Chen IS , Furutani K , Kurachi Y .

	Figure 1. Voltage-dependent response of pilocarpine-induced GIRK currents in rat atrial myocytes and Xenopus oocytes. (a) Pilocarpine (100 µM)-induced GIRK currents in rat atrial myocytes were recorded with a step pulse protocol as shown above the current traces. Basal currents were subtracted. (b) I-V curve of pilocarpine-induced GIRK currents in atrial myocytes. I p (filled circles) indicates the peak current at an activated state of GIRK channel; I s (open circles) indicates the steady-state current at the end of the voltage pulse. Red arrows in a were used to reconstruct the I-V curve in b. (c–f) Pilocarpine (1, 10, 100 μM)-induced GIRK currents in Xenopus oocytes expressing M2 muscarinic receptor, Kir3.1/Kir3.4 with (c,d) and without (e,f) RGS4 were recorded with a step pulse protocol shown above. Basal currents were subtracted. Amplitudes of I p and I s indicated by red arrows in c and e were used to reconstruct the I-V curve in d and f.
	Figure 2. Effects of RGS4 on modulation of GIRK current in a pilocarpine concentration-dependent manner. (a,b) Pilocarpine (1, 10, 100 μM)-induced GIRK currents in Xenopus oocytes expressing M2 muscarinic receptor, Kir3.1/Kir3.4 with (a) and without (b) RGS4 were recorded at −140 mV with the test pulse protocol shown above. The I p evoked by different concentrations of pilocarpine (1, 10, 100 μM) were normalized to the same level (red dashed line). Different levels of I s in the presence of different pilocarpine concentrations were observed. (c) The ratio of I s to I p (I s/I p) at hyperpolarized potentials (−20 mV to −140 mV) in the applications of pilocarpine (1 µM, black circles; 10 µM, blue squares; 100 µM, green triangles) in oocytes expressing RGS4. Data are means ± s.e.m., n = 6–10. (d) Concentration-response curves of I s (filled circles) and I p (open circles) of pilocarpine-induced GIRK current in the presence of RGS4 were fitted with the Hill equation. The maximal value of I p of pilocarpine-induced GIRK current was normalized to 1. The –logEC50 was 6.23 ± 0.12 M for I s; 6.11 ± 0.02 M for I p. Data are means ± s.e.m., n = 3–6.
	Figure 3. Effects of RGS4 on the recovery of GIRK currents from desensitization. (a,b) Pilocarpine (100 µM)-induced GIRK currents in oocytes expressing M2 muscarinic receptor, Kir3.1/Kir3.4 with (a) and without (b) RGS4 were recorded with a protocol shown above. Current were recorded with two hyperpolarizing pulses from 0 mV to −100 mV for 2 s which were separated by an interpulse of 0 mV for a duration range of 0.1, 0.3, 0.5, 0.7 and 0.9 s. The fully recovery of GIRK current from the desensitized state can be observed when interpulse (0 mV) duration is longer than 0.5 s. Time constant for the recovery was fitted with exponential curve and discussed in the result section.
	Figure 4. Charged residues in the DRY motif of the M2 muscarinic receptor contribute to the voltage-dependent response of GIRK currents. (a) Alignment of amino acids of different species of M2 muscarinic receptors and other human rhodopsin-like GPCRs. Triangles indicate the position of charged residues D692.50, D1033.32, D1203.49 and R1213.50. (b) M2 muscarinic receptor model was based on the crystal structure (PDB accession 4MQS)27. The charged residues that we investigated in this study are colored. (c–f) Pilocarpine (100 µM)-induced GIRK currents in oocytes expressing the WT M2 muscarinic receptor (c) or mutants D120N (d), R121N (e) and D120N/R121N (f) with (left panels) and without (right panels) RGS4 were recorded at −100 mV as shown above. (g) The ratio of I s to I p (I s/I p) of pilocarpine-induced GIRK current at −100 mV in oocytes expressing M2 muscarinic receptor WT and mutants. (h) Concentration-response curves of pilocarpine-induced GIRK current were fitted with the Hill equation. The maximal value of pilocarpine-induced GIRK current was normalized to 1. The −log EC50 was 6.51 ± 0.01 for WT; 6.81 ± 0.01 for D120N; 5.66 ± 0.01 for R121N; 6.50 ± 0.01 for D120N/R121N. Data in g and h are means ± s.e.m., n = 6–8. ***Indicates a statistically significant difference (P ≤ 0.001).
	Figure 5. Effects of mutations in the intracellular loop 3 of M2 muscarinic receptor on the voltage-dependent response of GIRK currents. (a) Pilocarpine (100 µM)-induced GIRK currents in oocytes expressing M2 muscarinic receptor mutant ELAAL with (left panels) and without (right panels) RGS4 were recorded at −100 mV as shown above. (b) The ratio of I s to I p (I s/I p) of M2 muscarinic receptor WT and ELAAL at –100 mV in the presence of pilocarpine (100 µM). (c) Recoveries of pilocarpine (100 µM)-induced GIRK currents from hyperpolarization-mediated desensitization of channels in oocytes expressing WT and ELAAL M2 muscarinic receptors were recorded with a protocol shown above the current traces. (d) The time constant (tau) of I p recovery times in c were calculated with exponential curve fitting. (e) Concentration-response curves of pilocarpine-induced GIRK currents were fitted with the Hill equation. The maximal value of pilocarpine-induced GIRK current was normalized to 1. The −logEC50 was 6.51 ± 0.02 for WT; 6.15 ± 0.02 for ELAAL. Data in b, d and e are means ± s.e.m., n = 6. ***Indicates a statistically significant difference (P < 0.001).

Abramow-Newerly, RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins. 2006, Pubmed

Abramow-Newerly, RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins. 2006, Pubmed
Ballesteros, Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. 1998, Pubmed
Barchad-Avitzur, A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor. 2016, Pubmed , Xenbase
Ben Chaim, Voltage affects the dissociation rate constant of the m2 muscarinic receptor. 2013, Pubmed , Xenbase
Ben-Chaim, The M2 muscarinic G-protein-coupled receptor is voltage-sensitive. 2003, Pubmed , Xenbase
Ben-Chaim, Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor. 2006, Pubmed , Xenbase
Bernstein, RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. 2004, Pubmed
Chen, RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents. 2014, Pubmed , Xenbase
Croft, A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity. 2013, Pubmed
Dekel, Depolarization induces a conformational change in the binding site region of the M2 muscarinic receptor. 2012, Pubmed , Xenbase
Dohlman, RGS proteins and signaling by heterotrimeric G proteins. 1997, Pubmed
Fujita, A regulator of G protein signalling (RGS) protein confers agonist-dependent relaxation gating to a G protein-gated K+ channel. 2000, Pubmed , Xenbase
Furukawa, Conformation of ligands bound to the muscarinic acetylcholine receptor. 2002, Pubmed
Greasley, Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation. 2002, Pubmed
Haga, Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. 2012, Pubmed
Hague, Selective inhibition of alpha1A-adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop. 2005, Pubmed
Hepler, Emerging roles for RGS proteins in cell signalling. 1999, Pubmed
Inanobe, Interaction between the RGS domain of RGS4 with G protein alpha subunits mediates the voltage-dependent relaxation of the G protein-gated potassium channel. 2001, Pubmed , Xenbase
Ishii, Ca(2+) elevation evoked by membrane depolarization regulates G protein cycle via RGS proteins in the heart. 2001, Pubmed
Koelle, A new family of G-protein regulators - the RGS proteins. 1997, Pubmed
Krapivinsky, The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins. 1995, Pubmed , Xenbase
Kruse, Activation and allosteric modulation of a muscarinic acetylcholine receptor. 2013, Pubmed
Lai, Chimeric M1/M2 muscarinic receptors: correlation of ligand selectivity and functional coupling with structural modifications. 1992, Pubmed
Liu, Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis. 1996, Pubmed
Mahaut-Smith, A role for membrane potential in regulating GPCRs? 2008, Pubmed
Moreno-Galindo, Relaxation gating of the acetylcholine-activated inward rectifier K+ current is mediated by intrinsic voltage sensitivity of the muscarinic receptor. 2011, Pubmed
Moreno-Galindo, The agonist-specific voltage dependence of M2 muscarinic receptors modulates the deactivation of the acetylcholine-gated K(+) current (I KACh). 2016, Pubmed
Navarro-Polanco, Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding. 2011, Pubmed
Neubig, Regulators of G-protein signalling as new central nervous system drug targets. 2002, Pubmed
Ross, GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. 2000, Pubmed
Rovati, The highly conserved DRY motif of class A G protein-coupled receptors: beyond the ground state. 2007, Pubmed
Seifert, Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations. 2001, Pubmed
Shukla, Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. 2011, Pubmed
Tamirisa, RGS4 inhibits G-protein signaling in cardiomyocytes. 1999, Pubmed
Tamuleviciute, Muscarinic receptors: electrifying new insights. 2011, Pubmed
Vickery, Membrane potentials regulating GPCRs: insights from experiments and molecular dynamics simulations. 2016, Pubmed
Vickery, Structural Mechanisms of Voltage Sensing in G Protein-Coupled Receptors. 2016, Pubmed
Wong, G protein selectivity is regulated by multiple intracellular regions of GPCRs. 2003, Pubmed
Wong, Chimeric muscarinic cholinergic:beta-adrenergic receptors that are functionally promiscuous among G proteins. 1994, Pubmed
Zhang, RGS3 and RGS4 are GTPase activating proteins in the heart. 1998, Pubmed
Zohar, New mechanism for voltage induced charge movement revealed in GPCRs--theory and experiments. 2010, Pubmed , Xenbase