Point mutation of a conserved aspartate, D69, in the muscarinic M 2 receptor does not modify voltage-sensitive agonist potency.

The muscarinic M2 receptor (M2 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 M2 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 M2 R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M2 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 M2 R.