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Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.
Chen IS
,
Furutani K
,
Kurachi Y
.
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Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.
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).
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