Galantamine activates muscle-type nicotinic acetylcholine receptors without binding to the acetylcholine-binding site

Abstract

Galantamine (Reminyl; Janssen Pharmaceutica, Titusville, NJ) belongs to a class of acetylcholinesterase inhibitors approved for symptomatic treatment of Alzheimer's disease. The drug presumably acts by raising and prolonging the profile of acetylcholine (ACh) via an inhibitory effect on the esterase. However, there is also evidence demonstrating that galantamine can activate the nicotinic ACh receptor or modulate its activation by ACh. In this study, we have examined the ability of galantamine to directly activate the muscle-type nicotinic ACh receptor or to modulate receptor activation by selected nicotinic agonists. Studies of direct activation by galantamine demonstrated that this ligand is a low-efficacy agonist of the muscle-type ACh receptor. Point mutations in the M2-M3 linker (alphaS269I) and the M2 transmembrane domain (epsilonT264P) had similar effects on receptor activation by galantamine and nicotinic agonists, suggesting that the general features of receptor activation by galantamine are similar to that in the presence of ACh. Experiments performed in the simultaneous presence of galantamine and various nicotinic ligands showed that channel activation by the nicotinic ligands studied (ACh, carbachol, and choline) was not affected by the presence of galantamine at concentrations up to 100 microm. In addition, galantamine did not reduce the initial rate of binding for 125I-alpha-bungarotoxin. These results demonstrate that galantamine does not interfere with the occupation of the nicotinic agonist binding site by ACh, carbachol, or choline. We conclude that galantamine activates the muscle-type ACh receptor by interacting with a binding site that is distinct from the site for nicotinic agonists.

Figure 2.

Single-channel currents from wild-type and mutant receptors activated by galantamine. A, Galantamine is a low-efficacy agonist of the wild-type receptor. Receptor activity takes place as isolated openings without any indication of single-channel clusters. Occasional overlaps seen in the records indicate that at least two activatable channels were in the patch, suggesting that the true opening frequency of a single ion channel is less than what can be seen in the figure. For closed-time (CT) and open-time (OT) analysis, portions of the records without overlaps were used. The CT and OT histograms were fitted to a single exponential with 0.1 μm galantamine (CT, 6.0 ± 0.1 ms; OT, 0.80 ± 0.02 ms). In the presence of 10 μm galantamine, two components were seen in the CT histogram (0.21 ± 0.04 and 8.3 ± 0.3 ms) and one component in the OT histogram (0.70 ± 0.17 ms). The short-lived component in the CT histogram likely corresponds to dwells in the blocked state (see Results). Because of the uncertainty in the number of channels in the patch, the value of the longer-lived CT component cannot be interpreted. B, The αS269I mutation significantly enhanced OT durations and the opening frequency. In the presence of 0.1 μm galantamine, the mean CTs were 0.09 ± 0.03 and 34.5 ± 1.2 ms. The mean OTs were 0.26 ± 0.02 and 12.7 ± 0.8 ms. We believe that the shorter-lived openings arise from a monoliganded open state. With 10 μm galantamine, the mean OT duration was 2.6 ± 0.05 ms. The CT histogram was fitted with the sum of two exponentials (0.34 ± 0.01 and 30.3 ± 0.9 ms). C, The ϵT264P mutation greatly enhances gating efficacy. In the presence of 0.1 μm galantamine, three components were observed in the OT histogram (0.05 ± 0.01, 0.63 ± 0.30, and 41.7 ± 2.8 ms). We believe that the three components arise from unliganded, monoliganded, and diliganded receptors, respectively. The best fit of the CT histogram was achieved with four components (0.02 ± 0.01, 0.21 ± 0.04, 5.8 ± 3.3, and 32.3 ± 11.5 ms). No attempt to assign the individual components to specific processes of receptor activation was made. With 10 μm galantamine, the mean open duration was 3.6 ± 0.02 ms, likely reflecting openings from diliganded components. The disappearance of unliganded and monoliganded openings is a result of a higher agonist concentration. The relatively shorter duration of openings compared with the longest-lived component at a 0.1 μm concentration of galantamine is probably a result of channel block. The CT histogram was fitted to the sum of two exponentials (0.32 ± 0.01 and 40 ± 6.4 ms). The shorter component likely reflects dwells in the blocked state.

Figure 4.

Galantamine blocks single-channel currents from the wild-type receptor activated by ACh. For each condition, single-channel currents under two time resolutions as well as closed-time (CT) and open-time (OT) histograms are shown. An increase in galantamine concentration and/or hyperpolarization results in an increase in the relative number of blocked-related channel closures. A, Single-channel currents elicited by 5 μm ACh at -50 mV. The mean CT was 11.0 ± 0.4 ms, and the mean OT was 0.94 ± 0.03 ms. B, At a 10 μm concentration of galantamine, the CT histograms were fitted by the sum of two exponentials. The mean CTs were 0.35 ± 0.06 ms (20%) and 7.9 ± 0.3 ms (80%). The shorter-lived closed dwells arise from the blocked state. The mean OT was 0.72 ± 0.02 ms. C, The relative number of block-related channel closures is greater at 100 μm galantamine, but the duration is unaffected. The mean CTs were 0.42 ± 0.02 ms (65%) and 13.7 ± 0.6 ms (35%). The mean OT was 0.28 ± 0.01 ms. D, Hyperpolarization (-100 mV) resulted in an increase in the relative number of block-related dwells and the prolongation of the lifetime of the blocked state. The mean CTs were 1.3 ± 0.04 ms (78%) and 12.2 ± 1.0 ms (22%). The mean OT was 0.27 ± 0.01 ms.

Figure 5.

Effect of galantamine on wild-type receptors activated by carbachol. A, Single-channel clusters recorded in the presence of 1 mm carbachol, carbachol plus 1 μm galantamine, or carbachol plus 100 μm galantamine. The presence of 100 μm galantamine leads to a slight reduction in the open probability, because of channel block. The closed-time (CT) and open-time (OT) histograms from the respective patches are shown to the right. The CT histograms were fitted with two components for carbachol and carbachol plus 1 μm galantamine and with three components for carbachol plus 100 μm galantamine. The OT histograms were fitted to a single exponential. The results of the fit are as follows: At a 1 mm concentration of carbachol, the CTs were 0.22 ± 0.003 and 2.6 ± 0.2 ms, and the mean open duration was 0.68 ± 0.01 ms. The shorter-lived component in the CT distribution results from activation-related channel closures, and the longer-lived component corresponds to dwells in the A₂D state (see Scheme 1). With carbachol plus 1 μm galantamine, the CTs were 0.18 ± 0.002 and 2.3 ± 0.2 ms, and the OT was 0.52 ± 0.004 ms. With carbachol plus 100 μm galantamine, the CTs were 0.21 ± 0.01, 0.65 ± 0.06, and 4.5 ± 0.6 ms, and the mean open duration was 0.31 ± 0.003 ms. The additional (0.65 ms) component in the CT histogram is attributable to dwells in the blocked state, and the reduction in the mean open time reflects the block process. B, The effective opening rate at 1 mm carbachol in the absence and presence of 0.2-100 μm galantamine. Each data point corresponds to one patch. Overall, the data indicate that galantamine does not affect the effective opening rate. C, The effective opening rate curves for carbachol in the absence and presence of 100 μm galantamine. Each point corresponds to data from one patch. No data are shown for 500 μm carbachol plus 100 μm galantamine because of the similarities in the lifetimes for closed states arising from channel activation and block by galantamine. The curves were fitted with Equation 1. The results of the fit areas follows: β, 7176 ± 664 s^-; EC₅₀, 960 ± 117 μm, 2.1 ± 0.1 (control); β, 7553 ± 708 s^-; EC₅₀, 903 ± 147 μm; 2.1 ± 0.2 (plus 100 μm galantamine). The results demonstrate that galantamine does not affect the binding or opening properties of the receptor, because neither the EC₅₀ nor β estimates are affected.

Figure 1.

Structures of nicotinic receptor activators. The structures of the nicotinic receptor activators used (galantamine, ACh, carbachol, and choline) are shown. The results show that galantamine activates the receptor without interacting with the traditional ACh-binding site, also shared by carbachol and choline.

Figure 3.

Activation of the αS269I receptor by galantamine. A, Sample currents elicited by 0.2, 0.5, or 1 μm galantamine. Open-time (OT) and closed-time (CT) histograms. The dead time used in fitting the CT histograms at a 0.2-1 μm concentration of galantamine was 1.5 ms to eliminate the reduction in duration of activation-related channel closures by monoliganded openings (see Materials and Methods). The mean CTs were 135 ± 16 ms (0.2 μm), 83 ± 7 ms (0.5 μm), and 24.4 ± 1.2 ms (1 μm). The dead time used in fitting the OT histograms was 36-45 μs. The mean OTs were 0.18 ± 0.01 and 9.8 ± 0.7 ms (0.2 μm), 0.38 ± 0.04 and 14.7 ± 1.1 ms (0.5 μm), and 0.20 ± 0.03 and 6.0 ± 0.2 ms (1 μm). B, The channel effective opening rate is plotted as a function of galantamine concentration. Each symbol corresponds to data from one patch. At a 0.2-1 μm concentration of galantamine, openings of <1-1.5 ms were omitted from the calculation of the effective opening rate to avoid contamination by monoliganded openings (see Materials and Methods). The line was fitted using Equation 1. The best-fit parameters are as follows: β, 51 ± 10 s^-; EC₅₀, 0.7 ± 0.3 μm and 1.6 ± 0.5.

Figure 6.

Effect of galantamine on αS269I receptors activated by choline. A, Single-channel clusters recorded in the presence of 500 μm or 5 mm choline or in the absence and presence of 1 μm galantamine, as well as corresponding intracluster closed-time (CT) histograms. With 500 μm choline, the mean CTs were 0.08 ± 0.01 and 7.2 ± 0.2 ms. With 500 μm choline plus 1 μm galantamine, the mean CTs were 0.11 ± 0.01 and 6.7 ± 0.1 ms. With 5 mm choline, the mean CTs were 1. 3 ± 0.03 and 9.4 ± 2.0 ms. With 5 mm choline plus 1 μm galantamine, the mean CTs were 1.3 ± 0.02 and 8.2 ± 1.0 ms. The closed-time histogram with 500 μm choline alone shows two components. The longer-duration component includes both activation-related closures and dwells in the short-lived desensitized state. It is likely that the brief closed state primarily reflects immediate reopenings from the doubly liganded closed state. The longer-duration component was interpreted as the effective opening rate. With 5 mm cholinealone, the small contribution of the short-lived desensitized state can be resolved (mean duration, 9 ms), while the component reflecting activation-related closures has shifted to briefer durations (1.3 ms). Note that the presence of galantamine does not produce any change in the CT distributions. B, The mutant channel effective opening rate is plotted as a function of choline concentration. The presence of 1 μm galantamine has little effect on the effective opening rate value. For choline, the best-fit parameters are as follows: +---β, 3062 ± 1196 s^-; EC₅₀, 14.7 ± 12.6 mm and 0.9 ± 0.1. For choline plus 1 μm galantamine, the best-fit parameters are as follows: β, 4017 ± 3034 s^-;EC₅₀, 21.2 ± 31.1 mm and 0.9 ± 0.2.

Figure 7.

Galantamine does not reduce the initial rate of binding for I-αBGT. A, The reduction in the initial rate of I-αBGT binding for wild-type adult receptors (expressed in QA-33 cells) by carbachol (open circles) and galantamine (filled squares). The IC₅₀ for inhibition by carbachol is ∼38 μm, whereas that for galantamine is not defined in these data. B, Similar data for adult receptors containing the αS269I subunit. The IC₅₀ for inhibition by carbachol is shifted to ∼0.4 μm, but the IC₅₀ for inhibition by galantamine is not defined (>1000 μm). BTX, ¹²⁵I-α-bungarotoxin.