, algorithm | CCP4 | Winn et al., 2011 | ||
Software, algorithm | STARANISO | Tickle et al., 2018 http://staraniso.globalphasing.org | ||
Software, algorithm | XDS | Kabsch, 2010 | ||
Software, algorithm | Coot | Emsley et al. (2010) | ||
Software, algorithm | Buster | Smart et al. (2012) | ||
Software, algorithm | PDB-REDO | Joosten et al. (2014) | ||
Software, algorithm | MolProbity | Chen et al. (2010) | ||
Software, algorithm | PyMOL v2.3.0 | Schrödinger | RRID:SCR_000305 | |
Software, algorithm | HOLE | Smart et al. (1996) | ||
Software, algorithm | CAVER | Jurcik et al. (2018) | ||
Software, algorithm | PHENIX | Adams et al. (2010) | ||
Software, algorithm | HiClamp | Multi Channel Systems |
Crystallographic and refinement statistics.
Structures used for structure-based superposition and Ω-loop analysis.
Effects of MTSEA-biotin (MB) application on WT and mutant 5-HT3R.