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J Physiol (2003), 551.1, pp. 155-168
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.043885
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ABSTRACT |
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We have studied the activation and inhibition of the mouse muscle adult-type nicotinic acetylcholine receptor by tetraethylammonium (TEA) and related quaternary ammonium derivatives. The data show that TEA is a weak agonist of the nicotinic receptor. No single-channel clusters were observed at concentrations as high as 5 mM TEA or in the presence of a mutation which selectively increases the efficacy of the receptor. When coapplied with 1 mM carbamylcholine (CCh), TEA decreased the effective opening rate demonstrating that it acts as a competitive antagonist of CCh-mediated activation. Kinetic analysis of currents elicited by CCh and TEA allowed an estimate of receptor affinity for TEA of about 1 mM, while an upper limit of 10 s-1 could be set for the wild-type channel-opening rate constant for receptors activated by TEA alone. At millimolar concentrations, TEA inhibited nicotinic receptor currents by depressing the single-channel amplitude. The effect had an IC50 of 2-3 mM, depending on the conditions of the experiment, and resembled a standard open-channel block. However, the decrease in channel amplitudes was not accompanied by an increase in the mean burst duration, indicating that a linear open-channel blocking mechanism is not applicable. Upon studying block by other nicotinic receptor ligands it was found that block by CCh, tetramethylammonium and phenyltrimethylammonium can be accounted for by the sequential blocking mechanism while block in the presence of methyltriethylammonium, ethyltrimethylammonium or choline was inconsistent with such a mechanism. A mechanism in which receptors blocked by TEA can close would account for the experimental findings.
(Received 27 March 2003; accepted after revision 22 May 2003; first published online 24 June 2003)
Corresponding author G. Akk: Department of Anesthesiology, Washington University in St Louis, Campus Box 8054, 660 S. Euclid Avenue, St Louis, MO 63110, USA. Email: akk{at}morpheus.wustl.edu
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INTRODUCTION |
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It has long been known that tetraethylammonium (TEA), besides its ability to block currents from delayed rectifier channels, also strongly affects nicotinic acetylcholine (ACh) receptor function (Koketsu, 1958; Adler et al. 1979a,b). In the frog neuromuscular junction, application of TEA reduces the peak amplitude and accelerates the decay course of spontaneous endplate currents. The effect on the peak current is voltage dependent, and at TEA concentrations of 250 µM and higher, the slope of the current-voltage relationship becomes negative at hyperpolarized membrane potentials. The effect on the decay time course of synaptic events is also voltage dependent. While hyperpolarization of the membrane slows the decay time course in the neuromuscular junction (Magleby & Stevens, 1972), the application of TEA reduces, and at higher concentrations eliminates, this voltage dependence (Adler et al. 1979a). The reductions in amplitude and decay time constant have distinct concentration-effect profiles. The reduction in the decay time constant was noticeable at TEA concentrations as low as 10 µM and became fully saturated at approximately 100 µM. On the other hand, TEA at concentrations above 250 µM was needed to observe the reduction in the peak endplate current amplitude (Adler et al. 1979b).
Based on the available empirical, data Adler et al. (1979a) proposed a kinetic model in which TEA could bind to both closed (high-affinity site) and open (low-affinity site) configurations of the nicotinic receptor. TEA binding to the closed state did not affect the association of ACh to the agonist recognition site, allowing a normal opening of the channel gate. However, the lifetime of such an open-receptor complex was reduced compared to the lifetime of the open state which was not preceded by binding of TEA. According to this model, TEA could also bind to the receptor in the open state so long as TEA had not previously bound to the closed-channel form. In this case, the binding of TEA to the open-channel complex resulted in a reduction of the single-channel amplitude. Voltage sensitivity of TEA actions was explained in this model by voltage-dependent binding steps of TEA to both closed and open channels.
It should be mentioned that the above model did not incorporate interactions of TEA with the agonist binding site. However, it is reasonable to expect that TEA, with its quaternary amine group, can compete with ACh for access to this site. Indeed, it was demonstrated that TEA inhibited the binding of either ACh or perhydrohistrionicotoxin to the membranes from the Torpedo electric organ (Adler et al. 1979b).
Recently it was found that in cochlear outer hair cells, the application of TEA blocked ACh-evoked currents (Blanchet & Dulon, 2001). The effect had an EC50 of 30 µM and was present only when TEA was applied from the extracellular side of the membrane, suggesting that the high-affinity TEA interaction site is not accessible from the intracellular side of the membrane. Interestingly, block by TEA appeared to be voltage insensitive, because the magnitude of the blocking effect was similar at both positive and negative membrane potentials. It is not clear whether this type of block is mediated by interactions with the ACh binding site or the pore domain.
In this work, we have used single-channel recordings to examine the activating and blocking properties of TEA. The mouse muscle adult-type nicotinic receptor was exposed to solutions containing TEA or other quaternary ammonium derivatives, or mixtures of TEA and carbachol (CCh). The data were evaluated to characterize the kinetics and mechanism of action of TEA.
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METHODS |
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We used mouse cDNA encoding the wild-type 
, 
, 
and 
subunits subcloned into a CMV promoter-based expression vector (pcDNAIII; Invitrogen, San Diego, CA, USA). The S269I mutant clone was generously provided by Dr A. Auerbach (SUNY at Buffalo, USA). The receptors were expressed in HEK 293 cells using transient transfection as described previously (Akk, 2002). In brief, 3.5 µg of cDNA per 35 mm culture dish was used in the ratio of 2 : 1 : 1 : 1 ( : : : ). Cells were exposed to the DNA calcium precipitate overnight after which the medium was replaced. Electrophysiological experiments commenced 24 h later. A batch of transfected cells could be used for 2 days.
Chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Quaternary ammonium compounds were purchased from TCI-America (Portland, OR, USA).
Electrophysiological experiments were performed using the patch-clamp technique in the cell-attached configuration (Hamill et al. 1981). The bath solution was Dulbecco's phosphate-buffered saline containing (mM): 137 NaCl, 0.9 CaCl2, 2.7 KCl, 1.5 KH2PO4, 0.5 MgCl2, 6.6 Na2HPO4, pH 7.3. The pipette solution contained (mM): 142 KCl, 1.8 CaCl2, 1.7 MgCl2, 5.4 NaCl, 10 Hepes, pH 7.4. The agonists were added to the pipette solution. Unless indicated otherwise, the membrane potential was held at -50 mV based on the combination of the applied potential and the membrane potential of the cell. The latter was determined from the reversal potential of ionic currents. All experiments were performed at room temperature.
Single-channel currents were amplified with an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), digitized at 500 kHz, and saved on a PC hard disk using a Digidata 1322 Series interface (Axon Instruments). For long-term storage, the data were saved on DVD+R discs.
Event detection was carried out using program SKM (www.qub.buffalo.edu; Qin et al. 1996, 1997) at 2-7 kHz. Open and closed interval durations were estimated from histogram fitting using program MIL (Qin et al. 1996, 1997). When TEA was used in combination with CCh, the analysis was restricted to single-channel clusters (Sakmann et al. 1980). A cluster is a series of openings which are separated by relatively brief closed periods, while clusters are separated from other clusters by long-duration closed periods. In the present experiments (e.g. Fig. 2), clusters in the presence of strong agonists were obvious as periods of high probability of being open (Popen). Accordingly, clusters were selected by eye as periods of high activity with no simultaneous openings. In the experiments where single-channel clusters were observed, both closed and open interval durations were established. In patches where TEA alone was used as an agonist, the data consisted of isolated bursts such as seen in the presence of low concentrations of ACh (Colquhoun & Sakmann, 1985; Sine & Steinbach, 1986). Hence, in these experiments, only burst durations were examined.
We analysed interactions between 1 mM CCh and TEA using three approaches (see Results). The initial approach was to measure the duration of the predominant closed time within clusters of activity elicited by CCh, in the presence of increasing concentrations of TEA. The inverse of this duration provides an estimate of the effective opening rate for the channel.
The second approach used the standard linear model for receptor activation (see model 1). In applying this model to activation in the presence of both CCh and TEA, no additional kinetic steps for TEA action were added, and TEA action was studied in terms of its effect on receptor activation by CCh. In this analysis, the interaction between TEA and CCh was observed as an increase in the apparent KD value for CCh.
[A]k+1 [A]k+2 k+d
C ™ AC ™ A2C ™ A2O ™ A2D
k-1 k-2 k-d
In model 1, A represents an agonist molecule (CCh), C a receptor with a closed channel, O a receptor with an open channel and D a receptor in a short lived desensitized state. k+1 and k+2 are the agonist association rate constants, k-1 and k-2 are the agonist dissociation rate constants, is the channel-opening rate constant, and is the channel-closing rate constant. It appears that in the mouse, adult-type AChR, the two transmitter binding sites have essentially equivalent KD values, and the number of free parameters is reduced (Akk & Auerbach, 1996; Wang et al. 1997; Salamone et al. 1999). Therefore, k+1 = 2k+2, and k-2 = 2k-1, and the microscopic dissociation equilibrium constant of each site (KD) is k-1/k+2. The A2D state corresponds to a short-lived desensitized state with a mean duration of approximately 1-5 ms (Colquhoun & Sakmann, 1985; Salamone et al. 1999). This state is resolved under conditions when the closed intervals corresponding to the channel activation pathway are much shorter than the lifetime of A2D. Dwells in the A2D state were infrequent, making up less than 4 % of the total closed periods in a cluster. This state was omitted from analysis of records obtained in the presence of 1 mM CCh + 5 mM TEA, where the duration of the activation-related component was sufficiently similar to the mean predicted duration of A2D.
In the third approach, we studied TEA action using a model which takes into consideration separate, mutually exclusive binding of CCh or TEA to an agonist binding site. This approach allowed us to estimate the association and dissociation rates for TEA.
In model 2, an agonist binding site can bind either TEA or CCh. Binding of TEA (or CCh) to one binding site does not affect the binding of CCh (or TEA) to the other site. However, only receptors in which both ligand binding sites are occupied by CCh can open. Strictly speaking, TEA-occupied receptors and probably also heteroliganded receptors can open. However, due to the low gating efficacy of such receptors, the steps corresponding to opening from the CCh-TEA-C and TEA2-C states were excluded from the model. In the analysis, we assumed that the two sites were equivalent for TEA as well as for CCh (see above). The rate constants in models 1 and 2 were determined from the analysis of idealized intracluster interval durations using Q-matrix methods. A maximum-likelihood method incorporating correction for missed events was employed (Program MIL). Error limits were estimated from the curvature of the likelihood surface at its maximum using the approximation of parabolic shape (Qin et al. 1996). Simulated single-channel records were generated using the program SIMU (www.qub.buffalo.edu). Approximately 2000 events were synthesized, then analysed using SKM and MIL as described for experimental data.
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RESULTS |
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Activation of the adult wild-type and 1S269I receptors by TEA
We will use the term 'cluster' to refer to a series of many closely spaced openings. Each cluster is separated from other clusters by relatively long closed periods. For the muscle nicotinic receptor, a cluster reflects the activity of a single receptor as it emerges from a long-lived desensitized state (the start of the cluster), rapidly accesses a set of interconnected closed and open states (the cluster itself), and then re-enters a long-lived desensitized state (the termination of a cluster) (see Sakmann et al. 1980). The experimental resolution of a cluster, therefore, depends on several factors which include the duration of closed states within the cluster (reflecting agonist binding and unbinding as well as the intrinsic channel-opening rate) and the rates for entering and leaving desensitized states, as well as the number of activatable receptors in a patch. We will use the terms 'burst' and 'apparent opening' interchangeably, to refer to periods during which a channel is not resolved to be in a closed state. A burst, therefore, can be a single opening or series of very closely spaced openings separated by unresolved closed dwells. At least two mechanisms can produce unresolved closed dwells in our data: a dwell in A2C (see model 1) or a rapid blocking event (see model 3, in Results). A burst can occur within a cluster, or in the absence of resolved clusters.
Tetraethylammonium (TEA) is a weak agonist of the nicotinic receptor. Sample currents elicited by 100 or 2000 µM TEA are shown in Fig. 1A. Receptor activation takes place as isolated bursts over this concentration range. No systematic agonist-dependent changes in the closed interval durations were observed, suggesting that the affinity of the receptor to TEA is very low, the receptors occupied by TEA have a very low channel-opening rate constant, or both.
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Figure 1. Single-channel currents from wild-type (A) and The currents were elicited by 100 or 2000 µM TEA. Openings are shown downward. No clusters of single-channel activity were observed with any concentrations of TEA tested. Burst duration histograms are shown next to the traces. The data were best characterized by one- (at 2000 µM TEA) or two-exponential fits (at 100 µM TEA). The mean event durations were: wild-type, 100 µM TEA: 0.17 ms (93 %) and 0.77 ms (7 %); wild-type, 2000 µM TEA: 0.16 ms; | ||
Previously, Grosman et al. (2000) found that the S269I mutation results in a dramatic shift in the channel-gating equilibrium constant. This mutation leads to a ~2-fold decrease in the channel-closing rate constant and a 30-fold increase in the channel-opening rate constant for channels activated by choline. The mutation had a qualitatively similar effect on channel gating in ACh-activated receptors (Grosman et al. 2000), suggesting that gating is altered for all agonists. So, we used this mutant receptor to study whether a potential 60-fold increase in the gating equilibrium constant leads to the emergence of single-channel clusters in the presence of TEA.
The activity from the receptor containing the mutant subunit activated by 100 or 2000 µM TEA is shown in Fig. 1B. In the presence of 100 µM TEA, the mutant channel apparent open durations were increased by ~3-fold compared to the wild-type receptor. However, no clusters were observed at this concentration or even in the presence of 2 mM TEA, which has led us to the conclusion that the channel-opening rate constant in the presence of TEA is below a value at which single-channel clusters are observed using the approaches available to us.
Interactions between TEA and the nicotinic agonist binding site
We investigated the interactions of TEA with the nicotinic ligand binding site further by studying the effect of TEA on receptor activation by CCh. CCh is an intermediate-affinity, high-efficacy ligand of the nicotinic receptor. The receptor affinity to CCh is ~590 µM, and the channel-opening rate constant is 7630 s-1 (Akk & Auerbach, 1999; G. Akk, unpublished observations).
We studied the effect of TEA on the effective opening rate (') of receptors activated by 1 mM CCh. The effective opening rate is calculated as the inverse of the major, agonist-dependent component in the intracluster closed time histograms. The value for ' is determined by both receptor affinity and the channel-opening rate constant. In the absence of TEA, ' at 1 mM CCh is 3058 s-1. With the addition of TEA to the pipette medium, a decrease in ' takes place, concordant with our expectations of TEA acting akin to a competitive agonist in the presence of CCh. Figure 2A shows sample clusters elicited by 1 mM CCh in the absence and presence of 5 mM TEA. The data on the effect of TEA on channel effective opening rate are summarized in Fig. 2B. We used the following equation to fit the line in Fig. 2B: '([TEA]) = 'max/(1 + ([TEA]/KI)), where '([TEA]) corresponds to the effective opening rate at a given TEA concentration, 'max is the limiting value to which the effective opening rate approaches at low TEA concentrations, and KI is the TEA concentration at which half-maximal reduction in ' takes place. According to the fit, 'max is 3162 ± 418 s-1 which agrees well with our data obtained in the absence of TEA. The KI of the curve is at 1107 ± 326 µM. So, TEA at 1.1 mM inhibits half-maximally channel activation induced by 1 mM CCh. If we assume that the reduction in ' occurs because receptors with one or two binding sites occupied by TEA do not open, then this corresponds to an estimated KD of about 775 µM for TEA binding to the CCh binding site.
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Figure 2. Receptor activation in the presence of CCh and TEA A, single-channel clusters recorded in the presence of 1 mM CCh, 1 mM CCh plus 5 mM TEA or 100 µM CCh plus 5 mM TEA. Openings are shown downward. The presence of TEA leads to a reduction in the cluster open probability and the amplitude of channel openings. No clusters are seen in the presence of 100 µM CCh and 5 mM TEA. Burst and closed duration histograms are shown next to the traces. The results of the fits are: 1 mM CCh: burst, 0.66 ms; closed, 0.32 ms (99 %), 5.8 ms (1 %); 1 mM CCh + 5 mM TEA: burst: 0.54 ms; closed, 2.4 ms (67 %), 0.07 ms (33 %); 100 m M CCh + 5 mM TEA: burst, 0.42 ms; closed, 9.3 ms (91 %), 0.07 ms (9 %). B, effective opening rate of single-channel clusters obtained in the presence of 1 mM CCh and different concentrations of TEA. The presence of TEA reduces the effective opening rate. C, the apparent receptor affinity to CCh in the presence of TEA. The KD was determined according to model 1. The presence of TEA reduces the apparent affinity of the receptor to CCh. | ||
The effect of TEA on the reduction of ' is not voltage dependent. We examined the effect of TEA on the effective opening rate from receptors activated by 1 mM CCh at +50 mV. The relationship between ' and TEA concentration is given in Fig. 2B, demonstrating that membrane potential has a minor effect on the interactions of TEA with the nicotinic receptor binding site. The results of the fit yield estimates for 'max of 2259 ± 347 s-1 and for KI of 1111 ± 380 µM. The lack of voltage dependence agrees with previous observations on the inhibition of nicotinic receptor activation in the frog neuromuscular junction by tubocurarine (Colquhoun et al. 1979), or the activation of the nicotinic receptor by ACh itself (Auerbach et al. 1996).
We then analysed activity elicited by 1 mM CCh in the absence and presence of TEA at -50 mV using model 1. In the analysis, the channel-opening rate constant was constrained to the value obtained in the absence of TEA. Hence, it was assumed that the addition of TEA only affects the binding of CCh. As the concentration of TEA is increased, the apparent KD of the receptor for CCh increases. The plot in Fig. 2C shows the relationship between the apparent KD for CCh and TEA concentration where the continuous line is a fit to the equation KD([TEA]) = KD(0)/(1 + ([TEA]/KI)). According to the fit, KD(0) for CCh is 556 µM while a twofold increase in the apparent KD takes place at 2136 ± 976 µM TEA. This value would correspond to a KD of about 1650 µM for TEA binding to the CCh binding site.
The single-channel data obtained in the presence of 1 mM CCh and various concentrations of TEA were also analysed using model 2, which takes into consideration separate, mutually exclusive binding of CCh and TEA to the agonist binding site. To reduce the number of free parameters, we fixed the binding and gating steps for CCh to values obtained in the absence of TEA (k+ = 44 µM-1 s-1, k- = 25744 s-1, = 7630 s-1). We also assumed that TEA (as well as CCh) has identical microscopic association and dissociation rates at the two sites. According to the fit (1 patch at each of 0, 100, 200, 500 and 2000 µM TEA, number of events 2969, 4081, 11588, 5440 and 14262, respectively), the association rate constant for TEA is 4.0 ± 0.3 µM-1 s-1, and the dissociation rate constant is 6308 ± 315 s-1 (KD = 1577 µM). These results indicate that the equilibrium dissociation constant for TEA at the agonist binding site is about 1.5 mM.
It might be argued that the increase in the mean intracluster closed time durations in the presence of TEA and CCh is not due to the competition between the two agonists at the ligand recognition site, and that the interaction of TEA with the nicotinic receptor leads to the emergence of novel open-channel blocked states. If the blocked states were connected linearly, the composite lifetime in the blocked states would be TEA concentration dependent, increasing with the increased amount of the blocker. We, however, argue against such a notion. The emergence of novel states would result in an additional component in the intracluster closed time histograms. As the concentration of TEA is raised, the frequency of the blocked state would be increased and eventually dominate the distribution of closed time durations. However, our analysis of the intracluster closed times does not support the presence of such additional closed states: we do not see an additional component in the distributions (see Fig. 2A). Also, when TEA is used as agonist no sign of brief closed periods is seen (Fig. 1). Finally, if the change in the closed time distribution reflected an open-channel blocking action of TEA, it should be apparent at all concentrations of CCh. The data presented in the bottom row of Fig. 2A show the activity elicited by 100 µM CCh, in the presence of 5 mM TEA. There is no indication of bursting behaviour induced by TEA. In particular, there is no indication of a component in the closed time distribution with a duration of about 2 ms (see Fig. 2A), while this concentration of TEA results in a major closed time component in clusters elicited by 1 mM CCh (Fig. 2A). Accordingly, there is no indication that the change in closed durations induced by TEA in the presence of CCh results from channel block by TEA.
To further examine the origin of the closed time component, we simulated single-channel records using the kinetic scheme shown in model 2 and parameters derived from the analysis. As might be expected, when we simulated records for the combination of CCh and TEA, the predicted ' component agreed well with the experimentally obtained values (Fig. 3A). We then tested the competitive nature of the interaction by using a second agonist, ACh, at several concentrations in the absence and presence of 1 mM TEA. The concentrations of ACh (50, 100 and 200 µM) span the estimated KD value for ACh for the resting receptor (160 µM). The rate constants used for ACh were taken from published work (Akk & Auerbach, 1996), while the association and dissociation rates for TEA were established from the experiments using the combination of CCh and TEA (see above). There is a remarkably close agreement between the experimentally observed values for ' and the values predicted with no free parameters (Fig. 3B). This observation supports the interpretation of the interaction between TEA and agonists as a competitive interaction occurring at the agonist binding sites on the receptor. It also demonstrates that the closed times within clusters do not contain new components which might reflect additional non-conducting states induced by TEA.
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Figure 3. The observed values for A, the values obtained when 1 mM CCh is used as agonist, in the presence of various concentrtions of TEA ( | ||
TEA-mediated block of nicotinic receptor channels
The amplitudes of single-channel currents depend on the concentration of TEA. Sample currents elicited by 100 or 2000 µM TEA shown in Fig. 1 demonstrate that this 20-fold increase in TEA concentration leads to a reduction in the average current from -3.19 to -1.62 pA (at membrane potential (VM) = -50 mV). Figure 4A shows the relationship between the concentration of TEA and single-channel current, measured at -50 mV. As [TEA] is increased, the current decreases. The curve shown in Fig. 4A was fitted with i = imax /(1 + ([TEA]/IC50 )), where i is the mean amplitude at a given TEA concentration, imax is the extrapolated maximal amplitude and IC50 is the concentration of TEA producing half-maximal reduction in single channel amplitude. According to the fit, imax is 3.3 ± 0.3 pA and IC50 is 2.7 ± 1.0 mM.
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Figure 4. TEA affects channel event amplitudes and the current-voltage relationship A, the amplitudes of bursts for the wild-type receptor in the presence of TEA only ( | ||
We also looked at the relationship between single-channel current and [TEA] in S269I mutant receptors. These data have the additional, technical advantage of increased burst durations. The amplitude versus [TEA] relationship is shown in Fig. 4 where the fit gives imax = 3.8 ± 0.2 pA and IC50 = 3.0 ± 0.7 mM.
The reduction in the single-channel amplitude in the presence of TEA is not due to interactions between the TEA molecule and the agonist recognition site. Coapplication of 1 mM CCh with TEA reduces the total amount of time that the agonist recognition site can interact with TEA. Under these conditions, most channel openings arise as a result of CCh interacting with the agonist binding sites. If the reduction in single-channel amplitude was due to TEA interacting with the agonist binding site then the presence of CCh would shift the TEA inhibition curve towards higher TEA concentrations. The TEA amplitude inhibition curve obtained in the presence 1 mM CCh is given in Fig. 4A. The presence of 1 mM CCh had little effect on the maximal single-channel current (3.2 ± 0.2 pA) while shifting the midpoint of the inhibition curve slightly towards lower TEA concentrations (2.0 ± 0.6 mM).
The decrease in the single-channel amplitude in the presence of high concentrations of TEA is voltage dependent. Figure 4B shows the current-voltage (I-V) curves obtained at various TEA concentrations for the S269I receptor. Figure 4C presents inhibition data in terms of the reduction in normalized current as a function of [TEA] at various membrane potentials. If the voltage dependence is assumed to be exponential, it gives an apparent voltage sensitivity of 0.028 mV-1 (35 mV per e-fold change). If a simple blocking model by a monovalent cation is assumed, the cation at the blocking site senses about 70 % of the membrane field (Woodhull, 1973).
Action of TEA on channel open durations
An accepted kinetic model for the action of many drugs is simple linear open-channel block (Adams, 1976; Neher & Steinbach, 1978), model 3:
2A + R ™ A2R ™ A2O ™ A2OB
Here, A indicates an agonist molecule, B a blocker molecule (possibly the same as A), R a receptor with a closed channel, O a receptor with an open channel, and A2OB a receptor with an open channel which cannot conduct ions as a result of the binding of the blocker, B. A significant feature of this kinetic model is that a blocked channel both does not conduct ions and cannot close. If blocking events (the transitions A2O ™ A2OB) are very rapid, then individual blocking episodes cannot be identified and the mean conductance appears to be reduced proportionately to the fractional time spent blocked (
([B]) = ([0]) (1 - fraction of time blocked) = ([0]) (KB/(KB + [B])), where ([B]) is the single-channel conductance at blocker concentration [B] and KB is the dissociation constant for the blocker to the open-channel state (at a particular membrane potential). Similarly, the length of time that the receptor spends in the compound states A2O and A2OB will be increased proportionately to the time spent blocked. The inverse of the mean duration of such a burst is (1/
) = (1 - fraction of time blocked) ~( KB/(KB + [B]), where is the mean duration in A2O + A2OB and is the channel-closing rate. Accordingly, bursts will appear to have a lower conductance and an increased duration. Indeed, for a strict linear open-channel blocking mechanism, the total charge transfer during an activation event remains constant since, on average, a receptor spends that same amount of time in the open state before closing, regardless of the presence of blocker (Neher & Steinbach, 1978). Ogden & Colquhoun (1985) have demonstrated that this mechanism prolongs bursts of activity containing brief activation-related closures in an identical fashion.
However, TEA does not act in a fashion consistent with simple linear open-channel block. The mean duration of bursts does not increase with increasing [TEA], either for wild-type receptors activated by 1 mM CCh in the presence of various concentrations of TEA, or for receptors containing the S269I subunit and activated by TEA (Fig. 5). The lines in Fig. 5 illustrate the predicted inverse mean durations, based on the reduction in single-channel current at the different [TEA]s.
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Figure 5. The channel burst durations are minimally affected by TEA The inverse of the mean burst durations in the wild-type receptor ( | ||
It is known that the membrane potential affects the decay rate of endplate currents (Magleby & Stevens, 1972) due to voltage dependence of the channel-closing rate constant (Auerbach et al. 1996). The channel-closing rate constant of the nicotinic receptor is slowed e-fold by approximately 80 mV of hyperpolarization (Auerbach et al. 1996; Akk & Steinbach, 2000). In addition, the membrane potential affects the apparent affinity of TEA for the channel blocking site (Fig. 4B).
We looked at the effect of voltage on the burst durations elicited by various concentrations of TEA from S291I receptors. The inverse of the mean burst duration is plotted against the membrane potential in Fig. 6A. The lines through the data points were fitted according to: z(V,[TEA]) = z(0,[TEA]) exp(V/H) where z(V,[TEA]) represents the inverse of the burst duration at a specific voltage and [TEA], and H gives the voltage change required for an e-fold change in z at a given [TEA]. As the concentration of TEA is increased, the voltage sensitivity is reduced. Similar observations were made when wild-type receptors were activated by 1 mM CCh in the presence of various concentrations of TEA (Fig. 6B).
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Figure 6. Relationship between the inverse burst duration and membrane potential A, inverse burst duration vs. membrane potential in the | ||
This change in voltage dependence is contrary to what would be predicted by linear channel block. It would be expected that the intrinsic channel-closing rate would decrease at more negative potentials, and that the fraction of time blocked would increase. Both of these effects would increase the amount of prolongation as the membrane potential was made more negative in the presence of TEA. A possible confounding factor would be the appearance of a blocked state with a relatively long duration, which would produce a burst-terminating closed dwell. We have already presented arguments that such a closed dwell does not appear in the data, when we discussed the interpretation of the interactions between CCh and TEA. Accordingly, another explanation must be sought.
The simplest explanation is that a channel blocked by TEA can still close. In this case, z = (1 - fraction of time blocked) + 
(fraction of time blocked), where is the closing rate constant for the open-unblocked channel and is the closing rate for the open-blocked channel. If = 0, linear channel block would apply. If = , the presence of TEA would have no effect on the mean burst duration. Finally, if > , the presence of TEA would decrease the mean burst duration. Note that this is a simple occupancy model, in the sense that the channel-closing rate assumes one of two discrete values depending on whether TEA occupies the channel blocking site. We examined the utility of this model in its simplest form: the presence of TEA at the blocking site was assumed to have no effect on the voltage dependence of the channel-closing rate, but to result in a new closing rate at zero membrane potential. The fractional occupancy by TEA was estimated from the reduction in current, and the mean burst duration from the time constant of a single exponential distribution fitted to the burst duration data. To allow data at different potentials to be superimposed, the inverse burst durations were normalized to the burst duration at that potential, in the absence of TEA. Accordingly, the relative inverse burst duration is plotted. As shown in Fig. 6C, the inverse burst duration increased with higher fractional occupancy by TEA. The scatter of the data is sufficiently large that there is no indication of a voltage dependence of the relative increase (which might indicate that occupancy by TEA affects the voltage dependence of the closing rate). The continuous line through the data was predicted assuming that occupancy by TEA increases the closing rate by twofold ((0) = 1826 s-1, (0) = 3652 s-1, voltage dependence constant at e-fold per 123 mV). A similar analysis of the data obtained using 1 mM CCh plus various concentrations of TEA produced similar results (Fig. 6D). The predicted line was produced using the same value of as was used for the data obtained with TEA alone. The smaller relative increase in the rate of burst termination results from the observation that the closing rate for CCh is higher ((0) = 2740 s-1) than for TEA.
Block by other quaternary ammonium derivatives
Our data show that the reduction in single-channel current amplitudes observed in the presence of high concentrations of TEA is not accompanied by an increase in the burst durations. We examined the structural requirements of this phenomenon by looking at block by CCh, choline and by some related, simple quaternary ammonium derivatives: tetramethylammonium (TMA), ethyltrimethylammonium (ETMA), phenyltrimethylammonium (PTMA) and methyltriethylammonium (MTEA). Except for TMA and CCh, these compounds are all weak agonists of the nicotinic receptor. The single-channel activity elicited by PTMA and MTEA resembled that in the presence of TEA where only isolated openings were observed and a value for could not be estimated. Only in the presence of 1-5 mM ETMA were single-channel clusters observed. From these data, a lower limit of 280 s-1 for the channel-opening rate constant could be established for ETMA. Previous work has provided an estimate for for channels activated by choline (50 s-1 ; Zhou et al. 1999). Figure 7 shows sample currents from wild-type receptors activated by the various quaternary ammonium compounds.
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Figure 7. Single-channel currents from various receptor-ligand combinations in the presence of low and high agonist concentrations Low agonist concentrations were: 20 µM (wild type-CCh) or 100 µM (all remaining combinations). High agonist concentrations were: 1 mM (wild type-PTMA), 2 mM (wild type-MTEA), 5 mM (wild type-ETMA, wild type-choline), 10 mM (wild type-CCh, | ||
For CCh and TMA, currents were recorded from wild-type and mutant (CCh-Y93F, TMA-Y198F) receptors. The reason for using the mutant receptors was the following: the channel-opening rate constants for the wild-type receptor in the presence of these two agonists are relatively high. The opening rate constant for CCh is 7630 s-1 (see above), and for TMA 6100 s-1 (Akk & Auerbach, 1996; G. Akk, unpublished observations). This leads to the emergence of high-open-probability clusters, where the determination of burst durations can be complicated due to the short duration of the activation gap and the reduced amplitude and increased noise of the burst events. Hence, we compared the data obtained from the wild-type receptors in the presence of CCh and TMA with studies on two mutated receptors. The major consequence of these two mutations is a reduction in the channel-opening rate constant (Akk et al. 1999; Akk & Steinbach, 2000). This results in channel activity consisting of isolated channel openings where the durations of openings of bursts are relatively easy to measure.
The single-channel amplitudes were reduced in the presence of high concentrations of agonist for all compounds. An increase in the burst duration, as predicted by the linear blocking mechanism, was observed for CCh, TMA and PTMA. In the presence of choline, ETMA, MTEA and TEA, the burst durations were actually reduced when block was evident. The results are summarized in Fig. 8. For easier comparison, the data have been normalized according to the predictions of the linear blocking model. The mean burst duration and the mean current were determined at a low and a high concentration of the compound. In the figure, the relative change in inverse burst duration between the two concentrations is normalized to the relative change in current amplitude. For a linear blocking model the inverse burst duration should decrease linearly with the reduction in conductance, so the normalized data should give a value of 1. This corresponds to a situation in which a blocked channel does not close. A value of zero for the ratio would suggest that blocked channels close at the same rate as unblocked open channels, while a value below zero suggests that blocked channels close more rapidly than unblocked open channels.
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Figure 8. Reduction in channel amplitudes leads to an increase in channel burst durations for some but not all ligands The data shown are the relative change in inverse burst duration divided by the relative change in single-channel current. For a simple linear blocking model with rapid block kinetics, the apparent burst duration increases as the single-channel current is reduced and the ratio is one. The mean burst duration and mean single-channel current were estimated at two concentrations of agonist (termed low and high, see legend to Fig. 7), and the ratio of changes computed. The ratio was computed as: zlow - zhigh)/(zlow)/ilow - ihigh)/(ilow) Some data were obtained with receptors containing mutated subunits (CCh*: | ||
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Interactions of TEA with the nicotinic ligand binding site - activation and inhibition
We have examined the activation and blocking properties of TEA using the single-channel patch-clamp technique. Our data show that TEA is a weak agonist of the mouse adult muscle-type nicotinic receptor. While channel openings elicited by TEA were observed at concentrations as low as 20 µM, no single-channel clusters were evident at concentrations as high as 5 mM. Full kinetic analysis, as defined by determination of kinetic rate constants according to model 1, was not carried out for receptors activated by TEA.
In principle, the lack of clusters can be caused by a weakened binding reaction of TEA to the agonist recognition site (reduced affinity) or by an impaired coupling of the agonist binding site to the channel gate (reduced efficacy). Indirect evidence suggests that the channel-opening rate constant is extremely low for TEA-activated receptors. The activation of the S269I mutant receptor in the presence of 5 mM TEA did not lead to emergence of clusters. This mutation results in a 60-fold increase in gating efficacy for choline, and possibly for other agonists including TEA. The mean closed duration for the S269I receptor in the presence of 2000 µM TEA was 3.3 ms, which corresponds to an effective opening rate (') of 300 s-1. Given our estimate for the affinity of TEA for the binding site, the opening rate is similar to this value. Assuming that the mutation leads to a 30-fold increase in the channel-opening rate constant, the upper limit for in the wild-type receptor can be set at 10 s-1. However, the true must be even lower, as the ' value in the mutant receptor should be adjusted according to the number of active receptors in the patch.
The channel-opening rate constant in the presence of tetramethylammonium (TMA) is about 6100 s-1 (see above). Hence, the data show that a replacement of the methyl groups in TMA with ethyl side-chains leads to a significant (at least 3.8 kcal mol-1 (15.9 kJ mol-1)) increase in gating free energy.
On the other hand, it appears that the ligand binding reaction is less affected when TEA instead of CCh or TMA is used. Our estimate of the affinity of TEA is based on competition with a strong agonist (CCh) for activation of the receptor. The estimate is indirect, but our interpretation of the data is supported by two major observations. First, the effect of TEA on the effective opening rate seen in clusters is indistinguishable at -50 mV and 50 mV membrane potentials. This clearly differentiates this interaction from voltage-dependent channel block. Second, the analysis of interactions between TEA and CCh allows us to predict the effects of TEA on the effective opening rate over a range of ACh concentrations. The inferred KD for TEA is about 1.5 mM. For comparison, the KD for CCh is 0.6 mM while that for TMA is 3.1 mM.
There is evidence suggesting that the nicotinic receptor agonist binding site is hydrophobic by its nature or that the entrance leading into the docking site is lined with hydrophobic residues (Karlin, 2002). This would suggest that the apparent affinity of a ligand to the resting receptor binding site would be enhanced as the hydrophobicity of the ligand increases. Previous studies, in which the ability of monovalent inorganic cations to competitively inhibit the binding of ACh was studied, also showed that the affinity of the ligand increased with increasing hydrophobicity (Akk & Auerbach, 1996). Combining these data with the results from the present experiments gives us the following sequence of equilibrium dissociation constants: Na+ > K+ > Cs+ > TMA+ > TEA+ > CCh+. Such ranking is characteristic of a weak field-strength site where interaction between the site and the ion is determined mainly by the dehydration energy of the latter (Eisenman, 1962).
The coupling of the occupation of the ligand binding site to the movement of the channel gate seems to be more complex and to require specific interactions between the ligand and the binding site. Apparently, TEA, in contrast to TMA, is unable to make contact with the correct residues in the binding pocket that are required for efficient gating. The channel-opening rate constant follows the following rank order: ACh > CCh = TMA > ETMA > choline > (TEA, MTEA, PTMA). The available data do not allow us to distinguish among TEA, MTEA and PTMA.
Interactions of TEA with the nicotinic receptor channel domain - reduction of single channel current
The data presented in this manuscript demonstrate that TEA, at millimolar concentrations, inhibits the nicotinic receptor function through interactions with the ion channel domain of the nicotinic receptor. The effect is mediated by a reduction in the amplitude of single-channel currents. The voltage dependence of the blocking equilibrium suggests that the monovalent TEA molecule senses about 70 % of the membrane field at the blocking site, a value similar to that for ACh, CCh and suberyldicholine (Sine & Steinbach, 1984; Ogden & Colquhoun, 1985).
Adler et al. (1979b) studied the effect of TEA on neurally or iontophoretically evoked endplate currents. They concluded that their data did not show any significant effects of an interaction between TEA and the agonist binding site. However, they proposed a kinetic scheme where TEA can bind to a site on receptors with either closed or open channels, and have distinct consequences depending on whether the binding occurred to the closed or open channel state. According to the model, binding of TEA to a site on closed receptors, followed by subsequent binding of ACh to the agonist binding site, led to the emergence of ion channels with shorter open durations. On the other hand, binding of TEA to a site on the open channel resulted in a reduced conductance state of the channel. The model postulated that if binding occurred to a closed-channel state, then the binding to the (altered) open channel could not occur.
Our results agree with some of the features of the model proposed by Adler et al. (1979b) while disagreeing with others. For example, we observed a decrease in the single-channel amplitudes which became more marked at higher TEA concentration. The relatively high doses (mM) of TEA required to observe this effect suggest that it is similar to the much-described open-channel block by nicotinic ligands (Sine & Steinbach, 1984; Ogden & Colquhoun, 1985). Adler et al. (1979b) made similar observations, although in their observations the voltage dependence of block was less (suggesting that TEA sensed only 25 % of the membrane potential at the binding site).
Effect of TEA on the channel-closing rate
In contrast to Adler et al. (1979a), we did not observe a significant decrease in burst durations when low (micromolar) concentrations of TEA were coapplied with a strong agonist (ACh or CCh). This argues against the idea that the binding of TEA to a receptor with a closed channel results in the activation, by a strong agonist, of channels which have a shorter open duration. Since it could be argued that in the presence of 1 mM CCh, the receptor only rarely visits the unliganded state to which TEA can bind according to Adler's model (Adler et al. 1979a), we also studied the effect of 500 µM TEA on channel openings elicited by 10 µM ACh. This concentration of ACh corresponds to < EC1 in the effective opening rate curve (Akk & Auerbach, 1996). Our results show that the burst durations in the absence and presence of 500 µM TEA are almost indistinguishable (1.1 ± 0.1 ms, control conditions vs. 1.2 ± 0.1 ms, in the presence of TEA).
A number of blocking mechanisms have been proposed for the nicotinic receptor (Neher & Steinbach, 1978; Neher, 1983; Maconochie & Steinbach, 1995). The simplest of them involves binding of the blocker molecule to the open channel leading to a new state where the activation gate is in the open configuration, while current flow is reduced because of plugging of the open channel. Depending on the lifetime of the blocked state, blocking events may be resolved as interruptions between openings of normal amplitude or only as a reduction in amplitude associated with an increase in open-channel noise. In a strict linear block model (model 3), the activation gate cannot close when the channel is blocked. In this case, the total burst duration increases so that, on average, the total time spent in the unblocked open state stays constant. This mechanism has been used previously to describe nicotinic channel block by ACh, CCh and suberyldicholine (Sine & Steinbach, 1984; Ogden & Colquhoun, 1985), local anaesthetics (Neher & Steinbach, 1978; Ogden et al. 1981), epibatidine (Prince & Sine, 1998) and albuterol (Milone & Engel, 1996). Such linear open-channel block is, however, inadequate in describing our data as we saw no increase in burst durations. In fact, the burst durations got shorter at higher TEA concentration.
A model, similar to the one described above, has been discussed where the blocked receptor can close, with or without trapping the blocker molecule in its site (Neher, 1983; Gurney & Rang, 1984; Ogden & Colquhoun, 1985; Neely & Lingle, 1986). This model with a very fast blocking and unblocking reaction would adequately describe our data. If the blocked receptor can close with a rate somewhat higher than that of the unblocked open receptor, then the observed decrease in burst durations is predicted. Previous studies have reported results which indicate that other blocking agents have effects which are not consistent with a strict linear blocking model. Even in the case of the local anaesthetic QX-222, the burst duration does not increase as much as expected at either high [QX-222] or more negative membrane potentials (Neher, 1983). Isoflurane blocks nicotinic receptors via a mechanism where the blocker can bind to both open and closed channels, allowing the open but blocked channels to close (Dilger et al. 1992). Similar results have been obtained for other volatile anaesthetics such as enflurane, halothane and methoxyflurane (Wachtel, 1995), with ephedrine and pseudoephedrine (Milone & Engel, 1996) and with barbiturates (Dilger et al. 1997). While the binding of one blocker molecule is sufficient to cause block, the data for some of these compounds (barbiturates and isoflurane) suggest that more than one blocker molecule can bind to the site(s) as indicated by a concentration-dependent increase in the lifetime of the blocked state.
It is assumed that the binding sites for most nicotinic receptor blockers are located within the pore domain of the channel. These assumptions are supported by several lines of evidence. First, mutations to the pore-lining residues located near the central kink of the M2 transmembrane domain influence block by local anaesthetic (QX-222) and isoflurane (Charnet et al. 1990; Wenningmann et al. 2001). Second, the voltage dependence of the blocking reaction suggests that the blocker moves through the electric field. Finally, in the case of block by external ACh, very negative membrane potentials produce a relief from block, as though the ACh could be forced to exit to the interior side of the membrane (Sine & Steinbach, 1984). It should, however, be mentioned that for at least one nicotinic channel blocker, the voltage sensitivity may not be the result of interactions of the blocker with the electric field. Physostigmine, an acetylcholinesterase inhibitor, is mostly uncharged at physiological pH while its ability to block the nicotinic receptor currents is enhanced at hyperpolarized potentials (Wachtel, 1993). A possible explanation is that the local pH in the channel may differ from that in the bulk solution.
In the present study, we have examined a series of structurally related compounds in terms of deviations from the predictions of simple, linear block. Replacement of a single methyl group by an ethyl group (TMA to ETMA) greatly increases the deviation from linear block, suggesting that an increase in hydrophobic interactions with the channel might facilitate closing of blocked channels. We found that replacement of a methyl group with a phenyl group does not have the same effect as the ethyl replacement. A possible interpretation is that bulkier groups (e.g. carbamyl-, acetyl- or phenyl-) themselves interfere with closing. However, it is not clear how to accommodate the observation that choline (ETMA with a hydroxyl group added to the ethyl moiety) is more effective at promoting closure of the blocked channel than either TMA or PTMA. It has been reported previously that substitutions of the permeant alkali cations Li+, Cs+, K+ and Na+ can affect the channel-closing rate measured from endplate current decays or single-channel records (Gage & Van Helden, 1979; Akk & Auerbach, 1996). In general, more hydrophobic cations increase the closing rate.
In addition to such fast block, TEA may participate in another blocking mechanism of intermediate duration. We observed an increase in the number of resolved brief-duration closures as the concentration of TEA was increased at more negative membrane potentials (data not shown). Such short-lived gaps were resolvable in data idealized at 5 kHz but largely missed at 2 kHz. There is some precedence to these findings. Neher (1983) found that in the presence of high concentrations (> 40 µM) of QX-222, two kinetically distinct blocked states were present in the bursts. We have not pursued this observation further.
Block by TEA involves three actions on the muscle nicotinic receptor
In summary, our data indicate that block by TEA has three independent mechanisms. TEA binds to the agonist binding site with moderate affinity (1.5 mM) but gates the channel with very low efficacy (/ < 0.005). Hence, TEA acts as a competitive inhibitor. TEA also reduces single-channel current by a voltage-dependent mechanism, with a KD of about 12 mM at zero membrane potential which decreases e-fold for a 35 mV decrease in membrane voltage. Finally, a channel blocked by TEA closes at least as rapidly as an unblocked open channel, reducing charge transfer per activation event. These properties of TEA are apparently determined by independent interactions with the receptor protein.
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Acknowledgements
We thank Beth Mattingly for help with tissue culture. This material is based upon work supported by the National Science Foundation under Grant No. 0110282 (G.A.) and NIH NS-22356 (J.H.S.).
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). The small filled circles, connected by a line, show the values obtained when single-channel records were simulated using model 2 with the following values for CCh: k+1 = 44 µM-1 s-1, k-1 = 26000 s-1, 
). The lines and small symbols show values obtained from simulated data generated using model 2 with the same parameters for TEA and the following values for ACh: k+1 = 110 µM-1 s-1, k-1 = 18 000 s-1, 
), and for the 
). The currents were recorded at -50 mV. Increased concentrations of TEA result in a reduction of the single-channel current. The results of the fit are given in the text. B, I-V plots from the