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Activation and Block of the Adult Muscle-Type Nicotinic Receptor by Physostigmine: Single-Channel Studies

Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri

Received for publication March 10, 2008.

Accepted for publication June 2, 2008.

	Abstract

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The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (Lys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 µM^-1 s^-1 and 450 s^-1 (K_D = 23 µM). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.

A direct activating effect of physostigmine has been observed in Locusta migratoria neurons (van den Beukel et al., 1998). In contrast, no whole-cell currents in response to up to 1 mM physostigmine were detected in oocytes expressing rat neuronal 4β2 or 4β4 receptors (Zwart et al., 2000), or COS cells expressing mouse muscle embryonic (β) receptors (Svobodova et al., 2006). Single-channel patch clamp has shown that channel openings in the presence of physostigmine have a conductance identical to that for ACh, but, in contrast to channel activity elicited by high concentrations of ACh, physostigmine-induced openings are typically not condensed into single-channel clusters (Shaw et al., 1985; Wachtel, 1993; Cooper et al., 1996). Together, the whole-cell and single-channel findings are suggestive of low potency and/or low efficacy of physostigmine on the nAChR. Physostigmine-mediated potentiation of currents elicited by low doses of ACh has been shown for neuronal (Zwart et al., 2000) as well as muscle-type nicotinic receptors (Svobodova et al., 2006).

Exposure to high concentrations of physostigmine leads to channel block. In macroscopic recordings, coapplication of physostigmine with ACh results in accelerated apparent desensitization and a reduction of peak response (Storch et al., 1995; Zwart et al., 2000; Svobodova et al., 2006). In single-channel recordings, high concentrations of physostigmine produce a reduction in the mean open duration and an emergence of a novel 7- to 8-ms closed time component (Shaw et al., 1985; Wachtel, 1993). In BC3H-1 cells that express the embryonic type nAChR, the properties of block were consistent with an open channel blocking scheme with a single blocking site per receptor (Wachtel, 1993).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Structure of physostigmine. CAS number 57-47-6.

In the present work, we have examined the adult mouse muscle-type nAChR activation by physostigmine. We find that physostigmine is a low-efficacy agonist of the receptor. Channel activation by carbachol, a nicotinic agonist, was unaffected in the presence of physostigmine, consistent with previous data indicating different binding sites for nicotinic agonists and APLs. Mutations (S269I and T264P) shown previously to modify channel gating properties for nicotinic agonists act similarly on channel activation by physostigmine, whereas the single-channel conductance is indistinguishable for carbachol and physostigmine, suggesting that the channel gating mechanisms are generally similar for APLs and nicotinic ligands. But in contrast to channel activation by nicotinic agonists, openings elicited by physostigmine show little voltage dependence. Finally, mutations to the Lys125 residue, previously associated with the physostigmine binding site, resulted in briefer channel openings from receptors activated by physostigmine but not carbachol.

	Materials and Methods

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The mouse muscle adult-type receptor subunits (β) were subcloned into the cytomegalovirus promoter based-expression vector pcDNA3 (Invitrogen, Carlsbad, CA) and expressed in human embryonic kidney 293 cells using a standard calcium phosphate precipitation-based transient transfection technique (Akk, 2002). In brief, a total of 3.5 µg of cDNA per 35-mm culture dish in the ratio of 2:1:1:1 (:β::) was mixed with 12.5 µl of 2.5 M CaCl₂ and distilled water to a final volume of 125 µl. The mixture was then added slowly, without mixing to an equal volume of 2x N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered solution. The combined solution was incubated at room temperature for 10 min followed by mixing the contents and another incubation of 15 min. The precipitate was then added to the cells. The cells were incubated at 37°C with 5% CO₂ for 16 to 20 h at which time the medium was replaced. The electrophysiological experiments were performed 40 to 72 h after the start of transfection.

Single-channel activity was recorded in the cell-attached configuration. The bath solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The pipette solution contained 142 mM KCl, 1.8 mM CaCl₂, 1.7 mM MgCl₂, 5.4 mM NaCl, and 10 mM HEPES, pH 7.4. The agonist (carbachol and/or physostigmine; Fig. 1) was added to the pipette solution. In most experiments, the patch potential was held at -50 mV using a combination of cell membrane potential and applied pipette potential. The cell membrane potential was determined from the reversal potential of nicotinic receptor currents, several times during the course of a recording from a patch. Most human embryonic kidney cells had a membrane potential of -30 to -20 mV in the bath solution used. All experiments were carried out at room temperature (18-21°C).

Single-channel currents were amplified with an EPC7 (HEKA Instruments Inc., Port Washington, NY) or an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 10 kHz, and digitized at 50 kHz using a Digidata 1322 Series interface (Molecular Devices). Channel event detection was carried out using program SKM (QuB suite; Qin et al., 1996, 1997). In general, the currents were filtered at 5 kHz before idealization, and a minimal duration of 40 or 50 µs was imposed.

For currents recorded in the presence of carbachol, the analysis was restricted to clusters of single-channel currents (i.e., episodes of high open probability activity separated from other such episodes by prolonged silent intervals) (Akk and Steinbach, 2003). In terms of kinetic mechanism, a cluster is defined as series of openings separated from each other by dwells in the un-, mono-, and diliganded closed states. A cluster is initiated when a receptor returns from the long-lived desensitized state and is terminated by receptor entry into the long-lived desensitized state. The advantage of using single-channel cluster analysis is the near certainty that adjacent openings arise from the same receptor channel. This allows to examine the durations of intracluster closed times and how they react to changes in agonist or modulator concentrations, and to correlate these parameters with channel activation properties.

The clusters were identified by eye and isolated for further analysis. Clusters containing overlapping currents, which are an indication of two or more simultaneously active receptors, were discarded. In the absence of clear clusters (e.g., for receptors activated by physostigmine), stable sections of the record were selected so as to include enough channel events for dwell time analysis, but also to avoid overlapping currents. Physostigmine-elicited activity from the T264P receptors contained, in addition to single isolated openings, bursts or groups of activity. We focused our analysis on bursts of activity, but we note that the kinetic origin of bursts is unknown to us.

Open and closed interval durations were estimated using program MIL (QuB suite). The records were initially analyzed by fitting a simple CO model. The number of open states (or closed states, if estimating closed time durations) was then increased as long as the increase in the log-likelihood justified the addition of the extra free parameters (Horn, 1987). In general, an increase of >25 units was considered significant. The newly added states were assumed to be unconnected to each other. In some cases, the presence of an additional state led to a significant increase in the log-likelihood, but the new state had a relative frequency of <0.1%. In this case, data for that state were omitted. In some cases, parameters were estimated by simultaneous fitting of pooled data from several patches. Mean single channel current amplitudes were determined from the mean reported by the IDL module in QuB. Because some records were dominated by very brief events (e.g., 100 µM physostigmine), all amplitudes were estimated for events lasting longer than 200 µs, to allow full settling. Data are presented as mean ± S.D., except when error estimates are calculated for fit parameters, in which case the data are best fitting value ± estimated S.E. of the value.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Physostigmine directly activates the mouse adult wild-type nAChR. Single-channel currents and the respective open time histograms from patches exposed to 1 µM (A), 10 µM (B), or 100 µM physostigmine (C). The activity consisted of single, isolated openings without the characteristic clustering behavior observed with ACh or many other nicotinic agonists. The open time histograms were fitted to sums of two exponentials (1 and 10 µM physostigmine) or a single exponential (100 µM physostigmine). In the presence of 1 µM physostigmine, the open times for the data in this histogram were 0.22 ms (65%) and 0.74 ms. In the presence of 10 µM physostigmine, the open times were 0.12 ms (51%) and 0.49 ms. In the presence of 100 µM physostigmine, the mean open duration was 0.13 ms. Due to lack of clusters and the uncertainty in the number of active receptors in the patch, the closed times were not analyzed. The data are consistent with physostigmine being a low-potency or a low-efficacy agonist on the wild-type receptor.

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Physostigmine was stored in 1 mM aliquots at -20°C and diluted immediately before use.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Direct Activation of Wild-Type and Mutant Receptors by Physostigmine. Physostigmine directly activates the mouse adult-type muscle nicotinic receptor. We recorded single-channel currents in the presence of 1 to 100 µM physostigmine and found that physostigmine elicits single, isolated openings that are not condensed into high open probability episodes (clusters) of activity. We saw no indication of agonist-dependent changes in the durations of long closed times, suggesting that physostigmine is a low-affinity or low-efficacy (or both) agonist of the muscle-type receptor. Sample currents are shown in Fig. 2.

At 1 and 10 µM physostigmine, the open time histograms were best fitted by the sum of two exponentials. The brief openings (OT1) had mean durations of 0.30 and 0.22 ms at 1 µM physostigmine, and they made up 78 and 65% of all openings (data for two patches). The long-duration openings (OT2) had durations of 0.72 and 0.74 ms. At 10 µM, the parameters were 0.35 and 0.12 ms (74 and 51%) and 0.67 and 0.49 ms. At 100 µM physostigmine, only a single component was see in the open times, with a duration of 0.17 ± 0.04 ms (mean for three patches).

In the absence of single-channel clusters, the durations of the closed times between adjacent openings depend, in addition to receptor activation properties, on the number of active receptors in the patch. The latter parameter is typically unknown; thus, the durations of the closed times cannot be easily ascribed to a specific activation-related process. Accordingly, the closed intervals for physostigmine-activated wild-type receptors were not analyzed. The lack of clusters suggests that physostigmine is a low-affinity and/or low-efficacy agonist for wild-type muscle nicotinic receptors.

At -50 mV, the amplitude of single-channel events was similar at 1 and 10 µM (-3.6 ± 0.08 pA; four patches) and 100 µM physostigmine (-3.4 ± 0.2 pA; three patches). To obtain amplitudes of settled openings, only open durations longer than 200 µs were included in the analysis. Currents elicited by 1 mM carbachol exhibited a similar amplitude (-3.2 ± 0.4 pA; seven patches), suggesting that the ion conduction pathway is unchanged when physostigmine, instead of the nicotinic agonist carbachol, is used to activate the receptor.

Previous work on nicotinic channels has shown that specific mutations to the M2 transmembrane domain (e.g., T264P) or the linker region between the M2 and M3 domains (e.g., S269I) enhance open probability (P_o) of channels exposed to nicotinic agonists. The increase in the P_o is mediated by an increase in the channel opening rate constant and/or a decrease in the channel closing rate constant (Chen and Auerbach, 1998; Zhou et al., 1999). These two mutations were useful in further studying physostigmine-elicited activation of the nicotinic receptor for two reasons. First, we could test whether the mutations similarly affect channel activation by physostigmine. Similarities in the effects of the mutations on channel activation by ACh and physostigmine would be an indication that channel gating proceeds similarly in the presence of these two classes of agonists. Second, prolonged open events and, consequently, higher open probability may allow isolation of channel activity originating from a single ion channel (i.e., clusters of activity). This would allow us to attempt to associate the channel closed time components with specific components of receptor activation and give insight into physostigmine binding and gating properties.

The activation of the S269I mutant receptor was studied in the presence of 1 to 100 µM physostigmine (Fig. 3). Two classes of open events were observed with the mutant receptor. In the presence of 1 µM physostigmine, OT1 had a mean duration of 0.17 ± 0.03 ms (four patches) forming 75 ± 11% of all open events. The mean duration of OT2 at 1 µM physostigmine was 2.3 ± 0.6 ms. Similar to what we saw with the wild-type channel, the lifetime of OT2 was reduced at higher physostigmine concentrations. In the presence of 10 µM physostigmine, the mean duration of OT2 was 0.9 ± 0.3 ms (four patches), and when activated by 100 µM physostigmine the channels opened to a single class of open events with the mean duration of 0.14 ± 0.03 ms (five patches). Compared at a low physostigmine concentration (1 µM), the S269I mutation prolonged the mean OT2 duration by 3-fold. This is similar to the effect observed for channel openings elicited by the nicotinic agonist choline (Zhou et al., 1999).

View larger version (45K): [in this window] [in a new window]   Fig. 3. Activation of the S269I mutant receptor by physostigmine. Single-channel currents and the respective open time histograms from patches exposed to 1 µM (A), 10 µM (B), 100 µM physostigmine (C), or 10 µM carbachol (D). The open times were prolonged compared with the wild-type receptor, but no clustering behavior was observed. The open time histograms were fitted to sums of two exponentials (1 and 10 µM physostigmine and 10 µM carbachol) or a single exponential (100 µM physostigmine). The open times for these patches were as follows: 1 µM physostigmine, 0.15 ms (60%) and 1.43 ms; 10 µM physostigmine, 0.19 ms (75%) and 0.96 ms; 100 µM physostigmine, 0.11 ms (100%); and 10 µM carbachol, 0.23 ms (23%) and 2.34 ms. Carbachol produced activity grouped in clusters. Due to lack of clusters of activity elicited by physostigmine and the uncertainty in the number of active receptors in the patch, the closed times were not analyzed. The mutation enhances the channel opening rate constant for nicotinic agonists and the APL galantamine. The lack of clusters in the presence of physostigmine suggests that physostigmine is a low-efficacy agonist of the adult-type muscle nAChR.

As a control, we examined the activation of the S269I mutant receptor by 10 µM carbachol. Sample single-channel activity is shown in Fig. 3D. The channel open time histograms were fitted to the sum of two exponentials. The mean open times were 0.33 ± 0.14 ms (23 ± 5%) and 2.4 ± 0.2 ms (three patches). It is likely that the shorter lived component originates from monoliganded receptors, whereas the longer-lived component can be associated with diliganded receptors. The mean duration of diliganded open events was 3-fold greater than in the wild-type receptor (Fig. 7A) (see also Akk et al., 2005). Thus, the mutation similarly affects channel open durations in the presence of physostigmine and the nicotinic agonist carbachol.

View larger version (20K): [in this window] [in a new window]   Fig. 7. The presence of physostigmine does not interfere with channel activation by carbachol. Single-channel clusters and the respective open and closed time histograms from wild-type receptors exposed to 1 mM carbachol (A), carbachol + 1 µM physostigmine (B), or carbachol + 100 µM physostigmine (C). The activity consisted of easily identified clusters. In the presence of 1 mM carbachol, the mean intracluster open duration was 0.54 ms, and the closed times were 0.25 ms (96%) and 3.4 ms. In the presence of 1 mM carbachol + 1 µM physostigmine, the mean intracluster open duration was 0.39 ms, and the closed times were 0.24 ms (98%) and 9.6 ms. In the presence of 1 mM carbachol + 100 µM physostigmine, the mean intracluster open duration was 0.18 ms, and the mean closed times were 0.27 ms (40%) and 9.6 ms. The presence of 100 µM physostigmine led to a decrease in channel open probability through a decrease in the mean open duration and an increase in the prevalence and duration of the longer lived closed time component. The presence of physostigmine was ineffective at modifying the duration of the shorter-lived closed time component (inverse of the effective opening rate), which is a measure of agonist binding and channel opening. We conclude that physostigmine does not interact with the sites that mediate channel activation by carbachol.

Single-channel activity from the T264P mutant receptor activated by physostigmine exhibited complex behavior. In the majority of patches, physostigmine elicited 100- to 200-ms-long groups of openings (bursts) interspersed with single isolated openings. However, we note that some patches contained only isolated openings. We do not have an explanation for this observation. Sample currents are shown in Fig. 4.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Activation of the T264P mutant receptor by physostigmine. Single-channel currents and the respective open and closed time histograms from patches exposed to 1 µM (A), 10 µM (B), or 100 µM physostigmine (C). The channel activity consisted of episodes or bursts of activity (shown with lines above current traces) intermixed with brief, isolated openings. The bursts were isolated from the recording and analyzed for open and closed time durations. The open times were prolonged compared with the wild-type receptor. In the presence of 1 µM physostigmine, the open times were 0.14 ms (15%) and 13.1 ms, and the closed times were 0.10 ms (25%), 2.1 ms (58%), and 13.6 ms. In the presence of 10 µM physostigmine, the open times were 0.33 ms (12%) and 2.0 ms, and the closed times were 0.30 ms (6%) and 3.2 ms. In the presence of 100 µM physostigmine, the mean open duration was 0.21 ms, and the closed times were 0.05 ms (20%) and 4.9 ms. Due to the potent blocking action of physostigmine, we were unable to identify the activation-related closed time components. It is likely that the more prominent 3- to 5-ms closed time component arises from dwells in the blocked state.

In sum, when measured at low (1-10 µM) physostigmine concentrations, the presence of the T264P mutation increased the duration of the long openings by approximately 10-fold. This indicates that the mutation affects channel gating by cholinergic agonists and this APL in the same way. The reduction in open durations seen at higher physostigmine concentrations is indicative of the channel blocking properties of physostigmine (see below).

Physostigmine-Elicited Openings Are Not Modulated by Voltage. The experiments described above indicate that despite its low efficacy, channel activation by physostigmine proceeds in principle similarly to that by nicotinic agonists. The single-channel conductance is comparable for openings elicited by physostigmine and carbachol, and mutations previously shown to affect channel gating by nicotinic agonists also modify channel gating by physostigmine. As an additional measure of conformity in the mechanisms of gating we examined the voltage dependence of channel open durations. Previous work has demonstrated that, for a number of nicotinic agonists, the nAChR closing rate constant is reduced at more negative potentials, with an e-fold change per 80 to 100 mV hyperpolarization (Auerbach et al., 1996; Akk and Steinbach, 2000).

We investigated the effect of voltage on the durations of openings elicited from receptors containing the T264P mutation. The mutant, instead of the wild-type, receptor was used due to its longer open-time durations increasing the fidelity of dwell time estimates. The experiments were conducted in the presence of a low concentration (1 µM) of physostigmine to avoid contribution from channel block to the apparent open time duration. We were particularly interested in the voltage sensitivity of the longer lived component, which presumably originates from fully liganded receptors and should thus exhibit most voltage sensitivity (Auerbach et al., 1996). Figure 5 shows the mean duration for the longer duration component estimated at -100-, -75-, -50-, -25-, and +50-mV membrane potentials. Overall, the data displayed minimal sensitivity to changes in membrane potential. At -100 mV, the mean lifetime of the longer lived open time component was 7.6 ± 4.5 ms (three patches), whereas at +50 mV, the mean duration was 7.3 ± 3.0 ms (three patches). This gives a voltage sensitivity of >3000 mV per e-fold change in open interval duration.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Voltage does not affect channel open durations in the presence of physostigmine. The long open time component from the T264P mutant receptor activated by 1 µM physostigmine (circles) was estimated at -100-, -75-, -50-, -25-, and +50-mV membrane potentials. No voltage sensitivity was observed. For control, we estimated the open interval durations from the T264P mutant receptor activated by 100 µM carbachol (squares) at -50 and +50 mV membrane potentials. Carbachol-elicited openings showed a voltage sensitivity of 93 mV per e-fold change. This value is similar to previous estimates for the wild-type receptor and a number of mutant receptors activated by nicotinic agonists (Auerbach et al., 1996; Akk and Steinbach, 2000). Points show mean ± S.D. for data from three to six patches.

As a control, we estimated the voltage sensitivity of durations of openings elicited by carbachol. At -50 mV, the T264P receptors exposed to 100 µM carbachol showed two open time components. The brief openings had a mean duration of 70 ± 40 µs, and the long openings had a mean duration of 25 ± 10 ms (three patches). When the membrane potential was changed to +50 mV, the mean duration of brief openings remained unchanged (70 ± 30 µs), but the mean duration of long-lived openings decreased by almost 3-fold (8.8 ± 1.4 ms). Based on previous work (Auerbach et al., 1996), we believe that the voltage-insensitive short-lived openings arise from unliganded or monoliganded receptors. The change in the mean duration of long openings (96 mV/e-fold change) is consistent with previously published data on voltage sensitivity of doubly liganded channels (e.g., Auerbach et al., 1996; Akk and Steinbach, 2000).

Effects of Mutations to the Lys125 Residue. Photoaffinity labeling studies with [phenyl-(n)-³H](-)physostigmine have shown that the compound associates with the Lys125 residue in the T. californica receptor subunit (Schrattenholz et al., 1993). The Lys125 residue is at a considerable distance from the acetylcholine binding pocket (Brejc et al., 2001), suggesting that the physostigmine binding site does not overlap with the ACh binding site. We examined the effects of mutations to the Lys125 residue on adult mouse nAChR activation by physostigmine. The positively charged lysine residue was mutated to a neutral glutamine (Q) and a negatively charged glutamate (E). For enhanced opening frequency as well as longer open time durations, the experiments were conducted on the background of the T264P mutation.

Figure 6A shows sample currents elicited by 10 µM physostigmine from receptors containing the T264P, K125Q + T264P, or K125E + T264P mutant subunits. In five cells expressing the wild-type subunit (along with the T264P mutant subunit), the open durations were 0.13 ± 0.02 ms (66 ± 19%) and 2.7 ± 1.4 ms, and the weighted average open duration was 1.0 ms. When the wild-type subunit was replaced with one containing the K125Q mutation, the mean duration of OT1 was 0.08 ± 0.02 ms (80 ± 19%) and the mean duration of OT2 was 0.9 ± 0.5 ms (five patches) resulting in a weighted mean open time of 0.25 ms. Finally, having a glutamate residue in the 125 position resulted in mean open durations of 0.13 ± 0.01 ms (79 ± 13%) and 1.4 ± 0.4 ms (three patches), with a weighted mean open time of 0.39 ms. Thus, a replacement of the positively charged lysine residue with a neutral glutamine or a negatively charged glutamate in the putative physostigmine binding site leads to shorter open durations, but only the effect of the K125Q mutation on the OT2 duration was statistically significant (two-tailed t test, p < 0.05).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Mutations to the Lys125 site reduce channel open durations in the presence of physostigmine but not carbachol. Single-channel currents and the respective open time histograms for T264P, K125Q + T264P, and K125E + T264P mutant receptors activated by 10 µM physostigmine (A), or 100 µM carbachol (B). In the presence of 10 µM physostigmine, the activity consisted of bursts of activity intermixed with brief, isolated openings. All the openings within long sections of the recording were analyzed for open time durations. The open times for the T264P receptor activated by physostigmine were 0.10 ms (80%) and 1.4 ms, weighted average mean open duration of 0.28 ms. The open times for the K125Q + T264P receptor activated by physostigmine were 0.07 ms (87%) and 1.2 ms, weighted average mean open duration of 0.22 ms. The open times for the T264P + K125E receptor activated by physostigmine were 0.13 ms (64%) and 1.7 ms, weighted average mean open duration of 0.54 ms. In the presence of 100 µM carbachol, the activity consisted of bursts of activity. The open times for the T264P receptor activated by carbachol were 0.08 ms (25%) and 28.6 ms. The open times for the K125Q + T264P receptor activated by carbachol were 0.07 ms (30%) and 48 ms. The open times for the T264P + K125E receptor activated by carbachol were 0.08 ms (45%) and 60 ms.

Physostigmine Does Not Interact with the Nicotinic Agonist Binding Site. We also examined the possibility that physostigmine interacts with the nicotinic agonist binding site. To do that, we investigated receptor activation by a subsaturating concentration of carbachol in the absence and presence of physostigmine. We reasoned that if physostigmine binds to the carbachol site, then, because of its low efficacy, it would act as a competitive inhibitor of carbachol-elicited activity. This would manifest as a reduced effective opening rate because the receptor spends a fraction of time liganded with the low-efficacy agonist physostigmine (Akk and Steinbach, 2003).

Single-channel currents were elicited by 1 mM carbachol from wild-type receptors in the absence and presence of 1 to 100 µM physostigmine. For currents elicited by 1 mM carbachol alone, we observed the characteristic clustering behavior as described previously (Akk and Auerbach, 1999). The intracluster open time histograms were fitted to a single exponential with a mean open duration of 0.72 ± 0.22 ms (seven patches). The intracluster closed time histograms contained two components. The brief closed time component (CT1) had a mean duration of 0.33 ± 0.07 ms and the long-lived closed time component (CT2) had a mean duration of 4.4 ± 1.5 ms. It is likely that CT1 corresponds to sojourns in the unliganded and monoliganded closed states (i.e., reflects agonist rebinding and channel opening). The less-frequent CT2 component (7 ± 5% of all intracluster closed events) corresponds to a short-lived desensitized state (Salamone et al., 1999). Sample single-channel currents are shown in Fig. 7.

To test for physostigmine interactions with the carbachol binding site we examined the effect of physostigmine on the intracluster closed time distributions. We were particularly interested in changes in the duration of the CT1 component, because an increase in the duration of CT1 would be indicative of physostigmine-induced inhibition of channel activation. The intracluster closed time durations were estimated for 1 mM carbachol coapplied with 1, 10, or 100 µM physostigmine. Under all conditions the closed time histograms were best fitted to the sum of two exponentials with one component similar in duration to CT1 and the other to CT2 for currents recorded in the presence of 1 mM carbachol alone. When 1 µM physostigmine was coapplied with carbachol, the closed times were 0.29 ± 0.01 and 3.9 ± 1.5 ms (three patches). The relative contributions of the two components were similar to that in the absence of physostigmine—the CT2 component contributed 5 ± 4% of intracluster closed events. Coapplication of 10 or 100 µM physostigmine with carbachol similarly failed to alter the duration of CT1. The mean duration of CT1 was 0.41 ± 0.16 ms (three patches) or 0.35 ± 0.07 ms (six patches) in the presence of 10 or 100 µM physostigmine, respectively. We conclude that physostigmine, at up to 100 µM, is ineffective at competing with carbachol for the nicotinic agonist binding site.

In contrast, physostigmine had significant effects on the longer duration closed time component, CT2. The mean duration of CT2 was 6.5 ± 0.7 ms at 10 µM physostigmine and 10.6 ± 2.2 ms at 100 µM physostigmine. The increase in the concentration of physostigmine had a major effect on the prevalence of the CT2 component. The long-lived closed time component had a relative frequency of 22 ± 4 or 67 ± 6% in the presence of 10 or 100 µM physostigmine, respectively. These changes are likely associated with channel block that becomes the predominant contributor to the CT2 component at high physostigmine concentrations (see next section).

Channel Block in the Presence of Physostigmine. The data presented above suggest that physostigmine can block the nicotinic channel. The open durations were reduced and, when such analysis was possible, the mean intracluster closed times were progressively longer when the receptors were exposed to high (100 µM) concentrations of physostigmine. In this section, we present a systematic characterization of physostigmine-induced channel block under a variety of conditions.

In the first set of experiments, we examined physostigmine-induced block of physostigmine-activated channels. Although comparison of the durations of wild-type channel openings recorded at low versus high concentrations of physostigmine demonstrated the blocking action of physostigmine, we chose to conduct a more thorough examination on the T264P mutant receptor, for two reasons. First, physostigmine-elicited channel openings from the T264P mutant channel are longer than those from the wild-type receptor, allowing for less error and a greater dynamic range in studying the blocking actions of physostigmine. Second, we reasoned, the higher opening frequency and the presence of bursts of activity would allow us to isolate and study the closed time component that corresponds to sojourns in the blocked state.

An increase in the concentration of physostigmine resulted in shorter open time durations. We estimated the apparent rate constant for development of block (k_+B^*) from the slope of the relationship between the inverse of the mean of the long duration open time component and physostigmine concentration according to 1/(mean duration) = (channel closing rate constant) + k_+B^* x [physostigmine] (Fig. 8A). This approach gave k_+B^* = 44 ± 2 µM^-1 s^-1 for block of the T264P receptor. An independent estimate for k_+B^* was obtained by examining the intraburst closed time distributions. The intraburst closed time histograms, which were fitted to the sums of three (at <10 µM physostigmine) or two exponentials, contained a closed interval component (nominally called CT2) that demonstrated a strong physostigmine concentration dependence. The rate of entry into this closed state (k_+CT2) was highly dependent on the concentration of physostigmine, suggesting that this closed duration component corresponds to dwells in the blocked state. Accordingly, we postulated that the rate of entry into this closed state can be used to estimate k_+B^*. The relationship between k_+CT2 and physostigmine concentration is shown in Fig. 8A, and a linear regression analysis of the data gave a k_+B^* = 39 ± 2 µM^-1 s^-1. This value is essentially identical to the estimate from the open time analysis and confirms our identification of the blocked state in the closed time histograms.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Properties of channel block by physostigmine. The increase in the concentration of physostigmine was found to result in reduced open duration and an increase in the rate of entry into an 3- to 7-ms closed state. Both observations are consistent with physostigmine-induced channel block. A, inverse of the open duration (circles and dashed line) and the rate of entry into the putative blocked state (squares and solid line) for T264P mutant receptors are plotted as a function of physostigmine concentration. The lines were fitted to rate = rate at no physostigmine + k+B* x [physostigmine], where k+B* is the apparent blocking rate. The estimates for k+B* were 44 ± 2 µM-1 s-1 when fitting the reduction in the open duration, and 39 ± 2 µM-1 s-1 when fitting the increase in the rate of entry into the putative blocked state. B, relationship between the inverse of the duration of the putative blocked state and physostigmine concentration in the T264P mutant receptor. The increase in the duration of the putative blocked state at higher physostigmine concentrations is consistent with the presence of two (or more) blocking sites per receptor. The line was fitted to 1/Blocked = k-B (2k-B/(2k-B + k+B x [physostigmine])). This equation assumes the presence of two, equivalent blocking sites per receptor. The fitting results are k+B = 15.6 ± 4.8 µM-1 s-1 and k-B = 458 ± 28 s-1. C, inverse of the open duration (circles) and the rate of entry into the putative blocked state (squares) for the wild-type receptor activated by 1 mM carbachol in the presence of physostigmine are plotted as a function of physostigmine concentration. The estimates for k+B* were 36 ± 2 µM-1 s-1 when fitting the reduction in the open duration and also 36 ± 2 µM-1 s-1 when fitting the increase in the rate of entry into the putative blocked state. D, changes in membrane potential strongly affected the rate of return from the blocked state, but they were ineffective at altering the rate of entry into the blocked state. The values for k+B* (solid lines) and k-B* (dashed lines) in the presence of 100 µM physostigmine were estimated for wild type receptors activated by 1 mM carbachol (circles) or 200 µM ACh (squares), T264P receptors activated by 100 µM carbachol (triangles), and T264P receptors exposed solely to physostigmine (crosses) at -50 mV and +50 mV membrane potential. For the rate of development of block, the H value (change in membrane potential needed for an e-fold change in parameter) was 1141 mV (wild type + carbachol), 268 mV (wild type + ACh), 261 mV (T264P + carbachol), or 227 mV (T264P + physostigmine alone). For the rate of recovery from block, the H value was 44 mV (wild type + carbachol), 45 mV (wild type + ACh), 31 mV (T264P + carbachol), or 32 mV (T264P + physostigmine alone). For A to C, each point shows data from one patch, whereas for D each point shows results from the combined analysis of data from two or three patches.

In a model with a single blocking site per receptor, the lifetime of the blocked state is independent of blocker concentration. In contrast, the duration of the CT2 (putative blocked) state was longer at higher physostigmine concentrations, suggesting that the receptor contains two or more blocking sites, whereas the occupancy of any one of the sites can produce block. If so, then the apparent blocking rate is in fact a composite rate reflecting the binding properties of all sites. For example, in a model with two equivalent sites the microscopic blocking rate constant (k_+B) is equal to half the composite blocking rate k_+B^*, and the relationship between the apparent blocked state lifetime (_Blocked) and the microscopic unblocking rate constant (k_-B) can be expressed as 1/_Blocked = k_-B x (2k_-B/(2k_-B + k_+B x [physostigmine])). To test whether a model with two equivalent sites can adequately describe the data, we fitted this equation to the experimental data. Figure 8B shows that physostigmine-induced block of the T264P mutant receptor is well described by a model with two equivalent blocking sites per receptor, yielding a k_+B of 15.6 ± 4.8 µM^-1 s^-1 and a k_-B of 458 ± 28 s^-1 for physostigmine-induced block of the T264P mutant receptor. The predicted aggregate blocking rate constant (31 µM^-1 s^-1) is gratifyingly similar to the values for k_+B^* (39-44 µM^-1 s^-1).

To confirm these findings on the wild-type receptor, we examined physostigmine-mediated block of currents elicited by 1 mM carbachol. The somewhat longer open times and the presence of single-channel clusters are more amenable to measuring changes in the open time duration and the identification of the closed time component associated with the blocked state(s). The receptors were additionally exposed to 1, 10, or 100 µM physostigmine, and the intracluster open and closed time durations were determined. In the absence of physostigmine, 1 mM carbachol produced openings with a mean duration of 0.72 ± 0.22 ms (seven patches). The addition of physostigmine resulted in a reduction in the mean open duration. From the relationship between the inverse of the mean open duration and physostigmine concentration we estimated a k_+B ^* of 36 ± 2 µM^-1 s^-1 (Fig. 8C). An independent estimate for the rate of development of block was obtained from the analysis of the rate of entry into the putative blocked state. Linear regression analysis of the relationship between the rate of entry into the blocked state and physostigmine concentration also gave a k_+B^* of 36 ± 2 µM^-1 s^-1.

Thus, the analysis of block of the wild-type and the T264P mutant receptors in essence gave identical results. Accordingly, we conclude that the T264P mutation does not interfere with the blocking actions of physostigmine. We estimate that the microscopic binding rate constant for physostigmine, k_+B, is 20 µM^-1 s^-1, and the dissociation rate constant for physostigmine, k_-B, is 450 s^-1 producing a K_D value of 23 µMat -50 mV.

We next tested the voltage dependence of physostigmine-induced block. These experiments were conducted under four principal experimental conditions. Block of wild-type receptors activated by 1 mM carbachol or 200 µM ACh, and T264P receptors activated by 100 µM carbachol was studied in the presence of 100 µM physostigmine. In addition, we examined voltage dependence of block of T264P receptors exposed to 100 µM physostigmine alone. In the latter case, physostigmine served as both agonist and blocker.

For each case, the apparent blocking (k_+B^*) and unblocking (k_-B^*) rates were determined at -50 and +50 mV. The closed time histograms were fitted to the sum of two exponentials, and the closed time component corresponding to the blocked state was identified based on comparison with control data. The rate of entry into the blocked component (k_+B^*) was calculated from the inverse of the mean open duration, whereas k_-B^* was calculated as the inverse of the duration of blocked state. The results are summarized in Fig. 8D.

The data show that a change in membrane potential had little effect on the rate of development of block. The H value (change in membrane potential needed for an e-fold change in parameter) ranged from 227 to 1141 mV for k_+B^* for different receptor-agonist combinations. In contrast, k_-B^* was highly dependent on membrane potential. In the wild-type receptor activated by 1 mM carbachol, the lifetime of the blocked state was 1.2 ± 0.04 ms at +50 mV but 12.0 ± 0.3 ms at -50 mV, giving an H value of 44 mV. Comparable voltage sensitivity was observed for the wild-type receptor activated by 200 µM ACh as well as the T264P mutant receptor.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have characterized the activation and block of the adult mouse muscle nAChR by physostigmine. Physostigmine is a low-efficacy agonist, which produces single-channel openings of the same conductance as the nicotinic agonist carbachol. Physostigmine-elicited activity is affected by the mutations S269I and T264P in a manner similar to channel gating by nicotinic agonists. Mutations to the putative physostigmine binding site (Lys125 residue) shorten the open dwell duration of channels in the presence of physostigmine but not carbachol. The channel effective opening rate in the presence of 1 mM carbachol was not affected by up to 100 µM physostigmine, indicating that physostigmine does not interact with the ACh binding site. Finally, we demonstrate that the blocking actions of physostigmine can be accounted for by two equivalent binding sites per receptor.

Physostigmine elicits only a low level of activity, but previous studies have generally been noncommittal regarding the underlying cause for the weak activating properties of physostigmine (Pereira et al., 1993; Storch et al., 1995; Jackson et al., 2002). Our single-channel data from the wild-type receptor activated by physostigmine showed low open probability activity that did not condense into recognizable clusters. The lack of clusters is typically attributed to low potency or low efficacy of the agonist on the particular receptor type. We therefore used a receptor containing a mutation previously shown to increase channel P_o through effects on the channel opening and closing rate constants. The S269I mutant increases the channel opening rate constant for receptors activated by a nicotinic agonist, choline, by 30-fold (Zhou et al., 1999; Grosman et al., 2000), and it enhances the opening rate constant for another APL, galantamine, resulting in easily recognizable single-channel clusters (Akk and Steinbach, 2005). In contrast, no clusters were observed for the S269I mutant receptor activated by physostigmine at concentrations up to 100 µM. Although it is possible that the mutation selectively acts on channel gating by nicotinic agonists and galantamine, but not physostigmine, we note that the mutation had a similar effect on the channel closing rate (an 3-fold reduction) for nicotinic agonists and physostigmine. We conclude from this finding that physostigmine is a low-efficacy agonist of the adult-type muscle nAChR, and we estimate that the channel opening rate constant for wild-type receptors activated by physostigmine is less than 2 s^-1. The studies were limited to 100 µM physostigmine, because potent block by physostigmine hindered studies at higher concentrations.

Results of several previous studies could be interpreted as demonstrating the involvement of the nicotinic agonist binding site in physostigmine-mediated channel activity. Cooper et al. (1996) found that in Xenopus oocytes expressing rat embryonic receptors, physostigmine-elicited single-channel currents were not observed after an incubation with -bungarotoxin, whereas the presence of mecamylamine or methyllycaconitine strongly reduced the frequency of single-channel events. Methyllycaconitine, -bungarotoxin, and d-tubocurarine were also shown to inhibit receptors activated by physostigmine in L. migratoria neurons (van den Beukel et al., 1998; Jackson et al., 2002), whereas in oocytes expressing rat neuronal 4β4 nicotinic receptors physostigmine competitively displaced ¹²⁵I-epibatidine, a high-affinity ligand to the ACh site (Zwart et al., 2000). In contrast to these studies, methyllycaconitine inhibited channel activation by ACh but not by physostigmine in clonal rat pheochromocytoma cells (Storch et al., 1995), and saturating concentrations of d-tubocurarine and -bungarotoxin were found to be ineffective at blocking the ability of physostigmine to activate the T. californica receptor (Okonjo et al., 1991). Likewise, physostigmine had low potency to reduce binding of -bungarotoxin to the T. californica receptor (IC₅₀ of 70 to 500 µM; Sherby et al., 1985). Accordingly, the existing data are not in agreement.

To re-examine this question, we tested the ability of physostigmine to alter the channel effective opening rate in the presence of a subsaturating concentration of carbachol. We reasoned that if physostigmine interacts with the nicotinic agonist binding site then its presence should lead to a reduction in the effective opening rate for carbachol because the binding site is occupied by the low-efficacy agonist physostigmine a fraction of the time. We have previously shown that the presence of low-efficacy agonists known to interact with the nicotinic agonist binding site, such as tetraethylammonium or choline, competitively reduces the effective opening rate for channels activated by carbachol (Akk and Steinbach, 2003; Akk et al., 2005). In contrast, we found that physostigmine, at concentrations up to 100 µM, was ineffective at modifying the effective opening rate for carbachol. These data are consistent with the idea that physostigmine activation is not mediated by interaction with the ACh binding site. Previous studies had shown that another APL, galantamine, also activated nAChR but did not interact with the ACh binding site (Akk and Steinbach, 2005).

We show that mutations to the Lys125 site reduce channel open durations for physostigmine but not carbachol. This seems to be consistent with the proposal that the Lys125 residue participates in the binding of physostigmine (Schrattenholz et al., 1993) insofar as changes in open interval durations in response to mutations can be used to judge an involvement of a residue in agonist binding. However, it seems unlikely that the Lys125 residue forms a critical element of the binding pocket because of the limited effect that the K125Q and K125E mutations had on channel open durations.

Despite interacting with a distinct binding site, many basic features of channel activation by physostigmine were similar to activation by nicotinic agonists. The channel conductance was indistinguishable when physostigmine instead of the nicotinic agonist carbachol was used for channel activation. Furthermore, mutations to the M2-M3 linker (S269I) and the M2 domain (T264P) affected channel gating by physostigmine and nicotinic agonists in qualitatively similar ways. Previous work has shown that channel openings elicited by nicotinic agonists as well as the APL galantamine are prolonged at hyperpolarized potentials. But, surprisingly, changes in the membrane potential were ineffective at modifying the channel open durations in the presence of 1 µM physostigmine. It seems unlikely that block could produce the lack of voltage sensitivity because the onset of block was largely independent of membrane potential, and, in any case, termination of the open event is dominated by channel closing at this low physostigmine concentration. We are unable to provide a definitive explanation for this lack of voltage sensitivity.

Physostigmine is a strong channel blocker. We estimate that physostigmine-induced block develops with an apparent rate of 40 µM^-1 s^-1. This is not drastically different from previous estimates for k_+B^* for fetal type muscle nAChR (6 µM^-1 s^-1, Bufler et al., 1996; 16 µM^-1 s^-1, Wachtel, 1993). However, our data on physostigmine-induced block of the adult muscle-type nAChR display several important distinctions from a study conducted on the embryonic-type nAChR in BC3H-1 cells (Wachtel, 1993). First, we observed voltage dependence for recovery from block but not development of block, whereas Wachtel (1993) saw voltage sensitivity of both reactions. We note that voltage dependence has been reported for physostigmine block of neuronal nicotinic receptors in rat hippocampal neurons (Pereira et al., 1993), and end-plate currents from frog muscle (Shaw et al., 1985). Second, our findings are best described by two equivalent physostigmine blocking sites per receptor, whereas the study on embryonic receptors was consistent with a single blocking site per receptor (Wachtel, 1993). Previous studies on block by other APL have shown that tacrine-induced block is best described by two blocked states connected to the open state (Prince et al., 2002), but block by galantamine could be accounted for by interactions with a single site (Akk and Steinbach, 2005). The voltage dependence for block by physostigmine corresponds to a movement of a single charge through approximately 60 to 80% of the membrane field. This suggests that when physostigmine occupies the blocking site, the charge reaches a point near the cytoplasmic end of the channel. A binding site for the classic open channel blocker QX-222 has been described at the same distance (Neher and Steinbach, 1978).

Given the pK_a value for physostigmine (8.1; Meloun and Cernohorsky, 2000), the majority of physostigmine is charged when dissolved in the pipette medium, pH 7.4. Voltage dependence of block is a strong indication that ionized species of physostigmine produce channel block. It may be interesting to determine in future studies whether channel activation by physostigmine is accomplished by charged or neutral molecules, and whether more complete studies on the activation properties of physostigmine can be carried out at higher pH values where block is reduced.

In sum, physostigmine activates the muscle nicotinic receptor but does not interact with the nicotinic binding site. The single-channel conductance in the presence of physostigmine is indistinguishable from that in the presence of the nicotinic agonist carbachol. But unlike channel activity elicited by nicotinic agonists, the durations of openings elicited by physostigmine are not affected by voltage. Physostigmine is also a potent channel blocker, and the findings are well described by two equivalent blocking sites per receptor.

	Footnotes

 
This work was supported by National Institutes of Health grant NS22356 to J.H.S. J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; APL, allosterically potentiating ligand; ACh, acetylcholine; P_o, open probability; CT, closed time; OT, open time.

Address correspondence to: Dr. J. H. Steinbach, Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: jhs{at}morpheus.wustl.edu

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