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The Journal of Neuroscience, December 15, 1999, 19(24):10611-10626
Probing of NMDA Channels with Fast Blockers
Alexander I.
Sobolevsky,
Sergey G.
Koshelev, and
Boris I.
Khodorov
Institute of General Pathology and Pathophysiology, 125315 Moscow,
Russia
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ABSTRACT |
Using whole-cell patch-clamp techniques, we studied the
interaction of open NMDA channels with tetraalkylammonium compounds: tetraethylammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium (TBA), and tetrapentylammonium (TPentA). Analysis of the blocking kinetics, concentration, and agonist dependencies using a set of
kinetic models allowed us to create the criteria distinguishing the
effects of these blockers on the channel closure, desensitization, and
agonist dissociation. Thus, it was found that TPentA prohibited, TBA
partly prevented, and TPA and TEA did not prevent either the channel
closure or the agonist dissociation. TPentA and TBA prohibited, TPA
slightly prevented, and TEA did not affect the channel desensitization. These data along with the voltage dependence of the stationary current
inhibition led us to hypothesize that: (1) there are activation and
desensitization gates in the NMDA channel; (2) these gates are distinct
structures located in the external channel vestibule, the
desensitization gate being located deeper than the activation gate. The
size of the blocker plays a key role in its interaction with the NMDA
channel gating machinery: small blockers (TEA and TPA) bind in the
depth of the channel pore and permit the closure of both gates, whereas
larger blockers (TBA) allow the closure of the activation gate but
prohibit the closure of the desensitization gate; finally, the largest
blockers (TPentA) prohibit the closure of both activation and
desensitization gates. The mean diameter of the NMDA channel pore in
the region of the activation gate localization was estimated to be
~11 Å.
Key words:
NMDA; gating machinery; tetraalkylammonium compounds; blockade; desensitization; kinetics; patch-clamp; whole-cell; hippocampal neurons
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INTRODUCTION |
Considerable progress has been
achieved over the last few years in studies of the molecular structure
of the NMDA subtype of glutamate receptors (for review, see McBain and
Mayer, 1994 ; Dingledine et al., 1999) (Kuryatov et al., 1994;
Kuner et al., 1996; Krupp et al., 1996, 1998; Laube et al., 1997, 1998;
Villarroel et al., 1998; Anson et al., 1998; Beck et al., 1999).
However, some fundamental questions concerning their gross architecture and gating have not yet been finally settled. Present-day views on the
functional architecture of voltage-sensitive
Na+ and K+
channels are primarily based on the data obtained in studies of the
mechanism of their direct blockade by various quaternary ammonium
cations (for review, see Hille, 1992; Armstrong and Hille, 1998).
Probing with blocking compounds has also been used in studies of the
functional architecture of some ligand-gated channels, in particular
nicotinic acetylcholine channels and NMDA receptor channels. The use of
this method clearly demonstrated that the activation gate of these
channels is located in the external vestibule (Neher and Steinbach,
1978). Then, by analogy with voltage-sensitive channels (Armstrong,
1971; Strichartz, 1973; Yeh and Narahashi, 1977; Cahalan, 1978;
Armstrong and Croop, 1982), it was found that, depending on the type of
interaction with the gating machinery, most of the blockers of open
receptor-operated channels can be subdivided into at least two groups,
namely, those that do not prevent the channel closure, yielding the
so-called trapping block (Neely and Lingle, 1986; Huetter and Bean,
1988; MacDonald et al., 1991; Johnson et al., 1995; Blanpied et al.,
1997; Chen and Lipton, 1997; Sobolevsky et al., 1998) and those that
prohibit the channel closure (Koshelev and Khodorov, 1992, 1995; Costa and Albuquerque, 1994; Vorobjev and Sharonova, 1994; Benveniste and
Mayer, 1995; Johnson et al., 1995; Antonov and Johnson, 1996).
A comparative analysis of blocking effects of a series of organic
cations on NMDA channels led Koshelev and Khodorov (1992, 1995) to
suggest that, along with the activation gate, the NMDA channel, like
the voltage-sensitive Na+ channel, is
equipped with a desensitization gate; the closure of the latter was
assumed to underlie the channel desensitization.
In the present study we investigated the interaction of
tetraalkylammonium compounds (TAA) with open NMDA channels using a set
of kinetic models. We found the criteria for distinguishing the
blockers with a kinetics faster than the channel closure (fast blockers), which prohibited or did not prohibit the channel closure, desensitization, and agonist dissociation. According to these criteria,
we analyzed the action of tetraethylammonium (TEA), tetrapropylammonium
(TPA), tetrabutylammonium (TBA), and tetrapentylammonium (TPentA). The
results of this analysis provide new evidence in favor of the
hypothesis on the existence of functionally and spatially distinct
activation and desensitization gates in the NMDA channel and offer a
radically new approach to the study of their reciprocal position. Thus,
TAA proved to be useful tools to study NMDA channel gating.
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MATERIALS AND METHODS |
Pyramidal neurons were acutely isolated from the CA-1 region of
rat hippocampus using "vibrodissociation techniques" (Vorobjev, 1991). The experiments were begun after 3 hr of incubation of the
hippocampal slices in a solution containing (in mM): NaCl, 124; KCl, 3; CaCl2, 1.4;
MgCl2, 2; glucose, 10; and
NaHCO3, 26. The solution was bubbled with
carbogen at 32°C. During the whole period of isolation and current
recording, nerve cells were washed with an
Mg2+-free 3 µM
glycine-containing solution (in mM: NaCl, 140; KCl, 5;
CaCl2, 2; glucose, 15; and HEPES, 10, pH 7.3).
Fast replacement of superfusion solutions was achieved by using the
concentration jump technique (Benveniste et al., 1990a; Vorobjev, 1991)
with one application tube. This technique allows substitution of the tubular for the flowing solution with a time constant <30 msec but
backward with the time constant of 30-100 msec (Sobolevsky, 1999).
Therefore, except where noted, the rate of the solution exchange was
fast at the beginning of any application and slightly slower at its
termination. The currents were recorded at 18°C in the whole-cell
configuration using micropipettes made from Pyrex tubes and filled with
an "intracellular" solution (in mM: CsF, 140; NaCl, 4;
and HEPES, 10; pH 7.2). Electric resistance of the filled micropipettes
was 3-7 M . Analog current signals were digitized at 1 kHz frequency.
Statistical analysis was performed using the scientific and technical
graphics computer program Microcal Origin (version 4.1 for Windows).
The data presented are mean ± SE; comparison of the means was
done by ANOVA, with p < 0.05 taken as significant.
The kinetic models used to simulate the action of the blockers (Fig.
1) were based on the conventional rate
theory and used independent forward and reverse rate constants to
simultaneously solve first-order differential equations representing
the transitions between all possible states of the channel. These
models were obtained from a completely symmetric model for the
open-channel blockade (model 5) by means of consecutive reduction of
the blocked states. The processes of NMDA channel activation, opening,
and desensitization were described in accordance with a kinetic model proposed by Lester and Jahr (1992). The choice of values of the kinetic
constants was made as described previously (Sobolevsky and Koshelev,
1998). Thus, the values of the kinetic constants for the agonist
binding and unbinding were l1 = 2 µM 1 · sec1 and
l2 = 25 sec1, respectively; the
entrance and recovery from desensitization were  = 1.2 and  = 0.8 sec1, respectively, and the kinetic
constant of the channel closure was  = 200 sec1. The value of the rate constant of the channel opening,
 , was chosen according to the value of the open
probability, P0 = /( + ), which was previously
defined in a wide range of 0.04-0.5 (Jahr, 1992; Lester et al., 1993;
Lin and Stevens, 1994; Benveniste and Mayer, 1995; Colquhoun and
Hawkes, 1995; Rosenmund et al., 1995; Lu et al., 1998). In the majority
of computer experiments, except where noted, the value of
P0 was taken to be rather low (0.09)
by the reason clarified in Results. The values of the blocking and
unblocking kinetic constants, k1 and
k2, respectively, were too fast to be
estimated. The value of the unblocking kinetic constant was taken to be
sufficiently high, k2 = 1000 sec1. The
value of k1 was taken arbitrarily (3.5 µM1 · sec1, as for TBA in the
previous study by Sobolevsky, 1999) but the blocker concentration was
measured in the values of the microscopic Kd = k2/k1.
As it will be shown below from variation of the values of
k2 and
k1, their arbitrary choice does not
affect the major conclusions of this paper.
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Figure 1.
Kinetic models used to simulate the open-channel
blocker action. C, D, O, Channel in closed,
desensitized, and open states, respectively; subscripts A, AA,
B, binding of one agonist and two and one blocker molecules to
the channel, respectively; asterisk, conducting state,
[A], [B], agonist and blocker concentrations,
respectively.
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Differential equations were solved numerically using the algorithm
analogous to that described previously (Benveniste et al., 1990b).
Tetraalkylammonium compounds were purchased from Aldrich (Milwaukee,
WI). The three-dimensional structures of the blockers were obtained
with the help of Molecular Modeling System HyperChem (release 3 for Windows).
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RESULTS |
Concentration and voltage dependence of the
TAA-induced blockade
At the holding potential of  100 mV, aspartate (ASP) (100 µM) elicited an inward current through the NMDA channels,
which after the initial fast rise ( < 30 msec) up to the
value, IC0, decreased gradually
( = 250-750 msec) to the stationary level, ICS. This current decay under the
continuing action of the agonist is interpreted as a result of NMDA
receptor channel desensitization. When coapplied with ASP, TAA
suppressed both initial, IB0 (measured at the termination of the initial fast current increase), and stationary, IBS, currents.
Representative superpositions of the currents elicited by ASP alone
(control) or by ASP coapplied with TEA, TPA, TBA, and TPentA used at
different concentrations are shown in Figure
2. Termination of ASP coapplication with
each of these blockers was followed by a transient increase in the inward current ("hooked" tail current), which was absent in the control. In all the experiments with TEA and TPA, the maximal value of
the hooked current, IP, was smaller
than ICS at any blocker concentration.
In contrast, for TBA at high concentrations
IP was greater than
ICS in 60% of the cells
(n = 32 of 53), and even greater than
IC0 in three cells (n = 3 of 53). The maximal value of the hooked current for TPentA used at
high concentrations was always greater than
ICS (n = 22), and in
six cells (n = 6 of 22) it was larger than
IC0. The amplitude of the hooked tail
current, IP IBS, increased with the blocker
concentration for all TAA. However, this increase was considerably
greater for TBA and TPentA than for TEA and TPA. Another important
difference between the hooked tail currents concerns their time course.
In the case of ASP coapplication with TEA or TPA, the hooked tail
current always lay below the control tail current. In contrast, for TBA
and TPentA the hooked tail current and the control tail current
intersected.
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Figure 2.
Coapplications of TAA with ASP. The control
current elicited by ASP (100 µM) application is
superimposed with the current induced by ASP coapplication with TEA,
TPA, TBA, or TPentA at different concentrations. A transient increase
in the inward current (the hooked-tail current) appears after
termination of the agonist and the blocker coapplication and is more
pronounced at high TAA concentrations. The same labels apply to all
calibrations.
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The blockade of NMDA channels by TAA was voltage-dependent. The current
responses to ASP application and to ASP coapplication with TBA (2 mM) at the holding potential,
Eh, which varied from 100 to 40 mV
(with the step of 20 mV), are shown in Figure
3A. The control and blocked
stationary I-V curves are shown in the inset. The degree of the stationary block, 1 IBS/ICS,
(as well as the amplitude of the hooked tail current; Fig.
3A) diminished with membrane depolarization (Fig.
3B). According to the model of Woodhull (1973), the voltage
dependence can be fitted with the following equation:
![1−I<SUB><UP>BS</UP></SUB>/I<SUB><UP>CS</UP></SUB>=1−1/(1+[<UP>B</UP>]/K<SUB>0.5</SUB>(0)×<UP>exp</UP>(&dgr;FE<SUB><UP>h</UP></SUB>/RT)),](/content/vol19/issue24/fulltext/10611/img001.gif)
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(1)
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where K0.5(0) = 5.34 ± 0.27 mM is the equilibrium dissociation constant
at Eh = 0, and  = 0.60 ± 0.02 (n = 7) is the fraction of the
electric field that contributed to the energy of the blocker at the
blocking site. F, R, and T have their usual
meanings. The values of and
K0.5(0) estimated for other compounds
are presented in Table 1. The value of
increased for TAA with a decrease in the alkyl chain
length from 0.29 ± 0.03 (TPentA) to 0.90 ± 0.04 (TEA). This
means that according to the Woodhull model the smaller TAA penetrate
deeper into the membrane electric field. All the experiments described
below were performed at the holding potential of 100 mV.
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Figure 3.
Voltage dependence of the stationary current
inhibition. The voltage dependence is illustrated with 2 mM
TBA as an example. A, Experimental curves. ASP (100 µM) was applied alone (left traces) or was
coapplied with 2 mM TBA (right traces) for 3 sec at different holding membrane potentials,
Eh = 100, 80, 60, 40, 20, 20, and 40 mV. The degree of the stationary current inhibition, 1 IBS/ICS,
diminished with membrane depolarization. Inset, Control
and blocked stationary I-V curves.
B, The mean 1 IBS/ICS
values were plotted against Eh. The fitting
with Equation 1 (solid line) gave the following values
of parameters: K0.5(0) = 5.34 ± 0.27 mM and = 0.60 ± 0.02 (n = 7).
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To study the effect of the TAA on NMDA channel closure,
desensitization, and agonist dissociation, we considered five kinetic models (Fig. 1; see Materials and Methods). The first model implies that the blocker prohibits both the channel closure and
desensitization. In the second model, the blocker can be trapped in the
closed channel but does not allow the channel to desensitize and the agonist to dissociate from the channel. The third model implies that
the blocker only prohibits the agonist dissociation from the blocked
channel. Model 4 describes the situation when the blocker prohibits the
channel desensitization but does not prohibit the channel closure and
the agonist dissociation from the blocked channel. The fifth model is
completely symmetric and implies that the blocker prohibits neither the
channel closure and desensitization nor the agonist dissociation.
We tried to classify the action of the fast NMDA channel blockers
according to models 1-5 assuming, for simplicity sake, that the rate
constants for the transitions between the blocked states of the channel
(', ', ', ', l2', and
l1') are equal to the corresponding rate
constants for the nonblocked channels (, , , ,
l2, and
l1). Multiple experimental and
modeling protocols will be used to associate each blocker with a model.
On the condition that the blocking kinetics is rather fast, all
five models predict the appearance of the hooked tail current immediately after the termination of the agonist and the blocker coapplication (Fig.
4A). The kinetic
analysis showed that the ascending phase of the hooked current reflects
the blocker dissociation from the channel (transition from
OAAB to OAA* state),
whereas the falling phase reflects the processes of the channel
closure, desensitization and the agonist dissociation. In Figure
4A the degree of the stationary current inhibition,
1 IBS/ICS,
is the same for all models (0.86). To achieve this degree of stationary current inhibition, the blocker concentration was taken equal to 175, 16, 7, 13, and 6.5 Kd for models 1, 2, 3, 4, and 5, respectively. The significant difference in blocker
concentration ([B]) for different models clearly demonstrates that
the apparent affinity of the blocker (1/IC50) is
defined not only by its association-dissociation kinetics (the
association and dissociation rate constants for different models were
the same) but, to a considerable extent, by the blocker effect on the
channel closure, desensitization, and agonist dissociation.
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Figure 4.
Simulated hooked tail currents. A,
The simulated currents in response to the agonist application are
superimposed with simulated currents in response to the agonist
coapplication with the blocker. All models 1-5 predict the appearance
of the hooked current after termination of the agonist and the blocker
coapplication. To obtain the same degree of the stationary current
inhibition,
IBS/ICS,
we used the blocker concentrations [B] = 175, 16, 7, 13, and 6.5 Kd for models 1, 2, 3, 4, and 5, respectively. Hereafter (except as noted) the time constant of the
solution exchange, wash, = 30 msec, the open probability, P0, = 0.09, and the kinetic constant of the blocker dissociation,
k2, = 1000 sec1.
Inset, The control tail current (dashed
line) and the hooked tail currents predicted by models 1-5
(solid lines) are superimposed. All the models except
for model 5 predict the intersection of the control and hooked tail
currents. B, Hooked tail currents predicted by model 1 at different P0 values. The hooked currents
at P0 = 0.04, 0.09, 0.2, and 0.5 were
plotted after the stationary levels of the control simulated current at
different P0 values were normalized. The
value of P0 was varied by means of change in
the value of the rate constant of the channel opening, . The degree
of the stationary block is the same at different
P0 values. = 8.33, 20, 50, and 200 sec1; [B] = 413, 175, 77, and 23.5 Kd for P0 = 0.04, 0.09, 0.2, and 0.5, respectively. C, Hooked tail
currents predicted by model 1 at different
wash values. The hooked currents at
wash = 1, 10, 30, 50, 100, and 200 msec are presented. [B] = 175 Kd.
D, Hooked tail currents predicted by model 1 at
different k2 values. The hooked currents at
k2 = 0.3, 2, 5, 20, 100, and 1000 sec1 (k1 = 1.05, 7, 17.5, 70, 350, and 3500 mM1 · sec1,
respectively) are presented. [B] = 175 Kd.
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The amplitude of the hooked current,
IP IBS, is different for different models
(Fig. 4A, inset). It would be tempting to choose this amplitude as a criterion by which the action of the blocker
can be attributed to one of models 1-5. However, we found that a
number of factors affect the amplitude of the hooked current. We
illustrated this with the simplest model (model 1) as an example.
The first factor is the value of the open probability,
P0. A rise in
P0 increases the magnitude of the
simulated control current and enhances the simulated current stationary
inhibition at a given blocker concentration. Thus, to achieve the same
degree of the stationary current inhibition, we took smaller [B] at
higher P0; the relative amplitude
of the hooked current, (IP IBS)/ICS, decreased with increasing P0. This can
be clearly seen in Figure 4B, where the stationary
levels of the control simulated current at different
P0 were normalized.
The time constant of the solution exchange (assuming that the solution
exchange is a single-exponential process; Benveniste et al., 1990b),
wash, is the next factor that crucially
affects the amplitude of the hooked current (Fig. 4C). The
hooked current becomes higher and thinner with diminishing
wash.
A qualitatively inverse dependence of the amplitude of the hooked
current on the value of the unblocking rate constant,
k2, is observed (Fig.
4D). The hooked current becomes smaller and wider
with the slowing of the blocking kinetics, and at
k2 = 0.3-0.5 sec1 it disappears completely.
The next factor is the blocker concentration, [B] (Fig.
5A). The higher the [B], the
deeper is the block and the greater is the amplitude of the hooked
current. Such an experimental dependence is clearly seen in Figure
2.
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Figure 5.
Plateau/peak ratio. A, The current
responses to the agonist application and its coapplication with the
blocker at different concentrations ([B] = 3.5, 14, 35, 105, and 350 Kd) predicted by model 1 are
superimposed. B, The experimental values of the
plateau/peak ratio normalized to the control,
(IBS/IB0)/(ICS/IC0),
are plotted against the degree of the stationary current inhibition,
1 IBS/ICS,
for different TAA. C,
(IBS/IB0)/(ICS/IC0)
curves predicted by models 1-5 and 5a. Values of parameters for
A and C:
P0 = 0.09, wash = 30 msec, and
k2 = 1000 sec1.
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The nature of the dependencies of the hooked current amplitude on
P0,
wash,
k2, and [B] will be considered
elsewhere. However, the variety of parameters that affect the amplitude
of the hooked current (as well as its latency) makes it doubtful to
consider this value as a criterion of choice among models 1-5.
Apparently, it would be much better to find a qualitative criterion.
For example, the intersection of the hooked tail current and the
control tail current (Fig. 4A, inset) is
predicted by models 1-4 (but not by model 5) at any
P0,
wash,
k2, and [B] values considered. Thus, we have obtained the first criterion, which allows us to select a model
for describing the action of a blocker. This criterion permits one to
distinguish the blockers whose action can be described by model 5 from
those whose action can be described by models 1-4. According to this
criterion, the TEA and TPA action can be described by model 5, whereas
the TBA and TPentA action can be described by one of models 1-4. Other
qualitative criteria should be found to make a choice between models
1-4. The first of these criteria is the plateau/peak ratio.
Plateau/peak ratio
As can be seen from Figure 2, the plateau/peak ratio for the
block,
IBS/IB0,
may differ significantly from that for the control, ICS/IC0.
To compare the plateau/peak ratio for the block and the control, we
calculated it at different blocker concentrations. The mean values of
the normalized plateau/peak ratio,
(IBS/IB0)/(ICS/IC0), for different NMDA open-channel blockers were plotted against the
degree of the stationary current inhibition, 1 IBS/ICS
(Fig. 5B), which increased monotonically with [B] (Fig.
2). The mean (IBS/IB0)/(ICS/IC0)
values for TPentA (n = 7) and TBA (n = 10) were greater than unity; those for TPA (n = 5) were
slightly lower than unity. However, individual measurements for TPA
revealed three cells in which the normalized plateau/peak ratio was
lower than unity and two cells in which the normalized plateau/peak ratio was slightly higher than unity. The
(IBS/IB0)/(ICS/IC0) values for TEA (n = 5) were considerably lower than unity.
Models 1-5 also demonstrate different plateau/peak ratios for the
block with respect to the control (Fig. 4A). For
example, the simulated currents at different blocker concentrations
(Fig. 5A) indicate that for model 1 the gradual current
decay during the agonist application diminishes with an increase in
[B]. The values of the normalized plateau/peak ratio calculated for
all models are plotted in Figure 5C. Evidently, these values
are higher than unity for the models that imply that the blocker
prohibits the channel desensitization (models 1, 2, and 4) and slightly lower than unity for the models that predict that the blocker does not
prohibit this process (models 3 and 5). Therefore, the reason, why
IBS/IB0 > ICS/IC0
for models 1, 2, and 4, is the absence of the
DAAB state in which the blocked channels can be
gradually accumulated during the agonist and the blocker coapplication. Thus, the greater the gradual decrease in the simulated currents during
the agonist and the blocker coapplication for models 3 and 5 in
comparison with that of models 1, 2, and 4 (Fig. 4A) indicates that in the first case both blocked and nonblocked channels desensitize, whereas in the second case it is only the nonblocked channels that desensitize.
According to the plateau/peak ratio criterion, TPentA and TBA
prohibited channel desensitization, whereas TPA did not. In the case of
TBA, the reason by which the
(IBS/IB0)/(ICS/IC0)
curve is bent down at high values of 1 IBS/ICS
(Fig. 5B) is not clear. Presumably, it can be explained by
nonspecific TBA-induced inhibition of NMDA receptors or a comparatively
slow TBA-induced blockade of the residual nonselective cation current
(Xiong et al., 1997). The fact that in some cells the normalized
plateau/peak ratio for TPA is slightly higher than unity indicates that
under certain conditions TPA can decrease the probability of NMDA
channel desensitization. The plateau/peak ratio criterion is valid at
any P0 (from 0.04 to 0.5) and
wash (from 0 to 300 msec) but only for
fast blockers (k2 > 10 sec1), because a high value of
IB0/IBS
can be a consequence of the noncomplete initial blockade of the
channels because of a slow development of the block.
The
(IBS/IB0)/(ICS/IC0)
curve for TEA proved to be much lower than even those predicted by
models 3 and 5 (Fig. 5B). This fact can imply (Sobolevsky,
1999) either (1) the existence of a slow blocking kinetics component,
or (2) that TEA promotes the channel desensitization by increasing the
number of desensitized states or because of a shift of the
CAAB DAAB equilibrium
toward the DAAB state. To examine the first
possibility, model 5 was modified by addition of a new blocking site,
site 2 (Model 5a)
Model 5a.
Model 5a does not contain any additional assumptions. In this sense,
this model is the simplest one. Thus, the properties of site 2 are
qualitatively similar to those of site 1. The blocker can bind to site
2 during the channel opening and does not prohibit the subsequent
channel closure, desensitization, and agonist dissociation. Sites 1 and
2 cannot be occupied simultaneously, because the amplitude of the fast
component in the recovery kinetics for TEA in the continuous presence
of ASP does not depend on the blocker concentration (Sobolevsky, 1999,
his Fig. 5). The main difference between these two sites is in their
respective rates of blocker association and dissociation. Thus, the
value of the dissociation rate constant from the new site 2 was taken
to be 250 times lower than k2:
k2' = 4 sec1. The value of
the association rate constant was lowered proportionally (k1' = k1/250 = 0.014 µM1 · sec1), so that the value of
the microscopic Kd = k2/k1 remained the same
(0.29 mM). The
(IBS/IB0)/(ICS/IC0)
curve predicted by model 5a is shown in Figure 5C. At high
blocker concentrations, the plateau/peak value becomes much lower than
unity in compliance with that observed in the TEA experiment (Fig.
5B). The modifications of model 5 implying that the blocker
favored channel desensitization predicted a similar change in
(IBS/IB0)/(ICS/IC0)
curve (data not shown). The criterion that allows one to distinguish
these modifications of model 5 from model 5a will be considered below.
Blocking kinetics in the continuous presence of the agonist
Investigation of the blocking kinetics in the continuous presence
of the agonist provides valuable information about the mechanism of the
blocker-channel interaction (Sobolevsky and Koshelev, 1998; Sobolevsky, 1999). The experimental protocol is shown in Figure 6A with TPentA as an
example. The blocker was applied when the ASP-induced current already
reached its stationary level, ICS. Examples of the recovery kinetics are shown in Figure
6B. The recovery kinetics for all TAA contained a
fast ascending component, which reflected the rapid dissociation of the
blocker from the channel (the transition from
OAAB to OAA*); the time
constant of this component is mainly determined by the process of the
solution exchange (Sobolevsky, 1999).
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Figure 6.
The TAA recovery kinetics in the continuous
presence of ASP. A, The experimental protocol. TPentA (2 mM) was applied for 2 sec in the continuous presence of ASP
(100 µM) when the inward current gained its stationary
level, ICS. The solution exchange at the
termination of TPentA application was fast. B,
Representative examples of the current recovery after termination of
TEA (5 mM), TPA (2 mM), TBA (2 mM),
and TPentA (2 mM) application in the continuous presence of
ASP. The solid lines are double-exponential fittings of
the recovery kinetics in the cases of TEA and TPentA
(fast = 40 msec,
slow = 440 msec, and
Afast = 0.68 for TEA;
fast = 74 msec,
slow = 987 msec, and
Afast = 0.68 for TPentA) and a
single-exponential fitting in the case of TBA ( = 368 msec).
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Along with a fast component (fast = 155 ± 27 msec; n = 8), the recovery kinetics for
TEA also contained a slow component with the time constant
slow = 2.04 ± 0.34 sec
(n = 8). The amplitude of the fast component,
Afast, measured as a relative weight
of the fast exponent in the sum of the fast and slow components, was
0.69 ± 0.04 (n = 8).
In the case of TPA, the slow component, if existed, was small. The
value of Afast for TPA was either
slightly lower (n = 4) or slightly higher
(n = 4; see the example in Fig. 6B)
but, on the average, was equal to unity.
In the case of TBA, the fast component was so large that after a rapid
increase the current reached a value exceeding considerably the
stationary current level. As in the previous study (Koshelev and
Khodorov, 1995), the recovery current exceeding the stationary level,
ICS, will be referred to as an
"overshoot." In the majority of experiments with TBA, the fast
ascending phase of the overshoot was followed by a slow
(slow = 389 ± 38 msec;
n = 10) current decrease back to
ICS. However, in three cells for which
the solution exchange was comparatively fast
(wash  10 msec), the descending phase
of the current contained, along with the slow component, also a fast component.
Such a fast component was present in the recovery kinetics for TPentA,
which also exhibited an overshoot. Double-exponential fitting of the
overshoot descending phase allowed us to determine the time constants
of the fast and slow components, fast = 54 ± 7 msec and slow = 596 ± 85 msec (n = 7), respectively; the amplitude of the
fast component, Afast, is 0.63 ± 0.04 (n = 7).
Computer simulation showed (Fig.
7A) that the overshoot in the
recovery kinetics is predicted by models 1, 2, and 4 but is not
predicted by models 3 and 5. Thus, the recovery kinetics predicted by
model 5 contains only a fast component (the involvement of the second
component is not justified statistically, Fischer's test; Korn and
Korn, 1974). There is a small slow component in the recovery kinetics
predicted by model 3. The fitting of the recovery curve predicted by
model 3 gave the values of the time constants,
fast = 80 msec and
slow = 1.2 sec, and the amplitude of the
fast component, Afast = 0.93. Therefore, the existence of an overshoot is the criterion
distinguishing fast NMDA channel blockers that prohibit channel
desensitization from those that do not. This criterion is valid at any
blocker concentration in the range of the
P0,
wash, and
k2 values identified in the legend to
Figure 4. According to this criterion, TEA and TPA do not prohibit channel desensitization, whereas TBA and TPentA do. The above-mentioned cases for TPA, when Afast was somewhat
larger than unity can be interpreted as cases when TPA slightly hinders
channel desensitization.
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Figure 7.
Modeling of the recovery kinetics in the
continuous presence of the agonist. The values of parameters are the
same as listed in the legend to Figure 4. A, Recovery
kinetics predicted by models 1-5 and 5a. B, Recovery
kinetics predicted by model 1 at different open probabilities,
P0. The simulated currents at
P0 = 0.04, 0.09, 0.2, and 0.5 are
presented. C, Recovery kinetics predicted by model 1 at
different time constants of the solution exchange,
wash. The simulated currents at
wash = 1, 10, 30, 50, 100, and 200 msec are presented. D, Recovery kinetics predicted by
model 1 at different kinetic constants of the blocker dissociation,
k2. The simulated currents at
k2 = 0.3, 2, 5, 20, 100, and 1000 sec1 are presented.
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Another important conclusion, which clearly follows from the
consideration of the recovery kinetics, concerns the effect of the
blocker on the NMDA channel closure. Model 1, which is the only one
implying that the blocker prohibits the channel closure, predicts the
existence of a fast component in the falling phase of an overshoot.
Thus, the falling phase of the overshoot predicted by models 2 and 4 contains only a slow component with the time constant,
slow = 600 msec. In contrast, the
falling phase of the recovery kinetics for model 1, along with a slow
component, contains also a fast component. The double-exponential fit
of the recovery kinetics illustrated in Figure 7A revealed
the time constant and the amplitude of this component:
fast = 35 msec; Afast = 0.4. Our simulations showed
that the slow component of the falling phase reflects channel
desensitization (the slow transition from CAA to
DAA) and does not depend on the agonist
association-dissociation kinetics. The latter conclusion was confirmed
by the observation that the time constant of the slow component for the
falling phase of the overshoot did not depend on the agonist type.
Thus, this time constant was 394 ± 65 msec for ASP and 377 ± 32 msec for NMDA (these values were not significantly different
(p > 0.7; n = 6). The fast
component of the falling phase of the overshoot reflects the closure of
the unblocked channels (the transition from OAA*
to CAA). The fast component for model 1 appears
if the channel closure is slower than the blocker dissociation and is not masked by a more slow solution exchange, i.e., < k2 and < 1/wash, respectively. These conditions
are fulfilled at any blocker concentrations if the channel has a low
open probability (P0 < 0.1; Fig.
7B), the solution exchange is not very slow
(wash < 50 msec; Fig. 7C),
and the blocker dissociation constant is fast enough
(k2 > 20 sec1; Fig.
7D) (for models 2 and 4 the fast component in the falling phase of an overshoot does not appear at any values of
P0,
wash, and
k2).
Therefore, we have considered TPentA as a blocker that prohibits the
NMDA channel closure. Our experiments with TBA, in which the value of
wash was comparatively low (10 msec), and
the falling phase of the recovery kinetics contained the fast component
may imply that TBA at least hampers the channel closure if not
prohibits it. The appearance of the fast component in the falling phase of the recovery kinetics for TPentA and TBA was that reason, which forced us to adopt the value of the open probability,
P0, to be rather low (0.09).
The experimental value of Afast for
TEA (0.69 ± 0.04) was noticeably lower than the values predicted
by models 3 (0.93) and 5 (1.00). The recovery kinetics predicted by
model 5a is shown in Figure 7A. The value of
Afast (0.67) is close to that observed in the experiment with TEA. As in the case of the plateau/peak criterion, we also examined the modifications of model 5, implying that
the blocker promotes channel desensitization (see above). These
modifications provide similar changes in the recovery kinetics as those
predicted by model 5a (data not shown). Only the following criterion
allows one to restrict the choice of model 5 modification, satisfactorily describing the blocking effect of TEA.
Dependence of the stationary current inhibition on the
agonist concentration
Tetraalkylammonium compounds demonstrated different dependencies
for the degree of the stationary current inhibition, 1 IBS/ICS,
on the agonist concentration. The superposition of the currents
elicited by ASP application and its coapplication with TPentA (1 mM) at different ASP concentrations is shown in
Figure 8A. As seen, the
degree of TPentA-induced stationary current inhibition increases with
ASP concentration. The mean values of 1 IBS/ICS for TEA (2 mM), TPA (1 mM),
TBA (0.15 mM), and TPentA (1 mM) depending on ASP concentration are shown in
Figure 8B. The degree of the stationary current
inhibition did not depend on the agonist concentration for TEA (the
mean values were not significantly different, p > 0.9;
n = 7) and TPA (the mean values were not significantly
different, p > 0.3; n = 6). In the
case of TBA, 1 IBS/ICS
decreased (the mean 1 IBS/ICS
values were significantly different, p < 0.003; n = 5), whereas in the case of TPentA it rose with the
agonist concentration (the mean 1 IBS/ICS
values were significantly different, p < 106; n = 6).
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Figure 8.
Experimental dependence of the stationary current
inhibition on the agonist concentration. A, Example of
experimental curves. ASP alone and together with 1 mM
TPentA was applied for 2.5 sec at concentrations of 6.25, 12.5, 25, 50, and 100 µM. The superposition of the control and blocked
currents at each ASP concentration is shown. B, The mean
values of the degree of the stationary current inhibition, 1 IBS/ICS,
for tetraalkylammonium compounds were plotted against the ASP
concentration. The 1 IBS/ICS
values for TEA (2 mM) and TPA (1 mM) were not
significantly different at different ASP concentrations. The mean
1 IBS/ICS
values for TEA (0.47 ± 0.02; n = 7) and TPA
(0.50 ± 0.01; n = 4) are represented by
horizontal lines and correspond to the agonist
dependence predicted by model 5 at [B] = 0.91 and 0.98 Kd, respectively. The 1 IBS/ICS
values for TBA (0.15 mM) and TPentA (1 mM) were
significantly different at different ASP concentrations. The degree of
the stationary current inhibition decreased with the ASP concentration
for TBA (n = 8) and increased for TPentA
(n = 6). The solid lines are the
predictions of model 4 at [B] = 1.02 Kd
for TBA and model 1 at [B] = 51 Kd for
TPentA (see Results).
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Models 1-5 also predicted qualitatively different agonist dependencies
(Fig. 9). 1 IBS/ICS
for models 1-3 increased with the agonist concentration. The
corresponding agonist dependencies coincided at the blocker
concentration, [B] = 28, 2.6, and 1.1 Kd for models 1, 2, and 3, respectively, and were well fitted with the following logistic
equation:
![1−<FR><NU>I<SUB><UP>BS</UP></SUB></NU><DE>I<SUB><UP>CS</UP></SUB></DE></FR>=<FR><NU>A<SUB>1</SUB>−A<SUB>2</SUB></NU><DE>1+([<UP>A</UP>]/[<UP>A</UP>]<SUB>0</SUB>)<SUP>∧</SUP>n<SUB><UP>Hill</UP></SUB></DE></FR>+A<SUB>2</SUB>.](/content/vol19/issue24/fulltext/10611/img002.gif)
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(2)
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The values of parameters were as follows:
A1 = 0, A2 = 0.515 ± 0.002, [A]0 = 8.18 ± 0.15 µM, and nHill = 1.39 ± 0.04. On the contrary, the 1 IBS/ICS
value for model 4 decreased with the agonist concentration. The
corresponding agonist dependence at [B] = 2.5 Kd was well fitted with Equation 2 at
A1 = 0.733 ± 0.003, A2 = 0.515 ± 0.001, [A]0 = 17.9 ± 0.6 µM, and nHill = 1.13 ± 0.03. The degree of the stationary current inhibition
for model 5 did not depend on the agonist concentration and was equal to 0.515 at [B] = 1.1 Kd. Therefore,
the models that imply that the agonist cannot dissociate from the
blocked channel (models 1-3) predict an increasing degree of block
with increasing agonist concentration, whereas the models that imply
that the blocker does not prevent the agonist dissociation predict a
decreasing degree of block with increasing agonist concentration (model
4) or no dependence of the degree of block on agonist concentration at
all (model 5). The agonist dependence criterion is valid at any values
of [B] in the range of the P0,
wash, and
k2 values identified in the Figure 4
legend. By this criterion, the action of TAA must be described by one
of models 1-3 in the case of TPentA, by model 4 in the case of TBA,
and by model 5 in the cases of TPA and TEA. The corresponding simulated
agonist dependencies for TEA, TPA, TBA, and TPentA are shown in Figure
8B by solid lines at [B] = 0.91 Kd (model 5), 0.98 Kd (model 5), 1.02 Kd (model 4), and 51 Kd (model 1), respectively. The
fitting parameters for TBA (model 4) and TPentA (model 1) were as
follows: A1 = 0.518 ± 0.002, A2 = 0.304 ± 0.001, [A]0 = 15.9 ± 0.3 µM, and nHill = 1.18 ± 0.02 for TBA and A1 = 0, A2 = 0.655 ± 0.003, [A]0 = 6.46 ± 0.10 µM, and nHill = 1.42 ± 0.04 for TPentA. The agonist dependence criterion is
sensitive to the blocker effect on desensitization. Thus, model 5, implying that the blocker does not affect channel desensitization,
demonstrates the absence of the agonist dependence, although the same
model without a desensitized blocked state (model 4), implying that the
blocker prohibits the channel desensitization, predicts that the degree
of the stationary current inhibition diminishes with the agonist
concentration. Correspondingly, all the modifications of model 5, implying that the blocker promotes channel desensitization predict an
increasing agonist dependence (data not shown). In contrast, model 5a,
implying the existence of two blocking sites, demonstrates the absence
of the agonist dependence as in the case of the nonmodified symmetric
model 5. Therefore, modifications of model 5, implying that the blocker promotes channel desensitization, cannot describe the TEA action, for
which the fraction of the stationary current inhibition did not depend
on ASP concentration (Fig. 8B). However, it
can be well described by model 5a with two binding sites that cannot be
occupied simultaneously by two different TEA molecules and differing by
the rates of the blocker binding to and dissociation from them. A
variety of two-site model modifications could be also offered to
describe the effects of TEA. Thus, the consequence of occupation of the
sites could be different (Sobolevsky, 1999): (1) any site can be
available from the external media, but the blocking molecule bound to
one of them cannot "jump" to the other; (2) only one site can be
available from the external medium, and the second site can be occupied
via a sequential jump of the blocker molecule from the first site; and
(3) both sites are available from the external medium, and the blocker
bound to one of them can jump to the other. A much greater number of
two binding site models could be obtained by possible variations of the
kinetic constants. Analysis of such a huge variety of two binding site models was not the aim of the present study, and here we will not
develop this topic any more. The only clear conclusion that can be made
from the consideration of model 5a is the existence of a fast-occupied
TEA blocking site in the NMDA channel, the blocker molecule binding to
which does not prevent the channel closure, desensitization, and
agonist dissociation.
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Figure 9.
Agonist dependencies of the stationary current
inhibition predicted by models 1-5. The degree of the stationary
current inhibition, 1 IBS/ICS,
rises with the agonist concentration for models 1-3, decreases for
model 4, and is constant for model 5. The agonist dependencies
predicted by models 1, 2, and 3 coincided at [B] = 28, 2.6, and 1.1 Kd, respectively, and were well
fitted with Equation 2 (solid line). The values of the
fitting parameters were as follows: A1 = 0, A2 = 0.515 ± 0.002, [A]0 = 8.18 ± 0.15 µM, and
nHill = 1.39 ± 0.04. The fitting
of the agonist dependence predicted by model 4 at [B] = 2.5 Kd (solid line) gave the
following values of the fitting parameters:
A1 = 0.733 ± 0.003, A2 = 0.515 ± 0.001, [A]0 = 17.9 ± 0.6 µM, and
nHill = 1.13 ± 0.03. The degree
of the stationary current inhibition for model 5 did not depend on the
agonist concentration and was equal to 0.515 at [B] = 1.1 Kd. The values of parameters were as
follows: P0 = 0.09, wash = 30 msec, and
k2 = 1000 sec1.
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Another important criterion for the effect of the blocker on agonist
dissociation is the kinetics of tail currents after termination of the
agonist application in the continuous presence of the blocker. This
criterion is not sensitive to the effect of the blocker on channel desensitization.
Tail currents in the continuous presence of the blocker
In contrast to the agonist and the blocker coapplication (Fig. 2),
the application of ASP in the continuous presence of the blocker was
not followed by the hooked current, as illustrated in Figure
10A with TBA (1 mM). The kinetics of the tail current after ASP
application in the continuous presence of the blocker (b)
was different in comparison with that of the control (c) for different blockers (Fig. 10B). Such blockers as TEA
and TPA did not affect the tail current kinetics: the b
decay was practically identical to the c decay. This fact is
clearly illustrated in the insets, where the normalized
c and b curves are superimposed. In contrast,
TPentA caused a pronounced delay in the current recovery kinetics,
which is manifested in the intersection of curves c and
b. Such an intersection was never observed in the case of TBA: curves c and b were tangent, or curve
b was clearly below curve c (Fig.
10B). However, there was a small delay in the
recovery kinetics, which can be revealed only after superposition of
the normalized tail currents (Fig. 10B, inset). In
the majority of cells (n = 14 of 16), the normalized
curve c was below the normalized curve b, but in
2 of 16 cells these curves coincided.
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Figure 10.
Effects of the continuous presence of the blocker
on tail currents. A, Experimental protocol with TBA as
an example. ASP (100 µM) was applied for 2 sec in the
control external solution or in the continuous presence of 1 mM TBA. B, The control tail currents
(c) are superimposed with the tail currents in
the continuous presence of TEA (2 mM), TPA (0.6 mM), TBA (1 mM), and TPentA (0.5 mM) (b). The same labels apply to all
calibrations. Insets, Superposition of the normalized
curves c and b.
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Computer simulation clarified the origin of all these effects. Figure
11 shows that the time course of the
tail current in the continuous presence of the blocker predicted by
models 4 and 5 is very similar to the control tail current: the
nonnormalized curves b4 and b5 do not intersect
with the control curve c, whereas the normalized curves
b4 and b5 coincide with curve c (see
inset). In contrast, intersection of curves b1,
b2, and b3 with curve c points to a
considerable blocker-induced delay in the tail current kinetics
predicted by models 1, 2, and 3, respectively. The common feature of
these three different models (1-3) is that they exclude the agonist
dissociation from the blocked channel. Thus, it is just the trapping of
the agonist in the blocked channel that is responsible for the delay in
the final channel closure in the presence of the blocker in the washout
solution. The criterion of the tail currents in the continuous presence
of the blocker is valid at any values of
P0 (from 0.04 to 0.5),
wash (from 0 to 300 msec), and [B] and
k2 > 0.3 µM/sec. According to this criterion, TEA, TPA,
and TBA,do not prohibit the agonist dissociation, whereas TPentA
does.
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Figure 11.
Tail currents in the continuous presence of the
blocker predicted by models 1-5. The experimental protocol is the same
as shown in Figure 10A. The control tail current
(c) is superimposed with the tail currents in the
continuous presence of the blocker for models 1-5 (curves
b1-b5, respectively). Curves
b1-b3 intersect with curve
c, whereas curves b4 and
b5 do not. To achieve the same degree of the stationary
current inhibition, the blocker concentration was different for
different models: [B] = 28, 2.55, 1.09, 2.07, and 0.98 Kd for models 1, 2, 3, 4, and 5, respectively. The values of the parameters are as follows:
P0 = 0.09, wash = 30 msec, and
k2 = 1000 sec1.
Inset, Normalized curves c and
b1-b5. The control tail current (curve c)
and the normalized tail currents in the continuous presence of the
blocker predicted by models 4 and 5 (curves b4 and
b5) practically coincide.
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The criterion under consideration can also be named as a criterion of
the blocker-induced prolongation of NMDA channel activation. Thus, the
blocker prohibiting the agonist dissociation induces prolongation of
NMDA channel activation. If during such prolongation we accelerate the
channel transition from the blocked state, OAAB, to the nonblocked state, OAA* (models 1-3), a
large-amplitude tail current will be generated. Such acceleration was
achieved in the experiments with 9-aminoacridine by termination of the blocker application (Benveniste and Mayer, 1995; Koshelev, 1995) or
membrane depolarization (Benveniste and Mayer, 1995). In the latter
case, the large-amplitude tail current had an outward direction. Our
computer experiments showed that the amplitude of such tail currents
increases with the blocker concentration (when the occupation of the
blocked states increases) and a decrease in the time between the
termination of the agonist application and the accelerating stimulus,
whereas their kinetics is mainly defined by the rate constant of the
blocker dissociation, k2 (data not shown).
Consideration of TAA action according to a set of criteria
Based on consideration of models 1-5, the present study reveals a
set of criteria that allow one to determine the effect of fast blockers
on the channel closure, desensitization, and agonist binding
(dissociation). These criteria are listed in Table
2.
According to criteria listed in Table 2 and taking into account
everything mentioned above, TEA action can be described by model 5a
with two blocking sites, to which two blocker molecules cannot bind
simultaneously. To explain the inability of the simultaneous occupancy,
these sites can be supposed to overlap or to be located so close that
electrostatic repulsion does not allow two TEA molecules to bind to
them simultaneously (Sobolevsky, 1999). The binding of the TEA molecule
to the fast occupied site (the main site, because
Afast = 0.67) does not prohibit the
channel closure, desensitization, and agonist dissociation from the
blocked channel. Elucidation of the properties of the second, slowly
occupied TEA blocking site requires further experiment.
The effect of TPA can be best described by model 5. Therefore, we may
conclude that TPA does not prohibit the channel closure, desensitization, and agonist dissociation from the blocked channel. The
cases when the Afast and
(IBS/IB0)/(ICS/IC0)
values were slightly higher than unity gave us the reason to suppose
that TPA can slightly prevent NMDA channel desensitization.
TPentA action can be well described by model 1. According to this
model, TPentA prohibits both the channel closure and desensitization and the agonist dissociation from the blocked channel.
According to the set of criteria listed in Table 2, TBA action should
rather be described by model 4. However, some observations point to the
necessity of its modification. These observations are as follows: (1)
in contrast with the prediction of model 4 with ' = , ' = ,
l2' = l2, and
l1' = l1 (see Fig. 4A), in the majority of experiments the hooked current exceeded the value of
the stationary control current, ICS
(see Fig. 2); (2) at low values of the time constant of the solution
exchange, wash 10 msec, the fast
component appeared in the falling phase of the recovery kinetics of TBA
in the continuous presence of ASP; (3) in accordance with modeling
prediction (Fig. 11), the control tail current and the nonnormalized
blocked tail current in the continuous presence of TBA did not
intersect (Fig. 10B). However, in the majority of
experiments the normalized blocked tail current lay above the control
tail current (Fig. 10B, inset); this
circumstance is in obvious contradiction with model 4, which predicted
their coincidence (Fig. 11, inset).
In principle, modification of model 4 can be fulfilled by means of
changes in the closure-opening transition
(OAAB-CAAB) or the agonist
binding-dissociation transitions
(CAAB-CAB-CB).
When we modified model 4 via changes in the agonist
binding-dissociation transitions, in compliance with the three facts
listed above, we were forced to predict that the blocker hampered the
agonist dissociation from the closed blocked channel. Such a
modification did not predict the fast component in the falling phase of
the recovery kinetics in the continuous presence of the agonist
(similar to model 2) and considerably changed the agonist dependence of the stationary block by transforming it from the "descending type" predicted by the nonmodified model 4 (Fig. 9) to the "ascending" one similar to the agonist dependencies predicted by models 1-3. However, in the cases when the solution exchange was comparatively fast, the descending phase of the TBA recovery kinetics contained the
fast component (see above), and the agonist dependence observed experimentally was descending (Fig. 8B). Therefore,
the only possibility to modify model 4 to simulate the experimental
observations was to correct the
OAAB-CAAB transition,
implying that the blocker increased the open probability of the blocked
channel. There are two ways to increase the open probability. The first
one is to increase the kinetic constant of the channel opening, '.
In this case, the blocker promotes the channel opening. The second one consists in reducing the rate constant of the channel closure, '. In
this case, the blocker slows the channel closure. This case is
especially natural because the larger tetraalkylammonium compound,
TPentA, was found to prohibit the channel closure. Both cases stipulate
similar changes in the modeling kinetics. An example of predictions of
model 4, implying that the blocker slows the channel closure, is shown
in Figure 12. The kinetic constant of the channel opening, , was assumed to be the same (20 sec1) for a blocked and a nonblocked channel. In contrast,
the value of the closure rate constant for the blocked channel was
assumed to be 10 times lower ( |
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ABSTRACT
INTRODUCTION
