Open-Channel Blockers at the Human a4b2 Neuronal Nicotinic Acetylcholine Receptor

To extend our knowledge of the pharmacological profile of human a4b2 neuronal nicotinic receptors, we investigated the action of hexamethonium on the major brain human nicotinic acetylcholine receptor (nAChR) stably expressed in human embryonic kidney 293 cells. This compound displays all of the characteristics of an open-channel blocker at the human a4b2 nAChR: a voltage-dependent inhibition (more pronounced at hyperpolarized potentials), absence of competition, and use dependence. Moreover, we observed that classic N-methyl-Daspartate open-channel blockers amantadine, 3,5-dimethyl-1adamantanamine (memantine), and dizocilpine [(1)-MK-801] and the calcium channel antagonist 8-(diethylamino)octyl3,4,5-trimethoxybenzoate are powerful inhibitors of the human a4b2 nAChR. Dose-inhibition curves yield, at 2100 mV, IC50 values in the micromolar range for all of compounds and Hill coefficients below unity. Whole-cell current-voltage relationships display a strong rectification profile at hyperpolarized potentials, and current blockades are fitted adequately by a mathematical model that describes the mechanism of an ion channel block. We conclude that these molecules are powerful human a4b2 open-channel blockers ranking in the following order of potency: amantadine . memantine 5 hexamethonium . 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate ; (1)MK-801. Neuronal nAChRs belong to the superfamily of ionotropic LGCs (Bertrand and Changeux, 1995). Using the crayfish muscle preparation, it has been shown that muscle nAChRs are activated within a microsecond time scale on binding of the agonist (Franke et al., 1987). Very fast opening of the ionic pore results from the particular structure of these proteins that form both the ligand binding site and transmembrane channel. Given its physical dimensions, this aqueous pore is readily blocked by small molecules. Hexamethonium is a compound initially identified for its ability to block the ACh transmission in autonomic ganglia while leaving muscle nAChRs unaffected (Paton and Zaimis, 1951). The work of Blackman et al. (1963) and Ascher et al. (1979) suggested that hexamethonium inhibited the ACh-evoked currents in ganglionic neurons by sterically blocking the ionic pore of the nAChR. Extensive investigations have indicated that hexamethonium can be considered as a prototype OCB of the ganglionic nAChRs (Gurney and Rang, 1984) or of the reconstituted chick (Bertrand et al., 1990) and rat (Charnet et al., 1992) a4b2 nAChRs because this small molecule fulfilled the following characteristics: (1) its blocking effect is voltage dependent (Ascher et al., 1979; Gurney and Rang, 1984; Bertrand et al., 1990; Charnet et al., 1992), (2) it displays a use-dependent mode of action (Gurney and Rang, 1984), and (3) its blocking effect is more pronounced at higher agonist concentrations (Ascher et al., 1979). We therefore apply the term OCB to any compound that complies with these criteria. Some of the physiological and pharmacological properties of the human a4b2 neuronal nicotinic receptor have been investigated using the patch-clamp technique (Buisson et al., 1996) and indicate that human nAChRs present a distinct profile compared with other vertebrate nAChRs. To further establish the pharmacological signature of the human a4b2 nAChR, we investigated the effect of hexamethonium and TMB-8, a calcium channel antagonist that has been identified as a noncompetitive nicotinic antagonist (Bencherif et al., 1995) (Fig. 1). In addition, we examined the properties of (1)-MK-801 (dizocilpine) at the human a4b2 nAChR and of two other classic NMDA OCBs: amantadine (1-amino-adamantane) and memantine (3,5-dimethyl-1-adamantana-