Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Biochem Biophys Res Commun
2018 Jan 29;4961:101-104. doi: 10.1016/j.bbrc.2018.01.005.
Show Gene links
Show Anatomy links
Point mutation of a conserved aspartate, D69, in the muscarinic M 2 receptor does not modify voltage-sensitive agonist potency.
Ågren R
,
Sahlholm K
,
Nilsson J
,
Århem P
.
???displayArticle.abstract???
The muscarinic M 2 receptor (M 2 R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M 2 R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M 2 R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC 50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M 2 R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M 2 R.
Fig. 1. Voltage-sensitive potency of GIRK channel activation by M2R D69N is similar to that of WT M2R. A) Representative current traces showing the GIRK current response to increasing concentrations of Ach, as indicated, in an oocyte expressing GIRK1/4 channels together with the M2R D69N mutant, at 0 mV (top) and at −80 mV (bottom). Both traces were recorded in the same oocyte. B) Mean currents (±S.E.M.) induced in oocytes expressing GIRK1/4 channels together with WT or D69N mutant M2R, by maximally efficacious concentrations of ACh (WT; 1 μM, D69N; 10 μM), at 0 mV (top) and −80 mV (bottom). At −80 mV, the maximal response to ACh at M2R D69N was −1.4 ± 0.5 μA compared to −4.3 ± 0.6 μA at M2R WT (p = .012, Student's t-test; B), whereas at 0 mV, the maximal response was 50 ± 15 nA at M2R D69N, and 151 ± 27 nA at WT M2R (p = .041, Student's t-test). Asterisks indicate statistical significance; *, P < .05. n (WT) = 17, n (D69N) = 7. C) Concentration-response curves for M2R-evoked GIRK activation at −80 and 0 mV, in WT and D69N mutant M2R receptors. The pEC50 for GIRK activation was 7.82 ± 0.04 (15.2 nM) and 7.52 ± 0.05 (30.4 nM) at −80 and at 0 mV, respectively, at WT M2R (n = 7–17), and 6.80 ± 0.07 (160 nM) and 6.47 ± 0.03 (343 nM) at −80 and at 0 mV, respectively, at the D69N mutant receptor (n = 2–6). Thus, the voltage dependent shift is 2.0-fold at the WT and 2.1-fold at the D69N mutant receptor. The pEC50s were significantly different between the two voltages at both the WT and the D69N mutant receptor (p < .001, ANOVA). D) Rates of current deactivation upon ACh washout. Rates of deactivation of GIRK currents evoked by ACh acting at WT M2R (left) and D69N mutant M2R (right) were significantly faster at 0 mV than at −80 mV (WT, −80 mV; 22.6 ± 1.1 s, 0 mV; 18.2 ± 2.0 s, n = 17; D69N, −80 mV; 58.5 ± 13.2, 0 mV; 30.1 ± 7.5, n = 7). *, P < .05; paired Student's t-test.
