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J Physiol
2014 Mar 15;5926:1237-48. doi: 10.1113/jphysiol.2013.269803.
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RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents.
Chen IS
,
Furutani K
,
Inanobe A
,
Kurachi Y
.
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Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist-receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K(+) inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist-receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine-M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses.
Figure 1. Effects of RSG4 on ACh- and pilocarpine-evoked KG currentsA, 100 μm pilocarpine-evoked KG currents (red) relative to 1 μm ACh-evoked KG currents (black) in the same rat atrial myocyte. Whole-cell currents were recorded with a voltage pulse protocol as showed below the current traces and basal currents were subtracted. Arrowheads indicate the zero current level. Pilocarpine (100 μm)-evoked currents were blocked in the presence of 0.7 μm atropine (B) or 0.1 μm tertiapin-Q (C) in atrial myocytes. Agonist-evoked currents were recorded in the same oocyte expressing M2Rs and the Kir3.1/Kir3.4 heteromultimer with (D) and without (E) RGS4 at −100 mV.
Figure 2. Concentration–response curve of pilocarpine and AChCurrents were recorded in the presence of pilocarpine (0.1–100 μm) and ACh (0.001–10 μm) with a holding potential of −60 mV in oocytes with (A) and without (C) expression of RGS4. The bars above each trace indicate the periods and concentrations of application of ACh or pilocarpine. Dashed lines indicate the zero current level and arrowheads indicate the basal current level. Concentration–response curves of pilocarpine and ACh in oocytes with (B) or without (D) expression of RGS4. The average maximal response of ACh-evoked KG currents was set as 100%. The vertical axis indicates the relative percentages of response that were induced by each concentration of agonists. Data show the means ± SEM, n = 6 at each concentration in both with and without RGS4 groups. All symbols have attached error bars, but some of them are smaller than the symbol.
Figure 3. Voltage-dependence of relative response of pilocarpine to AChACh (1 μm,
a) and pilocarpine (100 μm,
b)-evoked KG currents in oocytes with (A) and without (B) expression of RGS4. Whole-cell currents were recorded with a step protocol as showed above the current traces and basal currents were subtracted. Arrowheads indicate the zero current level. C, the pilocarpine (100 μm)-evoked KG currents as a percentage of ACh (1 μm)-evoked KG currents at each membrane potential were calculated from steady state. Filled circles indicate the oocytes expressing RGS4; open circles indicate absence of RGS. The graph shows the means ± SEM, n = 6 for each condition.
Figure 4. Requirements of RGS4 domain for partial agonism of the M2R-activated KG currentsA, ACh (1 μm)-evoked KG currents (black) and pilocarpine (100 μm)-evoked KG currents (red) in oocytes expressing wild-type RGS4 (a), without RGS4 expression (b) and expressing various RGS4 mutants (c–h) at –60 mV. B, formations of truncated mutants of RGS4 and a point mutation in RGS domain. Black portions represent the region of RGS domain. C, the response of pilocarpine-evoked KG currents relative to ACh-evoked KG currents was calculated from steady state at −60 mV. The bar graph shows the means ± SEM, n = 6–9. The filled bars indicate that the intact RGS domain is included in the mutants; the open bars indicate absence of the intact RGS domain and a significant difference (P < 0.05) from the wild-type RGS4 group.
Figure 5. Effects of a palmitoylation inhibitor 2–BP on agonist-evoked KG currentsACh (1 μm)-evoked KG currents were recorded in RGS4-expressing oocytes in vehicle (contained ≤0.1% ethanol) (A) or pretreatment with 2–BP (100 μm) for ≥2 h (B) with a continuous test pulse as showed on the right side of current traces. The interval of each pulse is 3 s. Relative KG currents of pilocarpine (100 μm) to ACh (1 μm) were recorded in the same oocyte at −60 mV in the presence of vehicle (C) or 2–BP (D). Basal currents were subtracted.
Figure 6. Suppression of dopamine-evoked KG currents by pilocarpine via RGS4 and M2RAgonist-evoked KG currents were recorded in oocytes expressing D2R and M2R (injected cRNA ratio of D2R to M2R = 100, D/M = 100) with (A) or without (B) RGS4 by a continuous test pulse as shown above the current traces. The bars above each trace indicate the application of dopamine, pilocarpine and K+ channel blocker Ba2+. C, the current recorded in an oocyte that did not express M2R. D, the current recorded in an oocyte that did not express D2R. E, the ratio of dopamine-evoked currents in the presence of pilocarpine (100 μm) to dopamine-evoked currents in the absence of pilcarpine at −60 mV. Basal and pilocarpine-evoked currents were subtracted. D/M = 100 indicates the high M2R-expressing group and D/M = 1000 indicates the low M2R-expressing group (injected cRNA ratio of D2R to M2R = 1000). Data show the mean ± SEM, n = 6. * indicates a significant difference (P < 0.05).
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