Trapping Channel Block of NMDA-Activated Responses By Amantadine and Memantine
Thomas A. Blanpied1,
Faye A. Boeckman2,
Elias Aizenman2, and
Jon W. Johnson1
1 Department of Neuroscience, University of Pittsburgh, 15260 and 2 Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
Blanpied, Thomas A., Faye Boeckman, Elias Aizenman, and Jon W. Johnson. Trapping channel block of NMDA-activated responses by amantadine and memantine. J. Neurophysiol. 77: 309-323, 1997. We investigated the mechanisms by which the antiparkinsonian and neuroprotective agents amantadine and memantine inhibit responses to N-methyl-D-aspartic acid (NMDA). Whole cell recordings were performed using cultured rat cortical neurons or Chinese hamster ovary (CHO) cells expressing NMDA receptors. Both amantadine and memantine blocked NMDA-activated channels by binding to a site at which they could be trapped after channel closure and agonist unbinding. For neuronal receptors, the IC50s of amantadine and memantine at
67 mV were 39 and 1.4 µM, respectively. When memantine and agonists were washed off after steady-state block, one-sixth of the blocked channels released rather than trapped the blocker; memantine exhibited "partial trapping." Thus memantine appears to have a lesser tendency to be trapped than do phencyclidine or (5R,10S)-(+)-5m e t h y l - 1 0 , 1 1 - d i h y d r o - 5 H - d i b e n z o [ 1 , d ] c y c l i h e p t e n - 5 , 1 0 - i m i n e(MK-801). We next investigated mechanisms that might underlie partial trapping. Memantine blocked and could be trapped by recombinant NMDA receptors composed of NR1 and either NR2A or NR2B subunits. In these receptors, as in the native receptors, the drug was released from one-sixth of blocked channels rather than being trapped in all of them. The partial trapping we observed therefore was not due to variability in the action of memantine on a heterogeneous population of NMDA receptors in cultured cortical neurons. Amantadine and memantine each noncompetitively inhibited NMDA-activated responses by binding at a second site with roughly 100-fold lower affinity, but this form of inhibition had little effect on the extent to which memantine was trapped. A simple kinetic model of blocker action was used to demonstrate that partial trapping can result if the presence of memantine in the channel affects the gating transitions or agonist affinity of the NMDA receptor. Partial trapping guarantees that during synaptic communication in the presence of blocker, some channels will release the blocker between synaptic responses. The extent to which amantadine and memantine become trapped after channel block thus may influence their therapeutic effects and their modulation of NMDA-receptor-mediated excitatory postsynaptic potentials.
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INTRODUCTION |
A large number of ions and drugs can block the channel of the glutamate receptor activated by N-methyl-D-aspartate (NMDA), and the effects of these blockers are diverse. Consider, for instance, the effects of Mg2+, the dissociative anesthetics phencyclidine (PCP) and ketamine, and 1-amino-3,5-dimethyladamantane (memantine). The block of the NMDA-activated channel by extracellular Mg2+ is critical to the normal functioning of the NMDA receptor in synaptic transmission and plasticity. However, PCP and ketamine, which also block this channel (MacDonald et al. 1991
), have severe and deleterious behavioral effects in humans (Krystal et al. 1994
; Luby et al. 1959
), probably due to their interaction with the NMDA receptor (Javitt and Zukin 1991
). Finally, memantine as well blocks the NMDA-activated channel (Bormann 1989
; Chen et al. 1992
), but is currently used in the treatment of Parkinson's disease (Fischer et al. 1977
), dementia (Ditzler 1991
), and several movement-related disorders (e.g., Weller and Kornhuber 1991
). During therapeutic use, the memantine concentrations found in cerebrospinal fluid suggest that its primary site of action is the NMDA receptor (Kornhuber and Quack 1995
), and yet it appears to induce fewer and less profound effects on perception or consciousness (Ditzler 1991
) than PCP or ketamine. The reasons for the surprisingly diverse behavioral effects of blockers of the NMDA-activated channel are not known. It is plausible that this variation arises in part from a diversity of mechanisms by which the channel may be blocked.
To explore in detail the range of mechanisms that may be exhibited by blockers of the NMDA-activated channel, we have examined the actions on NMDA responses of memantine and the closely related drug 1-amino-adamantane (amantadine). Amantadine is an NMDA antagonist used for many of the same therapeutic purposes as memantine (Brenner et al. 1989
; Kornhuber et al. 1993
; Schwab et al. 1969
). It inhibits [3H](5R,10S)-(+)-5-methyl-10,11-dihyd r o - 5 H - d i b e n z o - c y c l i h e p t e n - 5 , 1 0 - i m i n e ( [ 3 H ] M K - 8 0 1 )binding to human brain membrane homogenates (Kornhuber et al. 1991
) and reduces responses mediated by NMDA receptors with an apparent inhibition constant greater than memantine's (Parsons et al. 1996
).
We report here that channel closure and agonist dissociation is permitted while either of these drugs is bound in the NMDA-activated channel, resulting in trapping of the drug in the channel. This characteristic of the action of memantine and amantadine, which will be termed "trapping channel block" here, distinguishes them from "sequential blockers," which prevent the channel from closing while blocked (Adams 1976
; Antonov and Johnson 1996
; Neher and Steinbach 1978
). Sequential and trapping channel blockers differ greatly in several ways, including the dependence of their block at equilibrium on the agonist concentration and their inhibition of synaptic charge transfer. Many previously characterized blockers of the NMDA-activated channel act as trapping channel blockers, including MK-801 (Huettner and Bean 1988
), PCP, and ketamine (MacDonald et al. 1987
, 1991
). The molecular mechanisms for the trapping of blockers are at present unclear. The conformational changes involved in gating could sterically prevent movement of a trapping channel blocker out of the channel. On the other hand, the binding of a sequential blocker might prevent movement of the channel's gate (Antonov and Johnson 1996
). It seems likely that some drugs will exhibit a combination of these effects.
PCP and MK-801 appear to be trapped in virtually all blocked channels (Huettner and Bean 1988
; Jahr 1992
; Lerma et al. 1991
; MacDonald et al. 1991
); whether this is true of ketamine has not yet been determined. In experiments in which agonist and antagonist are simultaneously removed from the extracellular solution, we tested whether memantine becomes trapped in all channels that it blocks. We observed that memantine (and possibly amantadine) in fact was released from many of the blocked channels. This phenomenon of "partial trapping" may help to discriminate classes of drugs that otherwise have in common the mechanism of trapping channel block. In addition, partial trapping may significantly influence the effects of a blocker on synaptic transmission, because it guarantees that some channels will release the blocker between synaptic inputs.
Portions of these results have been presented elsewhere in preliminary form (Blanpied and Johnson 1994
).
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METHODS |
Preparations and solutions
Primary neuronal cultures were prepared as described previously (Antonov et al. 1995
) from enzymatically dissociated cortices of 16-day-old embryos of Sprague-Dawley rats. Cells were used for experiments after 12-40 days in culture.
The cDNAs for NMDA receptors subunits NR1, NR2A, and NR2B were subcloned into expression vectors for eukaryotic expression. NR2A and NR2B subunits were chosen for study because the mRNA for these subunits is expressed heavily both in neocortex in vivo (Watanabe et al. 1992
) and in primary cultures of cortical neurons (Zhong et al. 1994
). NR1-1a (nomenclature of Hollman and Heinemann 1994) and NR2A were subcloned previously (Boeckman and Aizenman 1994
, 1995
) into pRc/CMV (Invitrogen). The expression vector containing NR2B was created by ligating the 5.4-kb EcoRI fragment of pNR2B (Monyer et al. 1992
) into a partial EcoRI digest of pRc/CMV. Green fluorescent protein (GFP) (Chalfie et al. 1994
) expression was used as a marker of positive transfection in experiments for whole cell patch clamping. The 750-bp BssHII-EcoRI fragment from TU#65 (Chalfie et al. 1994
) was subcloned into the MluI-NotI sites of the expression vector pCI (Promega) to generate pCIGFP. NMDA-receptor subunit constructs were transiently transfected into CHO-K1 (ATCC CCL61) cells by lipid-mediated gene transfer. Cells were seeded 23-25 h before transfection in medium containing Ham's F-12 with 1 mM glutamine and 10% fetal bovine serum (CHO medium) at 3 × 105 cells per 35-mm well. Transfections were performed with 1.3 µg of total DNA to 6 µl of lipofectamine (GIBCO-BRL) in 1 ml of serum-free CHO medium per well for 4-5 h. Cells were transfected with a 1:4.3 ratio of marker plasmid (pCIGFP) to total NMDA subunit DNA; NMDA subunits were transfected at a 1:3 ratio of NR1 to NR2 (Cik et al. 1993
). Cells then were refed with serum-containing medium. Twenty-four hours posttransfection the cells were trypsinized and replated at a 1:2 dilution onto 12-mm glass coverslips in 1 mM 5,7-dichlorokynurenic acid (RBI). CHO-K1 cells were grown at 37°C in an atmosphere of 5% CO2-95% air and maintained in CHO medium.
During experiments, cells were bathed in a control solution, which contained (in mM) 140 NaCl, 2.8 KCl, 1.0 CaCl2, and 10N-(2-hydroxyethyl)piperazine-N
-[2-ethanesulfonic acid] (HEPES).pH was adjusted to 7.2 with NaOH. For all experiments, the solution also contained 0.2 µM tetrodotoxin, and in some experiments, 0.5 µM strychnine was added. The pipette solution contained (in mM) 120 CsF, 10 CsCl, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid (BAPTA), and 10 HEPES. pH was adjusted to 7.2 with CsOH. Concentrated drug stock solutions were prepared and kept frozen until use. Memantine kindly was provided by Merz (Frankfurt, Germany), 7-chlorokynurenic acid was purchased from RBI (Natick, MA), and all other chemicals were from Sigma (St. Louis, MO). For all experiments reported here, NMDA was applied at a concentration of 5 µM and was coapplied with 10 µM glycine; in descriptions of the experimental protocols, this combination of agonists is referred to as NMDA.
Whole cell recordings
Patch-clamp recordings from neurons or CHO cells were carried out at room temperature (20-25°C) on the stage of an inverted microscope (Zeiss Axiovert 10) equipped with Hoffman Modulation Contrast optics (Modulation Optics, Greenvale, NY). Pipettes were pulled from borosilicate thin-walled glass with filaments (Clark Electromedical, Reading, UK) and had resistances of 2-5 M
. Access resistance during experiments was typically 10-15 M
and was 80% compensated in many experiments. Currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 10 kHz, digitized at 44 kHz with a Neuro-Corder (Neuro Data Instruments, New York) and stored on video tape for later analysis. The liquid junction potential between the pipette and external solutions was measured to be near
7 mV, and this value has been subtracted from all holding potentials.
Drugs were applied through a five-barrel perfusion system, the outflow tubes of which were formed from square capillary tubing (0.7 mm OD, 0.5 mm ID; Longreach Scientific Resources, Orr's Island, ME). The tubes were connected to reservoirs that could be raised or lowered to adjust the speed of the gravity-fed solution flow over the cell. Solenoid valves (Neptune Research, W. Cadwell, NJ) below each reservoir were used to open or close the flow through each tubing line. After the formation of a gigaohm seal, a barrel perfusing control solution was positioned ~0.2 mm from the cell. To change the solution superfusing the cell, the array of tubes was moved laterally to position a different tube over the cell. The array of tubes was mounted on a linear ball slide assembly (DelTron Precision, Bethel, CT) and moved by a D.C. linear motor (Northern Magnetics, Van Nuys, CA) connected to the rail of the slide assembly. The current to the motor was supplied by an analog circuit that resembled a voltage-clamp circuit, designed to maintain the tubes at a command position. The circuit supplied a current proportional to the difference between the output voltage of a LD100 displacement transducer (Omega Engineering, Stamford, CT) that measured tube position and a position command voltage output by a DA700 digital-to-analog converter (Real Time Devices, State College, PA). The output of the digital-to-analog converter was under the control of a QuickBasic program running on an AT-compatible computer. Movements taking <5 ms could be made, but generally movement times of ~30 ms were used to minimize vibrations.
During solution exchanges, the solution flow rate was ~2.5 ml/min (~16 cm/s at the barrel exit) and between exchanges it was reduced to ~1.2 ml/min. We estimated a lower limit for the rate of solution exchange over the cell by measuring the current relaxation time constant following a step from 5 or 30 µM NMDA + 10 µM glycine to solution without agonists. The relaxation had an average time constant of ~80 ms. Solution exchange must take place more rapidly than this because the unbinding of NMDA time constant ~50 ms (Benveniste et al. 1990
; Lester and Jahr 1992
)] also contributes to the relaxation time course. Further analysis of the rate of wash-off of higher concentrations of antagonists is given in RESULTS (see Fig. 5C) and suggests that 98-99% of a solution can be washed off within 120 ms.

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| FIG. 5.
Noncompetitive inhibition of NMDA-activated responses by memantine and amantadine. A: 2 responses from a cell to which either 80 µM APV alone (left) or 1 mM amantadine + APV (right) was applied in absence of agonists. APV or APV + amantadine were washed off, and agonists then were reapplied after a 250-ms wash in control solution. Response preceded by APV + amantadine was transiently reduced. B: response from a cell held at 67 mV to which 50 µM memantine was applied in absence of agonists and in presence of APV. After memantine application, there was a 1-s wash in control solution, and then agonists were reapplied; resulting response was transiently reduced. Same cell as in Fig. 4. C: solution exchange rate was stringently tested by washing off 1.4 mM Mg2+ while holding the cell at 97 mV. At this Vm, IC50 of Mg2+ for whole cell responses is <7 µM (Jahr and Stevens 1990 ; Mayer and Westbrook 1987 ). Thick line shows the control response to NMDA 10 s after wash-off of Mg2+, dotted line shows response 1 s after wash-off of Mg2+, and thin line shows response 20 ms after wash-off of Mg2+. Each trace shows average of 5 responses; 3 protocols were delivered in an intermixed order. Twenty-millisecond wash reveals that although onset of the current was delayed while [Mg2+] fell to below saturating levels, current recovered to ~80% of control within ~100 ms. Thus within this time, [Mg2+] must have fallen to well below 7 µM, a 200-fold dilution. However, complete wash-off followed a biphasic time course, and remaining Mg2+ appeared to take ~1 s to wash away. Results consistent with these were also obtained washing off 2 mM DL-APV or 3.5 mM Mg2+. D: concentration-inhibition relation for memantine applied in absence of agonists at 67 mV. Memantine was applied for 20 s, and in nearly all cases, was applied with APV. Initial response to application of NMDA 1 s later was measured. Each point shows mean of data from 7 to 10 different cells. Solid line shows best fit of Eq. 1 to data pooled from all cells (IC50 = 179 µM; nH = 0.7).
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Analysis
Data were played back through an eight-pole Butterworth filter (Model 901, Frequency Devices, Haverhill, MA) and digitized using pClamp software and a Digidata 1200 analog-to-digital converter (Axon Instruments). Low-pass filter frequencies were chosen based on the fastest primary component of the response under analysis and typically were 50-200 Hz. If the NMDA response after applications of blocker did not recover to
80% of control, cells were not considered for further analysis except for measurement of the time course of recovery from block by memantine.
Concentration-response relationships were analyzed using the Origin plotting and analysis program (Microcal Software, Northampton, MA) by fitting the data with the equation
|
(1)
|
where [B] is the blocker concentration, IB is the steady-state response in the presence of blocker, INMDA is the steady-state response to agonists alone, and nH represents the Hill coefficient or slope factor. If recovery from block was not complete, then INMDA was calculated as the average of the response to agonists alone and the maximal response achieved after recovery. The values of nH and IC50 that yielded the best fit were determined using the Marquardt-Levenberg least squares method to minimize the
2 value of the fit. For trapping channel block by amantadine (e.g., Fig. 1), five to seven concentrations of amantadine were tested in each cell, and the IC50 in each cell was determined individually. For noncompetitive inhibition by memantine (e.g., Fig. 5), the data from all cells were pooled and fit simultaneously. Current relaxations during block or unblock were fit with single or double exponentials of the form I = A1e(
t/
1) + A2e(
t/
2) + C, where An and
a are the amplitudes and time constants of the exponential components. Fitting was performed either in Origin as described above or in Clampfit (pClamp, Axon Instruments) using the Simplex method to minimize the sum of the squared errors of the fit.

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| FIG. 1.
Concentration dependence of block by amantadine of N-methyl-D-aspartate (NMDA)-activated responses. A: currents evoked from a single cell by NMDA were reversibly blocked by amantadine coapplied at indicated concentrations. Lines above traces indicate times of drug application. B: concentration-inhibition relation for amantadine steady-state block in 6 cells to which 5 concentrations of amantadine were applied. Solid line shows best fit of Eq. 1 (IC50 = 38.1 µM; nH = 0.98) to data pooled from all cells. Mean IC50 calculated from fits to data from each cell individually was 38.9 ± 4.6 µM, with nH = 0.99 ± 0.02. Holding potential for all cells in A and B was 67 mV.
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The voltage dependence of inhibition was determined using the following equation
|
(2)
|
where IB and INMDA were measured at Vm, K0 is the apparent dissociation constant of the antagonist at Vm = 0 mV, and V0 is the change in Vm that results in an e-fold change in the apparent dissociation constant of the antagonist. Estimation of the voltage dependence of IB/INMDA was preferred to estimation of the voltage dependence of IB directly, because the INMDA-V relation was outwardly rectifying in 60% of the cells used for these experiments. The rectification could cause an overestimation of the degree of voltage dependence if V0 were calculated by fitting the IB-V relation assuming that the INMDA-V relation is linear.
In some experiments, the initial amplitude rather than the steady-state amplitude of test responses to NMDA was measured. To do this, the test response and the response to a preceding control application were both measured at a set time after the application of agonists. This isochronic point was at the time it took the control response to reach its peak amplitude, usually 100-150 ms after the time of the NMDA application.
The models presented in the Results were numerically evaluated using the program SCoP (Simulation Resources, Berrien Springs, MI). To optimize the fit of a model to the data, parameters were adjusted in SCoP using a principal axis method to minimize a least-squares error function. All data are presented as means ± SE, and comparisons were made using two-tailed Student's t-tests except as noted.
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RESULTS |
Voltage-dependent inhibition of NMDA-activated responses by amantadine and memantine
Responses to 5 µM NMDA + 10 µM glycine (hereafter referred to as NMDA) were reduced rapidly and reversibly by amantadine when it was coapplied at concentrations of 3-1,000 µM (Fig. 1A). Inhibition typically progressed in two phases: one fast phase (
~30 ms) that accounted for the majority (~85%) of the total inhibition and a second, slower phase that reached equilibrium within 10-20 s. To measure the equilibrium concentration-inhibition relation of amantadine, we applied the drug for
20 s in the constant presence of agonists. The IC50 of amantadine measured individually in six cells held at
67 mV was found to be38.9 ± 4.6 µM, with nH = 0.99 ± 0.02 (Fig. 1B).
The fractional inhibition produced by amantadine decreased at more positive Vms (Fig. 2, A-C), as would be expected of a postively charged channel-blocking molecule. Current-voltage relations comparing the equilibrium response evoked by agonists alone and the equilibrium response after the addition of amantadine are shown in Fig. 2B. Figure 2C shows these data transformed to a plot of fractional response, and the fit of Eq. 2 to these points. From fits to the fractional response remaining in the presence of 100 µM amantadine in four cells tested at 6-9 Vms, we calculated a mean K0 of 261 ± 10 µM and a mean V0 of 35.4 ± 1.8 mV.

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| FIG. 2.
Voltage dependence of block by amantadine and memantine. A: currents recorded from 1 cell held at indicated voltages while NMDA and 100 µM amantadine were applied for indicated periods. B: current-voltage relation for block by 100 µM amantadine in a second cell. Filled circle, steady-state responses to agonists alone; open circle, steady-state responses during addition of amantadine. C: data from cell used for B replotted as fractional response in the presence of amantadine. Solid line shows best fit of Eq. 2 (K0 = 252.2 µM, and V0 = 39.7 mV). D: current-voltage relation for block by 5 µM memantine in a third cell. Filled circle, steady-state responses to agonists alone; open circle, equilibrium responses after addition of memantine. E: data from cell used for D replotted as fractional response. Solid line shows best fit of Eq. 2 (K0 = 8.4 µM; V0 = 31.5 mV).
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The voltage dependence of inhibition by memantine is shown for one cell in Fig. 2, D and E. From fits to the data from four cells to which 5 µM memantine was applied at 5-8 Vms, we calculated a mean K0 of 14.2 ± 2.9 µM and a mean V0 of 29.3 ± 1.0 mV. The value of V0 for memantine was similar to but slightly greater than that of amantadine (P < 0.02). Given these values of K0 and V0, we calculated that the expected IC50 of memantine at
67 mV is 1.4 µM. This degree of voltage dependence and the IC50s of each drug are similar to that reported previously (Chen et al. 1992
; Frankiewicz et al. 1996
; Parsons et al. 1996
).
The depth of the blocking sites within the membrane electric field may be estimated from the values of V0 using the Woodhull model (Woodhull 1973
). Although the assumptions required for application of this model may be inaccurate for the NMDA receptor, estimation of electrical depth is useful for comparisons among blockers. According to this model, these antagonists sense a substantial fraction of the field, 72% for amantadine and 87% for memantine, at their blocking site. The similarity in the degree of voltage dependence is consistent with the hypothesis that the two drugs bind to overlapping or identical binding sites within the NMDA-activated channel.
Trapping of amantadine and memantine in theNMDA-activated channel
To explore further the nature of channel block by these drugs, we tested whether they blocked according to a trapping open-channel block scheme (Lingle 1983
)
where R is the receptor, A is an agonist molecule, AR and A2R are the receptor with the channel closed and one or two NMDA molecules bound, respectively, A2R* is the receptor with all agonists bound and the channel open, B is the channel blocking molecule, A2R*B is the receptor with the channel open and blocked by the drug molecule, A2RB is the receptor with all agonists and the blocker still bound but the channel closed, and RB is the receptor with the blocker still bound, but with the channel closed and agonists dissociated. The symbols we have used to indicate the rate constants of transitions between these states are shown. A similar model has been applied to explain block of NMDA-activated channels by ketamine, PCP, and MK-801 (Huettner and Bean 1988
; MacDonald et al. 1991
). The two agonist binding steps shown in this model both represent NMDA binding (Benveniste and Mayer 1991
; Clements and Westbrook 1994
; Lester and Jahr 1992
); glycine binding sites have been omitted from the model because all experiments were performed in a saturating concentration of glycine.
According to Model 1, if a channel becomes blocked and progresses to the trapped state RB, then it can become unblocked only after agonist rebinding. An alternative model of block is the "sequential" model (Adams 1976
; Antonov and Johnson 1996
; Neher and Steinbach 1978
), in which closure of the blocked channel is not permitted and in which the unbinding of the drug therefore proceeds independently of agonist binding. We tested whether amantadine and memantine are trapping channel blockers by determining if recovery from block depended upon the presence of agonists.
We first examined the kinetics of recovery after removal of 300 µM amantadine applied for
10 s in six cells held at
67 mV. These current relaxations were always better fit by two than by one exponential, and the two time constants (and relative amplitudes) were 0.17 ± 0.04 s (56 ± 8%) and 3.0 ± 0.2 s (44 ± 8%). Given these time constants, if amantadine block follows the sequential scheme, then after the wash-off of agonists, virtually all channels should be unblocked within 15 s. However, using the protocol shown in Fig. 3, we were able to demonstrate that a fraction of the drug molecules remained associated with the receptor for a much longer period of time. After a 20-s application, which produced steady-state inhibition, both agonists and amantadine were washed off with solution containing 80 µM DL-aminophosphonovaleric acid (APV). APV was included during the wash to reduce the possibility of channels opening due to contaminating agonists. The cell was perfused with this solution for 1-10 min, and then, after a brief wash of control solution (0.25-1 s to allow the dissociation of APV) (see Clements and Westbrook 1994
), NMDA was reapplied. In every cell tested, the response to NMDA reapplication was initially ~25% smaller than the control response but then recovered with a time constant of ~3 s (Fig. 3, B and C). In experiments on several cells, APV also was applied for 5-30 s and washed off 0.25-1 s immediately before the first application of NMDA (Fig. 3). APV preapplied alone in this manner never reduced the subsequent response to NMDA. These experiments indicate that after block, some amantadine remained associated with the channels for very long periods of time in the absence of agonists, consistent with the trapping channel block model.

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| FIG. 3.
Trapping of amantadine in NMDA channel after blockade. All traces are from same cell held at 67 mV. A: 87% block was produced by 300 µM amantadine applied for 20 s, and response recovered completely within 15 s. Scale bar applies to A and B. B: trapping was demonstrated by producing block as in A, then washing directly from amantadine + NMDA to solution containing 80 µM DL-aminophosphonovaleric acid (DL-APV) for 1 min. Reapplication of agonists then evoked a response that was reduced but that recovered to control levels within 15 s. Arrowhead and number indicate fractional response amplitude at time after application of agonists that control response had reached its peak. APV also was applied before 1st application of NMDA, and there was a 1-s wash in solution containing no APV before each step to agonists. Thus slow recovery of response to test application of NMDA does not represent unbinding of APV. In other cells, amantadine was shown to remain trapped for 10 min (longest period tested). Solid lines in A and B show output of model described in text using parameter values given in Table 1. C: recovery from blockade from each condition overlaid, showing that kinetics and amplitude of slow component of recovery under the conditions of A and B were very similar. Solid line shows trace from A, and dotted line shows trace from B.
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Trapping of memantine in the closed NMDA-activated channel also could be demonstrated using a similar protocol. In Fig. 4A, blockade of the response to NMDA by 50 µM memantine is shown; because the inhibition and recovery kinetics for memantine were quite slow, this concentration of memantine was chosen to produce rapid and nearly complete block. Figure 4, B-D, shows that, even after 10 min of wash in APV, a substantial proportion of the block by memantine remained, indicating that memantine also can be trapped in the NMDA channel. In addition, this figure demonstrates that virtually no escape from the "trapped" state was observed in the absence of agonists, because the degree of block remaining after 10 min was nearly identical to that remaining after only 1 min.

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| FIG. 4.
Trapping of memantine in the NMDA-activated channel after blockade. All traces are from the same cell held at 67 mV. A: 50 µM memantine produced 98% block that recovered within ~1 min. B-D: trapping was demonstrated using same approach as shown in Fig. 3. APV was washed off for 1 s after 1 min (B), 3 min (C), or 10 min (D), and then agonists were reapplied. Arrowheads and numbers in B-D indicate initial response amplitude to NMDA applied after wash-off of APV. Although this cell was unusual in small degree to which memantine was trapped, little or no unbinding appeared to occur in the absence of agonists. Solid lines in A and B show simulations using Model 1, as described in text and using parameter values given in Table 1. E: recovery from blockade in A and C overlaid. Solid line shows sum of 2 exponentials with s of 2.90 s (37% of total recovery amplitude) and 20.16 s. This sum fits well entire recovery from time of solution exchange, indicating that there was virtually no recovery that occurred with a faster than 2.9 s. Note change in time scale in E.
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Our observation that complete recovery from block is agonist dependent indicates that the sequential model of block is not sufficient to account for the actions of amantadine or memantine. The existence of a blocked state, recovery from which requires the presence of agonist, can be most simply explained by the "trapping" channel block model in which channels can close and agonists unbind even while the channel is blocked.
Approach to study partial trapping
The presence of a slow phase of recovery in the experiments shown in Figs. 3B and 4, B-D, indicates that some amantadine and memantine molecules were "trapped" in closed channels (state RB) after the agonists were washed off. However, the response after reapplication of agonists clearly contained both slowly and quickly rising phases. It is tempting to conclude that the presence of a quick phase indicates that some channels did not have the drug trapped at the time of agonist reapplication. If this is true and if the amplitude of this phase reflects the proportion of channels which did not trap the drug, then Figs. 3 and 4 would indicate that many blocked channels did not trap either drug. A result such as this would be surprising, given previous reports that trapping channel blockers such as PCP and MK-801 remain trapped in nearly all blocked channels under similar circumstances (Huettner and Bean 1988
; Jahr 1992
; Lerma et al. 1991
; MacDonald et al. 1991
). Furthermore, our use of a low concentration (5 µM) of NMDA should have promoted trapping of the blocker by minimizing the proportion of blocked receptors that are liganded at steady state.
Observation of partial trapping therefore may indicate that the presence of the blocker in the channel has a significant influence on the state transitions of the NMDA receptor. This possibility has not previously been explored and is important for understanding the mechanism and functional consequences of channel block. We therefore investigated the possibility that partial trapping can be explained by an effect of the presence of amantadine or memantine in the channel on receptor state transitions. We first tested the plausibility of this hypothesis by determining whether Model 1 can reproduce the observed partial trapping simply by permitting the rate constants between blocked states to differ from the corresponding rate constants between unblocked states. We then examined the validity of three alternative explanations for the biphasic recovery at the time of agonist reapplication: there may be two or more binding sites at which amantadine and memantine can antagonize NMDA responses, not all of which can trap the drugs; transitions leading to trapping may not have reached equilibrium, and during longer blocker applications, trapping could progress to completion; within the almost assuredly heterogeneous population of NMDA receptors in cultured cortical neurons, partial trapping may be observed because some subtypes of receptors are unable to trap the drugs.
Kinetic model of block by amantadine and memantine
To determine whether Model 1 alone can account for observations such as those in Figs. 3 and 4, we simulated these experiments by numerically solving a kinetic model based on Model 1. This model was designed primarily to illustrate the essential features of the blocking scheme, and in order to limit the number of free parameters, numerous states that are thought to be available to NMDA receptors have been omitted. For instance, the model includes no desensitized states, only one open state, and requires binding of exactly two agonist molecules for opening. Undoubtedly, such simplifications severely restrict the ability of the model to account for many aspects of receptor function. However, we have found that even this minimal model could reproduce many features of blockade by amantadine and memantine, including the partial trapping shown in Figs. 3 and 4.
Results of these simulations are shown in Figs. 3 and 4 as solid lines overlaying the physiological data points, and the rate constants which were used to produce this output are listed in Table 1. These simulations demonstrate that Model 1 can reproduce two important aspects of the data presented here. First, in both the simulations and current records, the slow phase of recovery following the reapplication of agonists (beginning at arrowheads in Figs. 3B and 4, B-D) did not start from the current amplitude observed during steady state block. Second, both the simulations and the current records exhibit multiexponential kinetics of blockade and recovery in the continuous presence of agonist. There are also notable differences between the current traces and fits; these differences will be considered in DISCUSSION.
In the simulated block by memantine, the initial jump in current after agonist reapplication suggests that trapping was not complete, and we confirmed that this was in fact the case. The simulated fractional occupancy of the trapped state RB was 45% during the steady state immediately before agonist reapplication, substantially less than the total fraction of channels that were blocked in the presence of antagonist (94%). The amplitude of the quickly rising phase of the simulated response was 48% of the control response amplitude, a value close to the percentage of blocked channels which had not become trapped.
On the other hand, the trapping of amantadine was not accurately reflected in the amplitude of the initial jump in current following agonist reapplication, as determined through use of the simulations. In the simulated experiment shown in Fig. 3B, the response to the reapplication of agonists rose quickly to 80% of the control amplitude and recovered slowly thereafter. Our initial interpretation of this observation was that the drug remained trapped in only 20% of the channels. In fact, the trapping of amantadine was much more substantial: 97% of blocked channels trapped the drug.
In general, we found that the quickly rising phase of the response to the reapplication of agonists reflects channels that had not trapped the drug only if the time course of recovery from block is much slower than the time course of response activation. To determine whether this is the case with memantine, we examined in detail the kinetics of the recovery from block by 20 µM memantine at
67 mV (Fig. 4E). In 12 of 13 cells, the current relaxations were better fit by two than one exponential, and the two time constants were 5.26 ± 1.10 s (31 ± 4% of the recovery amplitude) and 64.9 ± 20.0 s. Even the faster of these is substantially slower than either the time constant of solution exchange of our perfusion system (~30 ms) or of the onset of the control response to NMDA + glycine (time to peak 50-200 ms). We concluded that the relative amplitude of the recovery from block by memantine that proceeds slowly in experiments such as those shown in Fig. 4, B-D, can be taken as a measure of the proportion of channels that had trapped memantine.
Noncompetitive inhibition of NMDA-activated responses by amantadine and memantine
One clear premise of Model 1 is that the blocker has a single binding site, accessible only when the channel is open. However, if a second site existed at which amantadine or memantine could bind to inhibit NMDA responses but at which they could not become trapped, action at this site might reduce steady-state occupancy at the site of trapping channel block. Such an effect might interfere with our ability to determine whether memantine was trapped in all channels that were blocked. We tested the assumption that these drugs have only one site of action and found that both memantine and amantadine can inhibit responses to NMDA when the channel is closed, through access to a distinct, low-affinity binding site. As shown below, due to the low affinity of this interaction, the trapping of either drug in the channel should be nearly unaffected by action at this second site.
This effect on the closed channel is demonstrated in Fig. 5A, which shows responses from a cell to which NMDA was applied after application of either APV alone orAPV + 1,000 µM amantadine. The APV or APV + amantadine were washed off 250 ms before the applications of NMDA. Nevertheless, the initial response to the application of NMDA that was preceded by amantadine was considerably reduced, indicating that amantadine antagonized the response by acting when the channel was closed. Figure 5B shows responses from a cell to which 50 µM memantine + APV were applied in the absence of NMDA or glycine. Applications of NMDA before and 1 s after the application of memantine indicate that memantine acting on the closed channel inhibited the subsequent response to NMDA. Because the recovery from this type of inhibition by amantadine was quite fast, we concentrated in the following experiments on characterizing the inhibition by memantine.
The kinetics of recovery from inhibition in the absence of agonists resembled the kinetics of recovery from trapping channel block. We therefore were concerned that the inhibition shown in Fig. 5, A and B, could have resulted artifactually from trapping channel block. It is unlikely that this inhibition was due to block of channels activated by contaminating agonists because nearly all experiments on closed channel inhibition were performed in the presence of 80 µM DL-APV. In addition, when similar experiments were performed without APV or in the presence of 10 µM 7-chlorokynurenic acid, no difference in the degree of inhibition was observed. However, it is possible that the inhibition shown in Fig. 5 could have resulted from incomplete wash-off of the antagonist before reapplication of agonists. As a stringent test, we measured the rate of dilution of high concentrations of APV or Mg2+ (100-1,000 times their IC50s) in a protocol similar to that used to examine the closed-channel inhibition by the adamantanamines (Fig. 5C). In 10 cases examined using either APV or Mg2+, the mean time required for a 50- to 500-fold dilution was117 ± 10 ms. For the experiments with memantine presented here, we used primarily concentrations of 50-100 times the open channel IC50 and a wash duration of 1 s between memantine and agonist applications. For amantadine, because its unbinding time constant was considerably faster than memantine's, we used a 250- to 500-ms wash, but generally used 1 mM amantadine, only ~30 times its IC50. Thus in the following experiments, each drug should have had time to be washed off to at least several times below its IC50 before agonists were reapplied. Further evidence against artifactual open channel block due to incomplete wash-off of antagonist comes from experiments with another trapping blocker of NMDA-activated channels, ketamine (MacDonald et al. 1991
). When the protocol shown in Fig. 5B was used to apply ketamine at 100 or 150 µM (~200 times its open channel IC50), virtually no effects on the subsequent NMDA response were observed (data not shown). In one of three cells, there was no measurable effect, and in two other cells, only slight (<5%) and transient (<5 s) decreases in NMDA responses were observed, even after a 60-s ketamine application. On the basis of these results, we have termed this form of inhibition by amantadine and memantine "noncompetitive," because it occurred in the absence of agonist and was not influenced by the presence of competitive antagonists.
In four cells, the inhibition produced by memantine applications of 0.5-120 s indicated that steady-state noncompetitive inhibition was reached within 2-10 s. We therefore used memantine applications
20 s long in the absence of agonists to generate a concentration-inhibition curve (Fig. 5D). Fitting the pooled data from all cells with Eq. 1 yielded an IC50 of 179 µM and a Hill coefficient (nH) of 0.70. We tested the noncompetitive effects of amantadine by applying concentrations
10 mM. We found that the IC50 of amantadine for inhibition of NMDA responses in the absence of agonists was between 3 and 10 mM, ~100-300 times higher than its open channel IC50, as for memantine.
Two sites of action of amantadine and memantine
The two types of inhibition of NMDA responses produced by each drug appear to result from actions at two different sites. As shown in Figs. 3 and 4, complete recovery from trapping channel blockade required the presence of agonists. Thus if noncompetitive inhibition by memantine or amantadine were mediated by binding at the site of trapping channel block, then the drugs should be trapped at this site while the channel is closed and agonists are unbound. As shown in Fig. 6A, however, complete recovery from noncompetitive inhibition was observed even in the absence of agonists. Agonist-independent recovery was observed in each of four cells tested in this manner, and was also observed for inhibition by amantadine (n = 3).

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| FIG. 6.
Evidence that noncompetitive inhibition and channel block occur at different sites. A: memantine bound at the noncompetitive site is not trapped. Left shows noncompetitive inhibition by 100 µM memantine + APV and complete recovery during periodic applications of agonists. Gap preceding final application of NMDA represents 40 s. Right shows that complete recovery from an identical application of memantine also occurred in the absence of agonist. B: voltage dependence of noncompetitive inhibition. Responses from 1 cell to which 1,000 µM memantine + 80 µM APV were applied using protocol shown in Fig. 4B for 60 s at indicated Vms. C: voltage dependence of noncompetitive inhibition compared to that of trapping channel block. IMem/INMDA measured in cell shown in A is plotted. Solid line is best fit of Eq. 2 (K0 = 1.04 mM; V0 = 67.2 mV). Dashed line predicts amount of trapping channel block in this cell that would have been produced by memantine at a concentration that would cause same fractional block at 0 mV. Equation that describes dashed line is IMem/INMDA = 1/[1 + 13.7/(14.2 · e Vm/29.3)].
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Also consistent with action at two different binding sites, the voltage dependence of noncompetitive inhibition by memantine differed from that of trapping channel block. The responses shown in Fig. 6B are from one cell to which 1 mM memantine + APV were applied for 60 s, as in Fig. 5B, while the cell was held at three different Vms. The fractional block plotted as a function of Vm (Fig. 6C) shows that the degree of inhibition decreased at more positive potentials. From fits of Eq. 2 to such plots from six cells to which either 50, 300, or 1,000 µM memantine was applied for 60 s at 4-6 Vms, we calculated a mean K0 of 1.2 ± 0.5 mM and a mean V0 of 56.3 ± 3.5 mV. As illustrated in Fig. 6C, the voltage dependence of noncompetitive inhibition was significantly less than that found for trapping channel block by memantine or amantadine (P < 0.005 compared with block by memantine or amantadine). With this method, the measured voltage dependence of noncompetitive antagonism will be overestimated if any spontaneous channel openings allowed trapping channel block to occur during the antagonist application. Amantadine noncompetitive inhibition was also voltage dependent, but the effect was not quantified due to the high concentrations required to produce inhibition >50%.
Can the noncompetitive action of memantine explain its incomplete trapping at the channel blocking site? Although we do not know how the two sites interact, we can use a simple model to estimate the fraction of channels that would be unavailable for block (and therefore that could not trap memantine) if occupation of the two sites were mutually exclusive. If simultaneous occupation of both sites were possible, the effect of the noncompetitive site on trapping would be less than we will calculate here. The model can be represented by the reaction scheme RB
R + memantine
RN, where RB is the receptor with the channel blocked by memantine (apparent dissociation constant KB) and RN is the receptor noncompetitively inhibited by memantine (apparent dissociation constant KN). Using the data presented above, at
67 mV, KB = 1.4 µM and KN = 179 µM. The model predicts that the percentage of channels in state RB at equilibrium is 97.3% in the absence of noncompetitive binding and 96.5% when noncompetitive binding is permitted. Thus, because of the difference in the apparent affinity of memantine for the two sites, noncompetitive inhibition would have a negligible effect on trapping of memantine. We nevertheless attempted to minimize the potential influence of noncompetitive inhibition on drug trapping in subsequent experiments by using a relatively hyperpolarized holding potential (
97 mV) and a lower concentration of memantine (20 µM). Because the voltage dependence of trapping channel block by memantine is greater than that of noncompetitive inhibition, the difference between their apparent dissociation constants increases with hyperpolarization.
Partial trapping of memantine at equilibrium
To investigate whether partial trapping of memantine was due to slow progression of channels from the open-blocked into the trapped state, we next determined the steady-state proportion of channels in which the drug is trapped. We examined the time course of accumulation of channels in the trapped state by applying memantine in the presence of NMDA for various lengths of time (Fig. 7A). At the end of each application, the agonists and memantine were washed off at the same time, and APV was applied for 2-3 min. The fraction of channels that trapped the drug then was determined with a subsequent test application of NMDA. Any inhibition of the initial response to the test application in this protocol must be due to channels that had been in the trapped state.

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| FIG. 7.
Time course and steady state level of memantine trapping. A: responses from 1 cell held at 97 mV to which 20 µM memantine was applied for either 2 s (left) or for 60 s (right). Response was blocked 85% by short application and 98% by longer application. Arrowheads mark initial amplitude of responses to reapplication of agonists, measured as described in METHODS. After 2 s of block, only 29% of channels trapped memantine, whereas after longer exposure, 90% of the channels did so. Breaks in data represent 160 s. Open circle and filled circle measurements plotted in B. B: open circle, degree of block produced by each of 7 applications of memantine given in random order to a single cell. Block was measured at end of each application, and dashed line is a fit to these data of a single exponential with = 8.1 s. The shortest memantine application was 500 ms. Filled circle, initial amplitude of test response evoked after wash-off of APV. Solid line shows a single exponential fit to these data with = 8.5 s. Sixty-second application was sufficient to produce steady-state block and trapping. C: summary of data from 7 cells held at 97 mV to which 20 µM memantine was applied for 2 or 60 s. Open bars show fractional steady-state current during application of NMDA and memantine, and hatched bars show fraction of channels that did not trap the drug. * Significantly less trapping than block at this application duration (P < 0.005). # Significantly less blocking or trapping than following2-s application (P < 0.005).
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After short applications of memantine, the response was substantially inhibited, but very few channels trapped the drug. The onset of block of the whole cell current was well fit by a double exponential function (n = 5). The first exponential (
= 85 ± 2 ms) accounted for the majority (74 ± 2%) of the total inhibition, and the second slow phase approached equilibrium with
= 5.8 ± 1.6 s. A 2-s application of memantine thus inhibited the response by 80.1 ± 2.6%; however, the response subsequently tested after the period of APV application initially was reduced only by 15.8 ± 4.0% compared with the original response (n = 5). Longer applications of memantine revealed that blocked channels slowly accumulated in the trapped state after an approximately single exponential time course with
= 10.0 ± 1.1 s, as shown for one cell in Fig. 7B. Even after long applications in which both the current and the degree of blocker trapping had reached a steady-state level, the fraction of channels that trapped memantine was much less than the fraction that had been blocked. Figure 7C illustrates that memantine applied for 60 s produced 97.8 ± 0.5% block, yet only 84.0 ± 2.2% of the channels trapped the drug (n = 6). These values were significantly different (P < 0.005), indicating that 14.2 ± 1.9% of blocked channels did not trap the memantine. Additional experiments with 20 µM memantine revealed that at
67 mV, 19.7 ± 2.1% of blocked channels released rather than trapped the blocker (n = 7). The results at
67 and
97 mV were not significantly different (P > 0.08), and, taken together, suggest that in this voltage range, roughly one-sixth of channels blocked by memantine released the drug rather than trap it.
Time course of amantadine trapping
We next determined the time course with which channels accumulated in the trapped state when blocked with amantadine, using an approach similar to that used with memantine (Fig. 8A). However, recovery from block by amantadine, unlike recovery from block by memantine, included a large, rapid component (see also Fig. 3A). This component could not be distinguished from the fast phase of response activation after reapplication of agonist (see Fig. 3C). Thus during the fast phase of response activation, some channels that had trapped amantadine may have had time to unblock. Therefore, our approach should characterize reliably the time course of the approach to steady state trapping, but the proportion of channels that trap amantadine cannot be determined without additional information. In this cell, accumulation of channels in the trapped state progressed with an apparent
= 3.9 s (Fig. 8B). In six cells tested with 100-300 µM amantadine at
97 mV, the amplitude of the slow phase of recovery increased with a time constant of 8.7 ± 2.7 s, a value not different from that calculated for memantine. On average, 300 µM amantadine applied for 60 s at
97 mV blocked 95.5 ± 0.6% of the response, and the amplitude of the slow phase of recovery was 31.0 ± 2.8% of the control amplitude (Fig. 8C).

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| FIG. 8.
Time course of amantadine trapping. A: responses from 1 cell held at 97 mV to which 100 µM amantadine was applied for either 2 s (left) or for 60 s (right). Response was blocked 89% by short application and 92% by longer application. Protocol was same as in Fig. 5, except that APV was applied for 1 min. Arrowheads mark initial amplitude of response at time of application of NMDA. After 2 s of block, amplitude of slow phase of recovery was only 4% of INMDA, whereas after longer exposure, it was 23%. Breaks in data represent 50 s. Open circle and filled circle measurements plotted in B. B: open circle, degree of block produced by each of 6 separate applications of amantadine given in random order to a single cell. Dashed line is a fit to these data of a single exponential with = 3.82 s. Shortest amantadine application was 1 s. Filled circle, initial amplitude of test response evoked after wash-off of APV, and solid line shows a single exponential fit to these data with = 3.80 s. Sixty-second application was sufficient to produce steady-state block and trapping. C: summary of data from 8 cells held at 97 mV to which 300 µM amantadine was applied for 2 or 60 s. Open bars show fractional steady-state current during application of NMDA and amantadine, and hatched bars show initial amplitude of response evoked after wash-off of APV. * Significantly less than initial response after wash at this application duration (P < 0.005). # Significantly less than following the 2-s application (P < 0.005).
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Partial trapping of memantine within a homogeneous receptor population
The cortical neurons used in these experiments are likely to contain a heterogeneous population of NMDA receptors (Zhong et al. 1994
). Thus it is possible that all of these subtypes of receptors can be blocked by memantine and amantadine but that incomplete trapping was observed only because one or more subtypes do not trap the drugs. To test the hypothesis that a homogeneous population of receptors cannot exhibit partial trapping of a blocker, we examined block by memantine of recombinant NMDA receptors expressed in CHO cells. Cells were transiently transfected with mRNA for NR1-1a and either the NR2A or NR2B subunit. Except for possible differences in NR1/NR2 stoichiometry, this method should produce a uniform population of receptors.
We first determined the steady state level of memantine trapping in NR1-1a + NR2A receptors using a protocol identical to that used on the cortical neurons. We applied memantine for various durations, as in Fig. 7, and found that the proportion of receptors in which memantine became trapped reached steady state within 10 s, similar to but slightly faster than in the cortical neurons. We therefore used 60-s applications to measure the amount of trapping obtained in these receptors. Figure 9A shows a response evoked in one CHO cell by application of NMDA and block of that response by 20 µM memantine. In five cells, the current in the presence of memantine under these conditions was nearly indistinguishable from the baseline holding current, indicating that memantine almost completely blocked the response. After the wash-off of memantine and NMDA, the reapplication of agonists showed that memantine could be trapped in receptors of this type, because the response was initially reduced but then recovered in the presence of agonists.

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| FIG. 9.
Partial trapping demonstrated in recombinant receptors composed of NR1-1a + NR2A (A) or NR1-1a + NR2B (B) subunits expressed in CHO cells. Memantine applied as in Figs. 4 and 7 could be trapped in either type of receptor. Solid lines are double exponential curves fit to responses after reapplication of agonists. Insets: portion of response shown on an expanded time base. Recovery contained an initial, very quickly rising phase, corresponding to fraction of blocked channels that did not trap drug. Trace in B is average of responses to 2 identical applications. Cells were held at 97 mV.
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To measure the fraction of channels that had trapped the drug, we fit a double exponential function to the recovery and extrapolated this curve back to the time of agonist application. The amplitude of the fitted curve at this point was taken to represent the proportion of channels that had not trapped the memantine. In several cortical neurons tested, this analysis gave results very similar to those obtained by measuring, as for Figs. 7 and 8, the initial amplitude of the response to the reapplication of agonists. In principle, the method of fitting exponentials to the response is more accurate. However, in the cortical neurons, Ca2+-dependent inactivation (Legendre et al. 1993
) made it impractical to apply NMDA for a single period that was long enough to observe complete recovery from block by memantine. Instead, recovery from block was hastened typically by applying NMDA while holding the cell at a more depolarized Vm. Thus it was impossible in most neurons to measure the extent of trapping by fitting exponentials to the recovery phase. In the CHO cells, recoveries essentially were unaffected by Ca2+-dependent inactivation, possibly because the CHO cells tended to have smaller responses than did the neurons, and recovery was therefore taken to completion in the continuous presence of NMDA. In five CHO cells, the amplitude of the fitted curve at the time of agonist application was 17.2 ± 3.3% of the control response to agonists. Thus although nearly all receptors were blocked under these conditions, memantine became trapped in only 83% of the NR1-1a + NR2A receptors.
We performed similar experiments on CHO cells expressing only receptors composed of the NR1-1a + NR2B subunits (Fig. 9B). As in receptors that contained the NR2A subunit, the proportion of receptors in which memantine became trapped reached steady state within ~10 s. In five cells, 20 µM memantine applied for 60 s nearly completely blocked the response to NMDA, as it had in cells expressing NR1-1a + NR2A. A double exponential function again was used to fit the response to the reapplication of agonists. The amplitude of the fitted curve at the time of agonist reapplication indicated that memantine escaped from 12.0 ± 2.9% of the blocked channels, rather than become trapped in all of them. Thus even in a population of receptors containing just one variant of NR1 and one type of NR2 subunit, memantine becomes trapped in only a fraction of the blocked channels. The proportion of blocked channels in which memantine was trapped was not different in neurons or CHO cells expressing NR2A or NR2B (1-way ANOVA, F = 1.1, P > 0.3).
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DISCUSSION |
After block of NMDA-activated channels by amantadine and memantine, channel closure and agonist unbinding can result in trapping of the antagonist in the channel. The observation that trapping occurs implies that there must be no direct transition between states R and RB, consistent with Model 1. Although trapping of these drugs does occur, memantine appears macroscopically to be trapped only partially. Partial trapping was demonstrated by washing away blocker and agonists after block and trapping had reached steady state; memantine was observed to unbind from roughly one-sixth of blocked channels rather than becoming trapped in all of them. Such partial trapping may be due to an effect of drug binding on channel gating or agonist affinity. The feasibility of this hypothesis was demonstrated with simulations of a kinetic model that reproduced partial trapping. Alternative explanations of partial trapping were investigated and rejected. First, the time course was measured over which channels accumulate in the trapped state in the presence of NMDA and memantine, and it was found that memantine was trapped partially even at steady state. Second, memantine and amantadine each inhibited responses to NMDA by acting noncompetitively at a site distinct from the site of trapping channel block; however, the low affinity of this action made it unlikely to influence the degree of trapping. Third, memantine was trapped partially also in recombinant NMDA receptors composed purely of NR1-1a and NR2A or NR2B subunits, indicating that the heterogeneity of cortical NMDA receptors did not underlie partial trapping. We conclude that memantine and perhaps amantadine, when bound in the channel, greatly alter the gating of the channel or its interaction with NMDA.
Mechanisms of partial trapping
If the state transitions of channels blocked by memantine were the same as those of unblocked channels, release of the blocker from substantially fewer channels would have been expected in experiments such as shown in Fig. 7. When agonists and antagonists were removed after steady-state block, unblock is likely to have occurred only from fully liganded receptors. This suggests that
14% of blocked channels were liganded fully at the steady state preceding the solution exchange, a much greater proportion than expected for unblocked channels in the presence of 5 µM NMDA. Furthermore, this difference may be underestimated because unblock is likely under these conditions only if the blocker unbinding rate is not slow compared with the channel closing and agonist unbinding reactions. In fact, we found that the fastest component of memantine unblock (
~5 s) was much slower than channel gating and NMDA unbinding in the absence of blocker (
~80 ms in our experiments) (see Benveniste et al. 1990
; Lester and Jahr 1992
). In addition, the results of the simulations shown here predict the dissociation rate constant of memantine (~7 s
1) to be considerably slower than the closing rate constant of unblocked channels (~130 s
1) and the dissociation rate constant of the first molecule of NMDA (80 s
1).
There are a number of ways in which binding of memantine could increase the probability that a channel remains fully liganded. For instance, memantine binding could increase receptor affinity for NMDA. Alternatively, memantine could alter the equilibrium between open and closed liganded channels, drawing channels to the open state. Although these are straightforward interpretations within the context of Model 1, it is also possible that memantine modulates occupancy of desensitized states not included in the model. At present, we cannot confidently determine the effects that memantine has on channel state transitions. However, our simulations of Model 1 demonstrated that partial trapping arose primarily because blocked channels had a greater affinity for NMDA. Although memantine greatly altered the rates of channel gating, the simulated steady-state open probability of blocked channels in the presence of 5 µM NMDA was only 6% higher than that of unblocked channels.
The partial trapping of memantine functionally distinguishes it from block by PCP or MK-801, which remain trapped in nearly all blocked channels under analogous conditions (Huettner and Bean 1988
; Jahr 1992
; Lerma et al. 1991
; MacDonald et al. 1991
). It is conceivable that PCP has the same effect on channel gating and agonist unbinding as does memantine, but that memantine has the additional, functional consequence that it is trapped only partially simply because it unbinds more quickly. Although our data do not directly address this issue, we used further simulations of Model 1 to explore the trapping of a hypothetical blocker and to test possible differences between PCP and memantine. We supposed that the only difference between PCP (IC50 ~140 nM) (Lerma et al. 1991
) and memantine (~1.4 µM) might be a 10-fold difference in k
; that is, this fictional blocker had the effects of memantine but the affinity of PCP. To determine the extent to which such a blocker would be trapped, we simulated its application in a protocol identical to that shown in Fig. 4, at a concentration 10 times lower than used for memantine (to obtain the same steady state fractional block). After the blocker and agonist were removed, occupancy of state RB rose to much higher levels (5 times fewer channels unblocked rather than trapped); that is, the fractional trapping of this blocker was more nearly complete. Thus by this functional measure, the drug appeared to act much more as does PCP. In another simulation, we determined that if a blocker's k+ and k
are those predicted by the model of memantine's action, but its effect on agonist binding is eliminated, then the trapping of the blocker is complete. Based on these simulations, we conclude that both a drug's kinetics and its effects on channel gating or agonist binding are important in determining the extent to which it is trapped.
The simulations in Figs. 3 and 4 differ from the current traces in two notable respects. First, there are some discrepancies in the time course of blockade and recovery. These discrepancies may be due to omission from the model of closed states such as desensitized states. Second, all sets of parameters that produced adequate simulations of block by memantine also predicted a transient, inward "tail" current at the time the blocker and agonist were washed off. We examined the current relaxations following the removal of NMDA and memantine after equilibrium block, and saw no evidence of tail currents. The current relaxation time course was not substantially different from that expected based on relaxations after the wash-off of agonist alone, and there was never a peak greater than the steady state current level during block.
Tail currents have been observed after wash-off of tetrabutylammonium (Koshelev and Khodorov 1992
) or 9-aminoacridine (Benveniste and Mayer 1995
) and are thought to result from the requirement that blocked channels must pass through the open state to unblock. In our experiments, it is striking that even cells such as shown in Fig. 4 that trapped memantine in only a small proportion of channels still showed no tail current. These observations imply that there may be a long-lived blocked state from which unblocking is more likely than trapping. In this case, only few channels at a time would pass through the open state, and the tail current barely would be detectable. Alternatively, there may be a route from blocked states to unblocked states other than through the open state. In terms of Model 1, the only possibilities are direct transitions from state A2RB to A2R or ARB to AR (Benveniste and Mayer 1995
), although there may exist additional routes through states not represented in this model.
Evidence from this study and from numerous others suggests that the degree to which different blockers are trapped can vary through a continuum. Some blockers of NMDA-activated channels, such as IEM-1857 (Antonov and Johnson 1996
) and 9-aminoacridine (Benveniste and Mayer 1995
), either cannot be trapped or are trapped very infrequently; some blockers, such as memantine, exhibit substantial but partial trapping; some blockers, such as PCP and MK-801, appear to be fully trapped. There may be more than one mechanism by which a blocker can influence the degree to which it is trapped. Comparison of a variety of adamantine derivatives that block the NMDA-activated channel has suggested that blockers with an elongated structure can sterically inhibit channel closure and presumably therefore prevent trapping (Antonov and Johnson 1996
). The models used here suggest that trapping also can be affected if the presence of blocker in the channel causes a more general change in receptor conformation that allosterically alters agonist binding.
Noncompetitive inhibition by amantadine and memantine
The noncompetitive form of inhibition by amantadine and memantine differed from the trapping channel block in three important respects: as a noncompetitive inhibitor, each drug exhibited a lower affinity, a less steep voltage dependence, and an inability to become trapped. These three differences are most simply explained by the presence of two different binding sites on the NMDA receptor-channel complex for amantadine and memantine.
On the basis of the present experiments, we cannot determine whether the site of noncompetitive inhibition is in the channel or is outside the channel and inhibits current flow allosterically. It seems unlikely that the binding site is in the channel accessible through a hydrophilic pathway when the gate is closed, because one would expect the recovery from inhibition at this site to have been faster than observed, given the low affinity of each drug. Alternatively, because amantadine easily passes into phospholipid bilayers (Duff et al. 1993
), the site might be reached via a hydrophobic route. In this case, the relatively slow recovery kinetics from this mode of inhibition could reflect a slow exit of the antagonist from the membrane lipid. If the uncompetitive site can be reached through the membrane, it should be accessible from either the intracellular or extracellular solution. To test this possibility, we included 100-1,000 µM amantadine in the pipette solution. NMDA-activated currents recorded under these conditions were not visibly different from control currents in size or voltage dependence (Blanpied and Johnson 1994
). This argues against a hydrophobic location of the noncompetitive site. However, even 1,000 µM amantadine may not have been a sufficient intracellular concentration to cause significant noncompetitive inhibition, or the intracellular concentration of amantadine at the locations of the activated receptors may have been lower than that in the pipette. Localization of the site of noncompetitive inhibition will require further experiments.
Functional implications
Access to a trapped state may contribute to the effectiveness of amantadine and memantine as antiparkinsonian (Brenner et al. 1989
; Fischer et al. 1977
; Schwab et al. 1969
) or neuroprotective (Chen et al. 1992
; Seif el Nasr et al. 1990
; Weller et al. 1993
) agents by increasing their equilibrium affinity. Whether a blocker is a sequential or trapping channel blocker will not affect its microscopic KD, which by definition depends only on the rates of interaction with the open channel (KD = k
/k+). However, the blocker's macroscopic unbinding rate, and therefore also its IC50, decreases with increasing fractional occupancy of blocked states from which the blocker cannot unbind. Thus the observation that memantine and amantadine can be trapped implies that they inhibit NMDA responses more effectively than they would if channel closure were prevented while they are bound. During daily treatment for Parkinson's disease, cerebrospinal fluid concentrations of amantadine and memantine are estimated to reach 10 µM (Brenner et al. 1989
; Kornhuber et al. 1995
) and 0.3 µM (Kornhuber and Quack 1995
), respectively. These concentrations are sufficient to act at NMDA receptors, given the IC50s reported here.
Amantadine and memantine are practical for therapeutic use because they appear to induce fewer and less profound effects on perception or consciousness (Ditzler 1991
) than other NMDA channel blockers that can be trapped in the channel, such as PCP (Luby et al. 1959
) and ketamine (Krystal et al. 1994
). It has been proposed (Chen et al. 1992
; Rogawski 1993
) that some NMDA channel blockers may be safer because they effectively inhibit overstimulation of NMDA receptors by high tonic levels of glutamate, for instance during ischemia, while sparing synaptic responses. At least two features of blockers of NMDA-activated channels may relate to this hypothesis. First, faster kinetics may be correlated with increased clinical safety (Rogawski 1993
). This idea generally is supported by the sequence of unblocking rate constants (from slowest to fastest: MK-801 < PCP < ketamine < memantine < amantadine) (Blanpied and Johnson 1993
; Chen et al. 1992
; MacDonald et al. 1991
; Parsons et al. 1996
; and data presented here). Second, partial trapping may be important in determining the effects of a trapping channel blocker during repetitive synaptic transmission. A drug that is trapped in nearly all blocked channels will cause a relatively large fraction of channels to accumulate in the trapped state. However, a drug that is identical except for being partially trapped would cause fewer channels to accumulate in the trapped state; a portion of the channels that become blocked during a synaptic response would release the drug rather than trap it during the period between synaptic inputs. Therefore the properties of memantine may ensure that during its use a substantial fraction of channels remain available for synaptic activation. If the mechanism of channel block influences a drug's psychotomimetic effects, then the therapeutic utility of memantine and perhaps amantadine may be enhanced by a tendency to be only partially trapped.
 |
ACKNOWLEDGEMENTS |
We thank Dr. G. Quack at Merz and Co. for the gift of memantine, Dr. S. Nakanishi for NR1-1a cDNA, Dr. P. Seeburg for NR2A and NR2B cDNA, Dr. M. Chalfie for GFP cDNA, K. Newell and W. Potthoff for excellent technical support, and J. Dilmore and Dr. S. Antonov for helpful comments on the manuscript.
This work was supported by National Institutes of Health Grants MH-45817, MH-00944, and MH-45156 and a University of Pittsburgh Central Research Development Award to J. W. Johnson, by a Mellon Fellowship and National Research Service Award MH-10818 to T. A. Blanpied, by National Institute of Neurological Disorders and Stroke Grant NS-29365 and an unrestricted grant from Pfizer Central Research to E. Aizenman, and by training grant MH-18273 to F. Boeckman.
 |
FOOTNOTES |
Present address of T. A. Blanpied: Dept. of Neurobiology, Box 3209 Duke University Medical Center, Durham, NC 27710.
Address for reprint requests: J. W. Johnson, Dept. of Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260.
Received 29 April 1996; accepted in final form 29 August 1996.
 |
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