Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

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 D2 L 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.Ligand-gated ion channels or ionotropic receptors constitute a specific family of ion channels characterized by large extracellular ligand-binding domains physically and functionally connected to the transmembrane pore. They play essential roles in numerous physiological functions by translating extracellular biochemical messages into an electrical signal by modulation of the membrane potential. The family of vertebrate LGICs is divided in subgroups according to their sequence similarity and subsequently to their specificity to endogenous ligands. The ligands γ -aminobutyric acid (GABA), glycine (Gly), serotonin, acetylcholine and zinc are recognized by members of the Cys-loop subgroup, while glutamate and ATP are recognized by the glutamate and the P2X receptors, respectively. The discovery of invertebrate LGICs such as the nematode GluCl 1,2 extended the ion selectivity of glutamate-gated ion channels to Cl -, while prokaryotic LGICs extended the repertoire of endogenous extracellular ligand to protons 3,4 . The physiological role of those prokaryotic LGICs in unicellular organisms is not yet clearly

Functional mapping of the N-terminal arginine cluster and C-terminal acidic residues of Kir6.2 channel fused to a G protein-coupled receptor

“…When the same approach of incremental deletions was previously used between M2=K0-25 and M2=K-9-25, a linear correlation between the length of the M2 C-ter and the regulation of the channel was observed [3]. In Fig.…”

“…Thus, for example, the human M2 muscarinic receptor opens the ion channel upon acetylcholine (ACh) binding, while D2 L (D2) dopaminergic receptor closes it in the presence of dopamine. In a previous study [3], we demonstrated that the length of the receptor C-termini controls the sign and the amplitude of the channel regulation. In the muscarinic ICCR, the receptor C-ter was shortened by 9 residues in order to mimic the D2 C-ter length, resulting in an inversion of the channel regulation leading to an inhibition of the channel by ACh (Fig.…”

“…The n values represent the number of tested oocytes. (B) The data within the dashed-line box of panel (A) are overlayed onto data obtained previously by truncation of the receptor C-ter [3]. The abscissa axis represents the number of deleted residues from the construct M2=K0-25 either in the M2 C-ter (blue dots) or in the Kir6.2 N-ter (red dots).…”

“…Flow of K + ions is inward in our two-electrode voltage-clamp (TEVC) recordings at −50mV in 91 mM K + extracellular concentration. A previous study [3] showed that deletion of the last 9 residues of M2 (M2=K-9-25) inverted the channel regulation by ACh. In the present study, we characterize the isometric deletion in the ion channel N-ter (M2=K0-34).…”

“…Subsequently, we demonstrated that the length of the GPCR C-ter controls the sign of the signal independently of its primary sequence. Notably, the ability to finely tune the Kir6.2 channel regulation by engineering the C-ter of fused GPCRs opens perspectives in synthetic biology to control cellular responses to endogenous or exogenous ligands [3]. However, the molecular mechanisms of this allosteric regulation have not yet been characterized, and some GPCR are difficult to functionally couple to the ion channel.…”