Functional expression of heteromeric insect/human nicotinic acetylcholine receptor chimeras without accessory proteins.
- 1Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, LMU Munich, Munich, Germany.
Abstract
Nicotinic acetylcholine receptors (nAChRs) are primary targets of insecticides. While most mammalian nAChRs form functional channels in heterologous systems, insect nAChRs remain notoriously difficult to reconstitute. Here, we co-expressed insect/human subunit chimeras to enable functional analysis of heteromeric insect nAChR ligand‑binding sites in Xenopus laevis oocytes. First, EGFP-tagged human/cockroach α7 chimeras (Hα7/Pα7) were generated by exchanging the signal peptide (SP), extracellular (ECD), transmembrane (TMD), and intracellular (L3-4) domains. While all constructs were translated into protein, Hα7-like acetylcholine-evoked currents were only observed when human N- and C-terminal ECD and ICD were present, indicating that these domains impose insect-specific constraints on homomeric nAChR formation. Second, we generated heteromeric chimeric receptors by fusing insect-derived ECDs onto hα7 subunits. Co-expression of Pα6/Hα7L3-4 and Pα7ECD/Hα7 chimeras resulted in current amplitudes < 1 µA. Co-expression of the respective bee Aα5ECD/Hα7, Aα6ECD/Hα7, and Aα7ECD, CT/Hα7 chimeras yielded currents > 1 µA. α-Bungarotoxin inhibited diheteromeric Pα6α7 chimeras (IC₅₀ 49 nM) and triheteromeric Aα5/α6/α7 chimeras (IC₅₀ 51.2 nM). Third, this approach was applied to cockroach Pα2 and Pβ1 subunits. Replacement of the Pα2 ICD and the Pβ1 TMD/ICD with homologous regions from Hα2 and Hβ2 subunits and co-expression with Pα1 resulted in a ~ 100-fold current increase (2.6 µA) compared to the wild‑type Pα1α2β1 receptor. The chimeric receptor was activated by neonicotinoids (EC₅₀ clothianidin: 24.1 nM, EC₅₀ thiacloprid: 13.6 nM) and α-bungarotoxin-sensitive (IC₅₀: 36.6 nM). In conclusion, we demonstrate that replacement of insect ICD or ICD and TMD enables formation of functional insect-like heteromeric nAChRs without co-expression of chaperones.
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BibTeX
@article{lokaj2026functional,
title = {{Functional expression of heteromeric insect/human nicotinic acetylcholine receptor chimeras without accessory proteins.}},
author = {Gonxhe Lokaj and Annette Nicke},
journal = {Channels (Austin, Tex.)},
year = {2026},
month = oct,
doi = {10.1080/19336950.2026.2736911},
url = {https://europepmc.org/article/MED/42905803},
note = {CC BY},
}
RIS
TY - JOUR
TI - Functional expression of heteromeric insect/human nicotinic acetylcholine receptor chimeras without accessory proteins.
AU - Gonxhe Lokaj
AU - Annette Nicke
PY - 2026
DA - 2026/10/21
JO - Channels (Austin, Tex.)
DO - 10.1080/19336950.2026.2736911
UR - https://europepmc.org/article/MED/42905803
AB - Nicotinic acetylcholine receptors (nAChRs) are primary targets of insecticides. While most mammalian nAChRs form functional channels in heterologous systems, insect nAChRs remain notoriously difficult to reconstitute. Here, we co-expressed insect/human subunit chimeras to enable functional analysis of heteromeric insect nAChR ligand‑binding sites in Xenopus laevis oocytes. First, EGFP-tagged human/cockroach α7 chimeras (Hα7/Pα7) were generated by exchanging the signal peptide (SP), extracellular (ECD), transmembrane (TMD), and intracellular (L3-4) domains. While all constructs were translated into protein, Hα7-like acetylcholine-evoked currents were only observed when human N- and C-terminal ECD and ICD were present, indicating that these domains impose insect-specific constraints on homomeric nAChR formation. Second, we generated heteromeric chimeric receptors by fusing insect-derived ECDs onto hα7 subunits. Co-expression of Pα6/Hα7L3-4 and Pα7ECD/Hα7 chimeras resulted in current amplitudes < 1 µA. Co-expression of the respective bee Aα5ECD/Hα7, Aα6ECD/Hα7, and Aα7ECD, CT/Hα7 chimeras yielded currents > 1 µA. α-Bungarotoxin inhibited diheteromeric Pα6α7 chimeras (IC₅₀ 49 nM) and triheteromeric Aα5/α6/α7 chimeras (IC₅₀ 51.2 nM). Third, this approach was applied to cockroach Pα2 and Pβ1 subunits. Replacement of the Pα2 ICD and the Pβ1 TMD/ICD with homologous regions from Hα2 and Hβ2 subunits and co-expression with Pα1 resulted in a ~ 100-fold current increase (2.6 µA) compared to the wild‑type Pα1α2β1 receptor. The chimeric receptor was activated by neonicotinoids (EC₅₀ clothianidin: 24.1 nM, EC₅₀ thiacloprid: 13.6 nM) and α-bungarotoxin-sensitive (IC₅₀: 36.6 nM). In conclusion, we demonstrate that replacement of insect ICD or ICD and TMD enables formation of functional insect-like heteromeric nAChRs without co-expression of chaperones.
SN - 1933-6950
SN - 1933-6969
ER -