Abstract
Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.
Highlights
Kir6.2 hosts inhibitory binding sites for intracellular ATP and displays, within ion channel-coupled receptors (ICCRs), an IC50 in the range of hundred micromolars[6] leading to a partially opened channel at rest in a cellular environment. This basal activity allows the observation of the channel closing or opening evoked by GPCR ligands. These two opposite regulations have been observed in the first ICCRs composed of either the human muscarinic M2 receptor, which opened the channel in presence of acetylcholine, or the human dopaminergic D2L receptor (D2), which closed the channel in presence of dopamine[6] (Fig. 1a)
We postulated that M2 and D2 receptors undergo similar agonist-induced conformational changes, as observed in several crystallographic structures of class A GPCRs in their active states[7,8,9,10,11,12,13,14,15,16,17,18,19,20], and hypothesized that sequence differences in the ICCR region linking the receptor and the channel influenced the receptor-mediated regulation of Kir6.2 gating
To determine whether the difference in the M2 and D2 C-termini is implicated in the opposite regulation of the channel gating, we first removed the last 9 residues of the M2 receptor to match the D2 C-terminus length (Fig. 2a)
Summary
With the initial objective of designing versatile biosensors for various ligands, we created artificial LGICs by fusing metabotropic receptors (G protein-coupled receptors, GPCRs) to a mammalian potassium-selective channel (Kir6.2). In these fusion proteins called ion channel-coupled receptors (ICCRs), Kir6.2 gating is controlled by the GPCR conformational changes upon ligand binding. Kir6.2 hosts inhibitory binding sites for intracellular ATP and displays, within ICCRs, an IC50 in the range of hundred micromolars[6] leading to a partially opened channel at rest in a cellular environment This basal activity allows the observation of the channel closing or opening evoked by GPCR ligands. Our work demonstrates that artificial potassium-selective LGICs can be engineered to feature adjustable and versatile responses to natural ligands and opens the way for new applications in synthetic biology
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