Moreau CJ et al. (2017), Tuning the allosteric regulation of artificial ...

XB-ART-55414

Sci Rep 2017 Feb 01;7:41154. doi: 10.1038/srep41154.

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

Moreau CJ , Revilloud J , Caro LN , Dupuis JP , Trouchet A , Estrada-Mondragón A , Nieścierowicz K , Sapay N , Crouzy S , Vivaudou M .

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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.

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	Figure 1. Opposite regulation of muscarinic and dopaminergic Ion Channel-Coupled Receptors (ICCRs).(a) The artificial Ion Channel-Coupled Receptors are created by fusion of the Kir6.2 N-terminus to the C-terminus of G Protein-Coupled Receptors (GPCRs). Only one full-length subunit and the channel of a second subunit are shown. In M2 ICCRS, agonists upregulate Kir6.2 while in D2 ICCRs, they downregulate. Ext.: extracellular side, Mb.: membrane, Cyto.: cytoplasmic side. (b) Alignment of the C-termini of M2 and D2 receptors.
	Figure 2. Shortening the M2 C-terminus inverts the gating regulation.(a) M2 = K0-25 designates 0 residue deleted from the M2 C-terminus and 25 residues deleted from the Kir6.2 N-terminus. Under the diagram is shown a representative Two-Electrode Voltage-Clamp (TEVC) recording of the ICCR heterologously expressed in Xenopus oocytes at −50 mV in symmetrical K + concentration. ACh: 5 μM Acetylcholine. Ba2 + (3 mM) is a blocker of potassium channels. Deletion of 9 residues at the M2 C-terminus (M2 = K-9-25) inverted the regulation of the channel. (b) Basal currents showing significant expression of the muscarinic ICCRs. Histogram shows mean ± s.e.m. with numbers of experiments above bars. * indicates a significant difference (P < 10−9) from non-injected oocytes. (c) Average in % of current amplitude ± s.e.m. induced by 5 μM ACh. Positive and negative values reflect opening and closing of the channel respectively. M2 + Kir6.2Δ: coexpression of M2 with Kir6.2 truncated of its last 36 residues. Number above bars = number of experiments (n). *: significant difference (P < 10−7) from M2 + KΔ. (d) ATP concentration-effect curves of M2 = K0-25 (control) and M2 = K-9-25 obtained by application of increasing concentrations of ATP to the cytoplasmic face of excised inside-out patches. ATP is an endogenous blocker of the channel by direct binding to Kir6.2.
	Figure 3. Engineered M2 receptors retain the capacity to activate Gi/o proteins.(a) Diagram of the functional assay of G protein activation in Xenopus oocytes using the G protein-activated Kir3 channels. mRNA coding for the ICCR and the Kir3 channel are co-injected in Xenopus oocytes. Binding of acetylcholine onto M2 triggers activation of the endogenous Gi/o proteins. The release of the Gβγ subunits leads to opening of Kir3 channels. The current generated by the Kir3 channels being several-fold higher than that of the current generated by Kir6.2 in the ICCR, the inhibitory effect of the agonist on M2 = K-9-25 is masked by the activating effect on Kir3. (b) Representative TEVC recording from an oocyte co-expressing M2 = K-9-25 and Kir3.4* showing activation of the Kir3 channels by carbachol (CCh, 5 μM) (blue arrow) and the antagonist effect of 1 μM atropine (red arrow). (c) Average current change induced by 5 μM CCh (blue bar) and 5 μM CCh + 1 μM atropine (red bar). *: P = 4.10−3 between the two bars. n = 4.
	Figure 4. Extending the D2 C-terminus also inverts the gating regulation.(a) D2 = K0-25 is inhibited by 5 μM dopamine (Dopa). D2=K+9M2-25 contains an extended D2 C-terminus with the last 9 residues from M2 and is activated by 5 μM Dopamine. The first transmembrane domain (TMD0) of SUR1 is co-expressed for boosting the surface expression of the ICCR. (b) Basal currents of dopaminergic ICCRs show lack of surface expression of D2=K+9M2-25. Co-expression with the N-terminal transmembrane domain of SUR1 (TMD0) restores the surface expression. Extended C-terminus of D2 with 9 alanines is noted D2=K+9Ala-25 and with 9 C-terminal residues from the human β2 adrenergic receptor is noted D2=K+9β2-25. : compared to Non-Injected P < 0.02; ns (not significant) P = 6.10−2. (c) Average in % of current amplitude ± s.e.m. induced by 5 μM dopamine. : P < 10−3 (ref.: D2 + K∆). (d) Alignment of the C-terminal sequences of the human receptors M2 (blue), D2 (green) and β2 adrenergic (brown) receptors. The position of the helix VIII is shown above the sequences and the terminal cysteines are numbered and depicted as palmitoylated. The long C-terminus of the β2 adrenergic receptor is partially represented. The 3 different extensions of the D2 C-terminus are shown in the lower panel. (e) TEVC recording of D2=K+9M2-25 + Kir3.4* showing D2-mediated activation of Kir3.4* channel by 5 μM dopamine (green arrow) and the antagonism (red arrow) by 5 μM sulpiride. (f ) Average current change induced by 5 μM dopamine (green bar) and 5 μM dopamine + 5 μM sulpiride (red bar) on D2=K+9M2-25 + Kir3.4. P = 9.10−3 between the two bars. n = 6.
	Figure 5. The C-terminally altered M2 and D2 ICCRs retain their pharmacological properties.(a) Diagram of the M2 = K-9-25 ICCR. (b) Representative TEVC recording of M2 = K-9-25 showing 5 μM ACh-evoked inhibition (blue arrow) and the antagonist effect of 1 μM atropine (red arrow). (c) Average current change induced by ACh 5 μM (in blue) and ACh 5 μM + Atropine 1 μM (in red) in oocytes expressing M2 = K-9-25. : P = 3.10−4 (ref: Ach). n = 8. (d) Diagram of the D2 = K + 9M2-25 ICCR. (e) TEVC recording showing activation of D2=K+9M2-25 + TMD0 by 5 μM dopamine (green arrow) and its antagonism (red arrow) by 5 μM sulpiride. (f) Average effects of dopamine and sulpiride on D2=K+9M2-25 + TMD0. : P = 1.10−3 (ref.: dopamine). n = 5. (g) Concentration-effect curve of carbachol (CCh) of the indicated constructs. Each point is an average of 8 to 23 measurements. (h) Concentration-effect curves of dopamine of the indicated constructs. n = 4.
	Figure 6. The gating regulation of M2 = K is correlated with the length of the receptor C-terminus.(a) Sequences of the fusion zone of M2 = K ICCRs with incremental deletions in the M2 C-terminus (in blue). (b) With 2 exceptions, all M2 C-terminally truncated ICCRs are expressed into the oocyte plasma membrane. ND: Not Determined because of failure of their genetic engineering. *: significantly expressed compared to non-injected oocytes P < 10−2. (c) Average effects of 5 μM ACh on each construct. Activation is positive, inhibition negative. Each point was determined from 8 experiments or more. The dashed line represents the best linear correlation fit.

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Althoff, X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. 2014, Pubmed