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  • Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

Cell reports methods (2022-01-04)
M Dolores García-Fernández, Franck C Chatelain, Hugues Nury, Anna Moroni, Christophe J Moreau
ABSTRACT

Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
Dopamine hydrochloride
Sigma-Aldrich
MEK1 Protein, inactive, 50 g, Unactive, N-terminal GST & C-terminal His6-tagged, recombinant full-length human MEK1, for use in Kinase Assays.