Galantamine is not a positive allosteric modulator of human α4β2 or α7 nicotinic acetylcholine receptors

Abstract

Background and purpose: The alkaloid galantamine was originally isolated from the green snowdrop Galanthus woronowii and is currently marketed as a drug for treatment of mild to moderate dementia in patients with Alzheimer's disease. In addition to a well-documented proficiency to inhibit acetylcholinesterase, galantamine has been reported to increase neuronal nicotinic ACh (nACh) receptor function by acting as a positive allosteric modulator. Yet there remains controversy regarding these findings in the literature. To resolve this conundrum, we evaluated galantamine actions at α4β2 and α7, which represent the nACh receptors most commonly associated with mammalian cognitive domains.

Experimental approach: α4β2 [in (α4)₃ (β2)₂ and (α4)₂ (β2)₃ stoichiometries] and α7 nACh receptors were expressed in Xenopus laevis oocytes and subjected to two-electrode voltage-clamp electrophysiological experiments. Galantamine (10 nM to 100 μM) was evaluated for direct agonist effects and for positive modulation by co-application with sub-maximally efficacious concentrations of ACh. In addition, similar experiments were performed with α7 nACh receptors stably expressed in HEK293 cells using patch-clamp electrophysiology.

Key results: In concentrations ranging from 10 nM to 1 μM, galantamine did not display direct agonism nor positive modulatory effects at any receptor combination tested. At concentrations from 10 μM and above, galantamine inhibited the activity with a mechanism of action consistent with open-channel pore blockade at all receptor types.

Conclusion and implications: Based on our data, we conclude that galantamine is not a positive allosteric modulator of α7 or α4β2 receptors, which represent the majority of nACh receptors in mammalian brain.

Figure 1

Effects of galantamine and NS1738 at nACh receptors expressed in Xenopus laevis oocytes. Xenopus oocytes were injected with cRNA mixtures containing nACh receptor subunits and subjected to two‐electrode voltage‐clamp electrophysiology as described in Methods. (A–E) Representative traces are seen for galantamine at α7 (A), (α4)₃(β2)₂ (B), (α4)₂(β2)₃ (C), (α4)₃(β4)₂ (D) and for NS1738 at α7 (E). Following pipette insertion, the oocyte membrane potential was clamped at −60 mV and several rounds of ACh_control (1–30 μM), ACh_max (1–10 mM) were applied to ensure baseline stability and the ACh_max reference point (note only the ACh_max trace shown). Full concentration–response relationships for galantamine (10 nM to 100 μM) or NS1738 (0.316 to 31.6 μM) were next obtained using a pre‐incubation protocol. This entailed ~30 s application of galantamine/NS1738 alone [or saline solution (buffer) for the ACh_control reference trace] followed by co‐application of ACh_control with the same concentration of galantamine/NS1738 for ~30 s. The representative traces were baseline subtracted, and the bars above each trace represent the application periods and concentrations of galantamine/NS1738 and ACh. For clarity, the majority of the ‘wash‐out’ periods (2–5 min) between each trace are omitted.

Figure 2

NS1738 modulation and galantamine inhibition of ACh‐evoked nACh receptor currents from Xenopus oocytes. Peak current amplitudes from experiments illustrated by representative traces in Figure 1 were normalized to the amplitude of the respective prior reference ACh_control applications in the absence of galantamine/NS1738 as described in Methods. (A, B) Normalized current amplitudes were plotted as means ± SEM as a function of the galantamine/NS1738 concentrations for the receptors indicated and fitted to the Hill equation by non‐linear regression. Results from the fitting routines with galantamine were: α7, pIC₅₀ = 4.3 ± 0.03, n = 5; (α4)₃(β2)₂, pIC₅₀ = 4.1 ± 0.2, n = 6; (α4)₂(β2)₃ – the inhibitory level at the maximal concentration of galantamine is too low to allow for robust fitting – n = 7; (α4)₃(β4)₂, pIC₅₀ = 4.9 ± 0.03, n = 6. Results for NS1738 are indicated in the panel.

Figure 3

Effects of galantamine at the α7 nACh receptor expressed in Xenopus oocytes using alternative experimental conditions. The α7 nACh receptor was expressed in Xenopus oocytes and subjected to two‐electrode voltage‐clamp experimentation as described in brief in the Figure 1 legend. (A) Representative traces of ACh‐evoked currents in the presence or absence of galantamine (1 nM to 100 μM). In these experiments, the buffer contained Ca²⁺, and oocyte membrane potentials were clamped at −70 mV. The ACh_control concentration of 250 μM represented approximate EC₅₀ (average of n = 9). (B) Representative traces of ACh‐evoked currents in the presence or absence of galantamine (1 pM to 10 μM). In these experiments, the buffer contained Ca²⁺, and the membrane potential was clamped at −70 mV. The ACh_control concentration of 30 μM represented approximate EC₁₀ (n = 15). (C, D) The peak current amplitudes from experiments illustrated by representative traces in (A) and (B), respectively, were normalized to the respective prior reference ACh_control applications in the absence of galantamine as described in Methods. Normalized current amplitudes were plotted as means ± SEM or means ± SD as a function of the galantamine concentrations and fitted to the Hill equation by non‐linear regression. Results from the fitting routines are indicated in the panels. SD values were used in (D) as SEM values were smaller than the symbol size. (E) Representative traces of ACh‐evoked currents in the presence or absence of galantamine (100 nM to 1 μM). In these experiments, the buffer was Ca²⁺‐free, and the membrane potential was clamped at −60 mV. The ACh_control concentration of 10 μM represented approximate EC₅ (average of n = 9).

Figure 4

Examination of effects of galantamine and NS1738 in Xenopus oocyte experiments using net charge analysis. (A–D) Data illustrated in Figures 2A, B and 3C, D, respectively, were re‐analysed using net charge analysis (curve integration). Area under the curve from all experiments were normalized to the respective reference ACh_control applications in the absence of galantamine/NS1738 as described in Methods. Normalized values were plotted as means ± SEM or means ± SD as a function of the galantamine/NS1738 concentrations for the receptors indicated and fitted to the Hill equation by non‐linear regression. Results from the fitting routines are indicated in the panels except for (B) where the values for galantamine were: α7, pIC₅₀ = 4.1 ± 0.1, n = 5; (α4)₃(β2)₂, pIC₅₀ = 3.8 ± 0.2, n = 6; (α4)₂(β2)₃ – the inhibitory level at the maximal concentration of galantamine is too low to allow for robust fitting – n = 6; (α4)₃(β4)₂, pIC₅₀ = 4.8 ± 0.04, n = 6. SD values were used in (D) as SEM values were smaller than the symbol size. Note that for (A), (C) and (D), the plots obtained from peak current amplitude measurements are shown in grey.

Figure 5

Galantamine testing at the α7 nACh receptor expressed in HEK293 cells. Patch‐clamp electrophysiology recordings were performed on α7 nACh receptor stably expressed in HEK293 as described in Methods. (A, B) Representative traces of ACh‐evoked currents in the presence or absence of galantamine (A) or NS1738 (B) using the Dynaflow Resolve® system. Galantamine or NS1738 was initially applied for 40 s (60 s for the 0.01 μM concentration) and thereafter co‐applied with 80 μM ACh (~EC₂₀) for 250 ms. The membrane potential was clamped at −70 mV. (C, D) Baseline‐subtracted peak current amplitudes were normalized to the prior ACh_control application in the absence of galantamine/NS1738 as described in Methods. Normalized current amplitudes were plotted as a function of the galantamine/NS1738 concentrations and fitted to the Hill equation by non‐linear regression. Results from the fitting routine are indicated in the panels.

Figure 6

Mechanism of the inhibitory action of galantamine at nACh receptors expressed in Xenopus oocytes. nACh receptors were expressed in Xenopus oocytes and subjected to two‐electrode voltage‐clamp experimentation as described in brief in the Figure 1 legend. (A) ACh concentration–response relationships were obtained at the α7 nACh receptor in the presence and absence of 30 μM galantamine (Gal). The peak current amplitudes evoked were fitted to the Hill equation and normalized as described in Methods. The fitted data derived are indicated in the panel, n = 10–11. (B) ACh‐evoked currents from oocytes expressing the indicated receptors were obtained in the presence or absence of 30 μM galantamine (Gal) at membrane holding potentials ranging from −20 to −100 mV. Peak current amplitudes were normalized to that of the respective prior ACh_control applications and plotted as a function of the holding potential. Normalized data were well approximated by linear regression. The fitted slopes were −0.75, −0.32, −0.38 and −0.46 for α7 (n = 8), (α4)₃(β2)₂ (n = 6), (α4)₂(β2)₃ (n = 6) and (α4)₃(β4)₂ (n = 6) respectively.