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The Journal of Neuroscience, February 23, 2005, 25(8):1992-2001; doi:10.1523/JNEUROSCI.4985-04.2005
Previous Article | Next Article
Cellular/Molecular
Galantamine Activates Muscle-Type Nicotinic Acetylcholine Receptors without Binding to the Acetylcholine-Binding Site
Gustav Akk andJoe Henry Steinbach Department of Anesthesiology, Washington University, St. Louis, Missouri 63110
Abstract
Top
Abstract
Introduction
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 (
S269I) and the M2 transmembrane domain (
T264P) 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 µM. In addition, galantamine did not reduce the initial rate of binding for 125I-
Key words: acetylcholinesterase; channel; gating; binding; inhibition; block
Introduction
The muscle-type nicotinic acetylcholine (ACh) receptor is normally activated after binding two ACh molecules. The interactions of ACh molecules with specialized binding pockets lead to generalized movement in the protein structure and opening of the channel gate. Although ACh serves as the major agonist in vivo, the receptor can be activated by a number of compounds, including tetramethylammonium, nicotine, and choline. All of these drugs act by binding to the ACh-binding site.More recently, the activation of nicotinic receptors by a novel class of drugs, called allosterically potentiating ligands (APL), has been described (Pereira et al., 2002
). The most commonly used APL-type ligands are galantamine and physostigmine, which are both plant-derived inhibitors of acetylcholinesterase (AChE). Both compounds had been used for symptomatic treatment of Alzheimer's disease and schizophrenic patients acting, presumably, through a mechanism of an increase and prolongation of the ACh concentration profile. There is, however, evidence demonstrating that galantamine and physostigmine can directly activate or potentiate the nicotinic receptor function. In light of this, it has been proposed that a direct effect on the nicotinic receptor may contribute to the clinical actions of APLs (Maelicke and Albuquerque, 2000
The interaction sites on the nicotinic receptor for APLs are not known. APL-elicited openings have been reported to be insensitive to the actions of the nicotinic competitive antagonists methyllycaconitine, (+)-tubocurarine, or
In contrast, other studies have suggested that APLs interact with the ACh-binding site. For example, Zwart et al. (2000
4 receptors to ACh at low concentrations but inhibits at higher concentrations and (2) that physostigmine displaces an ACh-binding site ligand (epibatidine).
Most studies on nicotinic receptor activation by AChE inhibitors have been performed on the neuronal nicotinic receptor; relatively less is known about the effects of AChE inhibitors on the muscle-type receptor. The two types of receptors share a number of structural and functional properties. From the experimenter's point of view, however, the muscle-type receptor has several advantages. Most importantly, our understanding of the function of the muscle receptors is much more complete.
The present experiments were performed on adult and fetal-type muscle nicotinic receptors. The results demonstrate that galantamine is a low-efficacy agonist of the nicotinic receptor. The properties of channel activation produced by galantamine and nicotinic agonists are quite similar, indicating that the activated state is similar. However, both physiological and biochemical studies indicate that galantamine does not interact with the ACh-binding site. These observations suggest that activation of muscle-type nicotinic receptors can occur after ligand association with distinct sites on the receptor.
Materials and Methods
Mouse muscle nicotinic ACh receptor subunit cDNAs (,
,
Chemicals were obtained from Sigma (St. Louis, MO). One sample of galantamine was a gift from Memory Pharmaceuticals (Montvale, NJ), and a second sample was purchased from Tocris Cookson (Ellisville, MO).
Electrophysiological experiments were performed using the single-channel patch-clamp technique in the cell-attached configuration (Hamill et al., 1981
Single-channel currents were amplified with an Axopatch 200B amplifier (Axon Instruments, Union City, CA), digitized at 500 kHz, and saved on a computer hard disk using a Digidata 1322 Series interface (Axon Instruments) for additional analysis. For long-term storage, the original data files were saved on DVD+R disks.
Channel event detection was performed using the segmental k-means algorithm (QuB suite; www.qub.buffalo.edu) (Qin et al., 1996
The channel effective opening rate is defined as an inverse of the duration of dwells in activatable (not desensitized or blocked) closed states of the receptor. In the presence of nicotinic ligands, the effective opening rate is determined as the inverse of the major component of intracluster closed-time histograms. The effective opening rate depends on receptor affinity to the agonist and channel gating properties and is used to describe the agonist concentration-response profile. When galantamine was coapplied with a nicotinic ligand, two major closed-time components were observed, one corresponding to the effective opening rate and the second arising from dwells in the blocked state. The effective opening rate was then determined by identifying the component for which duration was dependent on the concentration of the nicotinic ligand. For wild-type receptors exposed to galantamine only, the channel openings were not condensed into easily identifiable clusters; thus, the effective opening rate for such currents was not determined.
It was more complicated to determine the effective opening rate for the
Effective opening rate curves (
(1)
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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
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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.
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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:
whereVoltage sensitivity was estimated from fitting the following equation:
(2) where Z0 is the parameter value at 0 mV membrane potential, V is membrane potential, and H is the change in membrane potential, which results in an e-fold change in duration. To convert H into f (the fraction of the electric field that would be traversed by a single positive charge), divide 25 mV by H.
The results presented here are not confined to a particular activation scheme. Still, when presenting the data, we assumed the following activation scheme for the nicotinic receptor (Magleby and Stevens, 1972
(Scheme1) According to Scheme 1, the closed, unliganded receptor (C) binds two agonist molecules, after which the channel opens (A2O). In Scheme 1, A is an agonist, k+ and k- correspond to the agonist association and dissociation rate constants,
10 µM) of galantamine resulted in the emergence of a new nonconducting state (A2B); the prevalence of this state, but not the duration, depended on galantamine concentration. Rate constants k+b and k-b describe the transitions between the open state and the blocked state.
In the presence of micromolar concentrations of galantamine, the rate of entry into the short-lived desensitized state (k+d) is usually much less than k+b or
The initial rate of binding for 125I-
Results
Direct activation of muscle-type nicotinic receptors by galantamine
The mouse adult wild-type nicotinic receptor is activated by galantamine (for structures of activators used, see Fig. 1). Sample currents elicited by 0.1 and 10 µM galantamine are shown in Figure 2A. Single-channel clusters were not observed, suggesting that galantamine is a low-efficacy agonist, that the Kd of the receptor to galantamine is much higher than 10 µM, or both. It has been noted previously that the nicotinic channel closing rate constant is relatively insensitive to the structure of the ligand used to activate the receptor, with the rates for most ligands lying between 1000 and 2000 s-1 (Akk and Auerbach, 199970 pS in these ionic conditions; data not shown), as has been reported previously for APLs (Pereira et al., 1993a
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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.
A number of mutations in the muscle-type nicotinic receptor have been shown to alter channel gating by nicotinic agonists. We asked whether mutations have similar effects on activation by galantamine. First, we examined the activation of theSample currents elicited by galantamine from receptors containing
The closed-time component for which duration depends on agonist concentration was identified, and the inverse of the mean duration was defined as the effective opening rate (
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Figure 3. Activation of the
The nicotinic receptor gating properties are known to depend on membrane potential, and the channel closing rate constant is slowed e-fold by0 = 6.08 ± 0.25 ms; H = 78 ± 5 mV (f = 0.32 ± 0.02; determined from one patch with 1476, 1217, 1699, and 817 events at VM =-100, -75, -50, and -25 mV, respectively). The degree of voltage sensitivity is similar to that obtained previously for nicotinic ligands (Akk and Steinbach, 2000
Next, we examined the effect of the
Finally, some cells were transfected with the
The results presented above demonstrate that galantamine is a low-efficacy agonist of the muscle-type nicotinic receptor. Channel gating in the presence of galantamine is not qualitatively different from that in the presence of nicotinic ligands. In particular, the open channel conductance and apparent closing rates are not greatly different, and the Hill coefficient for activation by galantamine is >1. Mutations known to affect gating by ACh have similar effects on gating elicited by galantamine, suggesting that the domains involved in gating by the two types of agonists are common.
Blocking actions of galantamine
At higher concentrations of galantamine, channel open durations were reduced, and a large number of brief dwells in a nonconducting state were seen (Fig. 4). These observations are consistent with an open channel block at higher concentrations of galantamine, as has been reported for a number of other positively charged nicotinic agonists (Adams, 1976The rate of entry into the blocked state in the presence of 100 µM galantamine (VM -50 mV) was 2035, 2837, or 2383 s-1 in the presence of ACh, carbachol, and galantamine, respectively. In the presence of 10 µM galantamine, the apparent blocking rate was 282 s-1 (ACh). Assuming a first-order reaction, the association rates are 2.0-2.8 x 107 M-1 s-1. The duration of the blocked state was also similar in the different conditions: 0.31, 0.35, and 0.39 ms in the presence of 100 µM galantamine and 0.35 ms in the presence of 10 µM galantamine. Galantamine did not affect the estimated apparent channel closing rates for activity elicited by ACh or carbachol (100 µM galantamine:
Channel block by ions often shows voltage sensitivity; in the case of block by cationic drugs, the block is enhanced at more negative membrane potentials (Woodhull, 1973
Galantamine-mediated modulation of nicotinic receptor activation
In the next set of experiments, we examined the modulating effect of galantamine on nicotinic receptor activation by three nicotinic agonists: ACh, carbachol, and choline. The approach relies on rapid competitive binding of two ligands at the same site, with one ligand having a much lower channel opening rate than the other. In this situation, the channel opening frequency is reduced below the value observed for the more efficacious agonist in the absence of the competing ligand, because the receptor spends a fraction of time liganded with one or two molecules of the poor agonist. For example, in studies of the weak traditional agonist TEA, the effective opening rate for activation by ACh or carbachol is reduced in the presence of TEA (Akk and Steinbach, 2003The first step was to study the effect of galantamine on single-channel clusters elicited by carbachol from wild-type receptors. As a measure of receptor affinity and channel opening, we examined the modulation of the channel effective opening rate with 1 mM carbachol by increasing concentrations of galantamine. We estimated the effective channel opening rate to determine whether galantamine produced changes in the apparent affinity for carbachol or the channel opening rate. Figure 5 shows the effective opening rate with 1 mM carbachol in the presence of 0.2-100 µM galantamine. This concentration of carbachol results in a half-maximal value for
We then determined the dependence of
We confirmed these results obtained with carbachol by examining the effects of galantamine on the effective opening rate produced by a low concentration of ACh. The channel opening rate for ACh is so large that the maximal value cannot be reliably estimated using our methods, while any potentiation might be most apparent at a low concentration of agonist. Receptor activation by a low concentration of ACh (5 µM) was studied in the absence and presence of 100 µM galantamine. The effective opening rate was 82 ± 35 s-1 (three patches; 4028 events) and 92 ± 18 s-1 (three patches; 18,422 events) for 5 µM ACh and 5 µM ACh plus 100 µM galantamine, respectively. Hence, there was no indication that galantamine affected receptor affinity to ACh.
Finally, we examined the effect of galantamine on receptor activation by choline. These experiments were performed on the
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Figure 6. Effect of galantamine on
It should be pointed out that there are some limitations to this approach. The approach requires that the ligands compete on a time scale appropriate to the duration of the relevant component of the closed-time distribution. Therefore, an inhibitor with a long dwell time, such as (+)-tubocurarine, would not show such interaction. However, this approach allows us to examine interactions between ligands with relatively low affinities.In sum, these experiments demonstrate that the presence of galantamine does not affect receptor activation by the nicotinic ligands ACh, carbachol, or choline. Exposure to galantamine did not result in receptor potentiation, as had been observed previously for neuronal-type nicotinic receptors. Furthermore, to our surprise, there was no indication of galantamine-induced competitive inhibition of activation by a nicotinic ligand. These findings suggest that the interaction sites for galantamine and nicotinic ligands do not overlap.
Galantamine effects on the binding of 125I-
A second approach to determine whether a drug binds to the ACh-binding site is to determine whether it reduces the initial rate of I-
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Figure 7. Galantamine does not reduce the initial rate of binding for I-
Discussion
The goal of the experiments described in this paper was to characterize the actions of galantamine on the muscle-type nicotinic receptor. Previous studies had indicated three possible effects: galantamine might directly activate the receptor, it might potentiate the ability of nicotinic agonists to activate the receptor, and it might inhibit receptor activation. Our results demonstrate that galantamine activates and inhibits the muscle-type nicotinic receptor yet has no potentiating effect on currents elicited by nicotinic agonists. More surprisingly, the data indicate that galantamine does not interact with the ACh-binding site on this receptor.Receptor activation by galantamine
There are several similarities between the receptor activation produced by galantamine and traditional nicotinic agonists. This study and previous ones (Pereira et al., 1993aThe effects of mutations on activation by galantamine were strikingly similar to the effects observed for receptors activated by nicotinic ligands. The
Overall, these observations indicate strong similarities between receptor activation by nicotinic ligands and galantamine. The conductance and the channel closing rate (and its voltage dependence) are similar. Channels open more readily when two agonist molecules are bound. Finally, mutations in the transmembrane domains that affect channel opening and closing rates have similar effects on activity elicited by nicotinic agonists and by galantamine.
Receptor inhibition by galantamine
Galantamine produces a brief, voltage-dependent channel block. Block of single-channel currents by AChE inhibitors has been observed previously (Shaw et al., 1985We did not see any evidence for inhibition mediated by competition for binding to the ACh-binding site or for allosteric effects on agonist affinity or channel opening rate.
Interaction between galantamine and the traditional nicotinic agonist binding site
We sought evidence for an interaction between galantamine and the nicotinic agonist binding site by examining the channel effective opening rate for the nicotinic agonist at various galantamine concentrations. In the present studies, we used three traditional nicotinic agonists. For two of them, we were able to obtain data for the full effective opening rate curve as a function of traditional agonist concentration, to show that there was no shift in the EC50 orOverall, these data demonstrate that galantamine has no interaction with receptor activation by classical nicotinic agonists, at least at concentrations up to 100 µM. Two interpretations follow from this observation. First, there is no indication that galantamine potentiates (or inhibits) the actions of nicotinic ligands by effects on affinity or opening rate. Second, there is no indication that galantamine binds to the ACh-binding site, at least in any manner that interferes with the binding of ACh, carbachol, or choline.
Because of the novelty of the second interpretation (that an activator of the muscle nicotinic receptor might act by binding to a site distinct from the ACh-binding site), we then examined the ability of galantamine to inhibit the initial rate of I-
Previous studies of muscle receptors have provided conflicting observations. The activation of fetal-type muscle nicotinic receptors by galantamine or physostigmine could be blocked by
Studies of neuronal nicotinic receptors have generally been interpreted to indicate that APLs do not bind to the ACh-binding site on those receptors. Activation of single-channel currents from several types of neuronal nicotinic receptors by a number of APLs has been reported to be insensitive to inhibition by several drugs that block the ACh-binding site (Pereira et al., 1993a
The most complete study (Zwart et al., 2000
Our results indicate that there are two (or more) classes of binding sites on each receptor, one for nicotinic agonists and another for galantamine. The occupation of either set of binding sites can lead to the opening of the channel gate. However, the data also show that channel activity elicited by even relatively low concentrations of a nicotinic agonist is not affected by the presence of a saturating concentration of galantamine (Fig. 6). This finding is rather surprising, because it might be expected, in the case of independent binding sites, that the presence of 1 µM galantamine (with an effective opening rate of 50 s-1) would have affected the overall effective channel opening rate elicited in the presence of 500 µM choline (with an effective opening rate of 140 s-1). Two possible explanations for the lack of apparent effect immediately come to mind. First, choline binding to the nicotinic binding site may prevent channel activation by galantamine. Such a mechanism would require the postulation of negative allosterism between an ACh binding site and a galantamine site. Alternatively, choline may competitively inhibit the binding of galantamine to the receptor yet be unable to elicit channel opening through the occupation of the galantamine site. Full resolution of the interactions between galantamine and nicotinic agonists will require more experiments.
Galantamine binding site(s)
Photoaffinity labeling with another AChE inhibitor, phenyl-3H-physostigmine, labels Lys125 in the Torpedo nicotinic receptor
Footnotes
Received Dec 7, 2004; revised January 12, 2005; accepted January 13, 2005.This work was supported by National Science Foundation Grant 0110282 (G.A.) and National Institutes of Health Grant NS-22356 (J.H.S.). J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology. We thank Bei-Wen Ma for help with tissue culture.
Correspondence should be addressed to Gustav Akk, Department of Anesthesiology, Campus Box 8054, Washington University, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: akk{at}morpheus.wustl.edu.
Copyright © 2005 Society for Neuroscience 0270-6474/05/251992-10$15.00/0
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