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The Journal of Neuroscience, October 15, 1998, 18(20):8175-8185
Activity-Dependent Modulation of Glutamate Receptors by
Polyamines
Derek
Bowie1,
G. David
Lange2, and
Mark L.
Mayer1
1 Laboratory of Cellular and Molecular Neurophysiology,
National Institute of Child Health and Human Development, and
2 Instrumentation and Computers Section, National Institute
of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
The mechanisms by which polyamines block AMPA and kainate receptors
are not well understood, but it has been generally assumed that they
act as open-channel blockers. Consistent with this, voltage-jump
relaxation analysis of GluR6 equilibrium responses to domoate
could be well fit, assuming that spermine, spermidine, and
philanthotoxin are weakly permeable open-channel blockers. Analysis of
rate constants for binding and dissociation of polyamines indicated
that the voltage dependence of block arose primarily from changes in
koff rather than
kon. Experiments with changes in Na
concentration further indicate that the voltage dependence of polyamine
block was governed by ion flux via open channels. However, responses to
1 msec applications of L-Glu revealed slow voltage-dependent rise-times, suggesting that polyamines additionally bind to closed states. A kinetic model, which included closed-channel block, reproduced these observations but required that polyamines accelerate channel closure either through an allosteric mechanism or by
emptying the pore of permeant ions. Simulations with this model reveal
that polyamine block confers novel activity-dependent regulation on
calcium-permeable AMPA and kainate receptor responses.
Key words:
polyamines; glutamate receptors; plasticity; channel
block; kinetic analysis; AMPA; kainate; ion channel block; ionic
mechanism
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INTRODUCTION |
Ionotropic Glu receptors
(GluR) generated from AMPA receptor subunits respond to the
transient presence of Glu in the synaptic cleft with rapid kinetics
such that, at most synapses, AMPA receptors relay the signal mediating
action potential initiation in the postsynaptic cell, whereas
NMDA serves a modulatory role (Jonas and Spruston, 1994
).
Functional roles for kainate receptors at presynaptic (Chittajallu et
al., 1996; Rodriguez-Moreno et al., 1997) and postsynaptic (Castillo et
al., 1997; Vignes and Collingridge, 1997; Mulle et al., 1998) sites
have also been identified in recent studies. RNA editing at the Q/R
site of AMPA and kainate receptors (Sommer et al., 1991; Higuchi et
al., 1993) regulates Ca2+ permeability (Jonas and
Burnashev, 1995), anion versus cation selectivity (Burnashev et al.,
1996), single-channel conductance (Howe, 1996; Swanson et al., 1996,
1997), and voltage-dependent block by cytoplasmic polyamines (Bowie and
Mayer, 1995; Donevan and Rogawski, 1995; Isa et al., 1995; Kamboj et
al., 1995; Koh et al., 1995). In each case, the positively charged Arg
or neutral Gln residues at the Q/R site presumably influence the
electrostatic environment of the pore differently and thus accounts for
the distinct permeation properties of the Q and R forms.
There is increasing evidence for cell-specific expression of individual
Glu receptor subtypes such that, in interneurons that lack GluR-B such
as basket cells of the dentate gyrus, AMPA receptors have high
Ca2+ permeability and rapid gating (Geiger et al.,
1995, 1997) and hence should be subject to modulation by cytoplasmic
polyamines. It has been proposed that the gating properties of synaptic
receptors are pivotal in defining the functional roles fulfilled by
interneurons in hippocampal network activity (Jefferys et al., 1996;
Buzsáki and Chrobak, 1995). The physiological role fulfilled by
polyamines and the possibility that polyamines could modulate the
gating of Glu receptors has been neglected, despite their ubiquitous presence in all cells (Pegg, 1986) at cytoplasmic concentrations sufficient to produce strong block of AMPA and kainate receptors (Bowie
and Mayer, 1995; Kamboj et al., 1995; Koh et al., 1995).
For inward rectifying potassium (Kir) channels,
polyamine block has been proposed to stabilize the membrane potential
at rest around the reversal potential
(Vrev) for K+ ions
(Hille, 1992; Nichols and Lopatin, 1997). Under conditions of
excitation, depolarization increases block by polyamines of Kir channels, thus lowering the overall membrane
conductance and reducing the metabolic expenditure of the cell
for ionic homeostasis (Hille, 1992). A similar physiological role of
polyamines for AMPA and kainate receptors would help to reduce influx
of Na+ and efflux of K+ during
action potential firing evoked by Glu-activated synaptic responses and
could also reduce shunting of the action potential amplitude by EPSCs.
We have shown previously that kainate receptors generated by GluR6(Q)
and AMPA receptors generated by GluR-A both show birectifying responses
in the presence of cytoplasmic polyamines, suggesting a common blocking
mechanism (Bowie and Mayer, 1995). We now propose that polyamine block
is bimodal in nature and attributable to both open- and closed-channel
block mechanisms. A 12-state cyclic gating model was developed to
satisfy our experimental observations. The model accounts for our
observation that the rate of activation of responses to Glu is slowed
in the presence of polyamines and reveals novel activity-dependent
plasticity of AMPA and kainate receptor responses.
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MATERIALS AND METHODS |
Cell culture and expression of recombinant receptors.
HEK 293 cells (CRL 1573; American Type Culture Collection, Manassas, VA) were maintained at a confluency of 70-80% in minimal
essential medium with Earle's salts, 2 mM Gln, and 10%
fetal bovine serum. Twenty-four hours after plating at low density
(2 × 104 cells/ml) onto the center of 35 mm
Petri dishes, cells were transfected using the calcium phosphate
technique; cotransfection with the cDNA for green fluorescent protein
(S65T mutation) helped to identify transfected cells during experiments
as described previously (Bowie and Mayer, 1995). Cells were washed with
PBS 12-18 hr after transfection and used for
electrophysiological recordings after another 24-48 hr. We used a cDNA
clone for GluR6(Q) incorporated into a cytomegalovirus expression
vector (a gift from Dr. P. Seeburg, Max Planck Institute, Heidelberg,
Germany).
Recording conditions. Unless indicated, all experiments were
performed in solutions with symmetrical 150 mM Na
containing low concentrations of external divalents to minimize weak
voltage-dependent block by Ca and Mg ions (Bowie and Mayer,
1996). The external solution was composed of 150 mM
NaCl, 5 mM HEPES, and 0.1 mM each of
CaCl2 and MgCl2. The internal solution
contained 120 mM NaCl, 10 mM NaF, 5 mM HEPES, 5 mM Na4BAPTA, and 0.5 mM CaCl2 to which spermine (Spm), spermidine
(Spd) (Sigma), and philanthotoxin 343 (PhTX 343) (Research
Biochemicals, Natick, MA) were added as required. In both cases, pH was
adjusted to 7.3, and osmolarity was adjusted to 295 mOsm with sucrose.
In experiments shown in Figure 4, the external solution was adjusted
with NaCl to give 405 mM total Na content; the
corresponding internal solution contained 375 mM NaCl.
Na2ATP (10 mM) was added to the internal
solution in experiments performed in the absence of internal blocker to
chelate endogenous polyamines present after patch excision
(Bähring et al., 1997). In this case, NaCl content was reduced to
110 mM and 360 mM NaCl to maintain the free Na
concentration at 150 and 405 mM, respectively. The program
BAD (Brooks and Storey, 1992) was used to calculate complex
formation with ATP and hence the free Na+
concentration.
All recordings were made with an Axopatch-200A amplifier (Axon
Instruments). Outside-out patches were excised from HEK 293 cells using
fire-polished thin-walled borosilicate glass pipettes (2-5 M
)
coated with dental wax to reduce electrical noise. Series resistance
(3-10 M) was routinely compensated by 95%. Current records were
filtered (eight-pole Bessel filter) at 25 kHz, digitized at 50 kHz, and
stored on a Macintosh IIfx or a Power Macintosh 7600/132 using a 16 bit
analog-to-digital converter (ITC-16; Instruteck Corp., Elmont,
NY) under control of the program Synapse (Synergy Research,
Silver Spring, MD). Two types of experiments were performed in this
study. In the first, patches were treated with concanavalin-A (0.1 mg/ml for 1-1.5 min) to reduce desensitization, and the high-affinity GluR6 agonist domoate was applied via a stepper motor-based fast perfusion system (Vyklicky et al., 1990) at a saturating concentration (50 µM) to promote a high probability of channel opening.
The rate of onset of block (see Fig.
1A,B) was then studied using a
series of voltage steps (5-15 msec duration) from a holding potential
of 
100 mV, at which almost no block occurs, stepping to more
depolarized potentials (85 to +125 mV, 15 mV increments) at which
polyamine block is stronger. The rates of recovery from block (see Fig.
2F) were measured in patches held at +40 mV, at which
strong block occurs, stepping to a range of potentials which increased
the rate of dissociation or permeation as appropriate (100 to +125
mV). Data obtained from each experimental paradigm were
leak-subtracted. In the second experiment type, excised patches were
placed near the interface of a four-bore glass flow pipe with control
and 10 mM Glu-containing solutions fed by gravity. The
solution was rapidly exchanged by displacement of the flow pipe using a
piezoelectric stack (Physik Instrumente). The solution exchange rate
was routinely determined at the end of each experiment by measuring the
liquid junction potential between the solution containing 10 mM Glu and control solution in which total
Na+-content was reduced by 5%. Typically, 100-150
trials were averaged to obtain the junction currents. Data were
discarded from patches in which the liquid junction currents exhibited
slow rise times. Experiments were performed at room temperature
(24-26°C).
Kinetic analysis of voltage-jump relaxations.
Model-independent analysis, such as exponential fitting and 10-90%
rise times, were performed using the Synapse program (Synergy
Research). The latency in channel opening observed in the presence of
Spm was determined by aligning records with the piezoelectric stimulus artifact and subtracting the delay between the stimulus artifact and
the rising phase of the junction current from the stimulus artifact and
the rise of Glu-activated currents at 100 mV. In all experiments,
block by polyamines exhibited first order kinetics, suggesting that
transitions between the two binding sites in a previously developed
Eyring rate theory model can be reasonably well approximated by a
single state (Bähring et al., 1997). To estimate block rate
constants from voltage-jump experiments (see Fig.
2A), a function describing the time- and
voltage-dependent nature of block for a single site model was defined
and, as described below, appropriate algebraic substitutions were made
to ensure that fit parameters converged on unique values. Code was
written that permitted the simultaneous fitting of multiple current
records at the various voltages tested (100 to +125 mV) with a
nonlinear, steepest descent algorithm ("NonlinearFit") provided in
the program Mathematica (Wolfram Research). The first 100 µsec of
experimental data were masked to eliminate the rising phase of the
voltage step, and current amplitudes were normalized to the response at 100 mV. Fits of a typical experiment required 200-300 megabytes of
random access memory and 40-60 min to allow convergence of the
fitting procedure. For convenience, fits were semiautomated in batches
on a 167 MHz UltraSPARC Enterprise 3000 (Sun, Inc.).
The current relaxations observed with polyamines in voltage-jump
experiments were fit by the blocking scheme shown below:
where polyamines on the inside bind to a single site with a rate
determined by the blocker concentration [B] and
kon. Once bound, the blocker can leave the
channel by either of two reactions: it may return to the inside
(koff) or permeate through the channel to
the external solution (kperm). Each rate
constant is a function of voltage (V) defined
as:
|
(1)
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(2)
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(3)
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Current relaxations were fit by the function
I(t), which describes membrane
current at any time point and voltage:
![I<SUB>(<UP>t</UP>)</SUB>={(I<SUB>0</SUB>−I<SUB>∞</SUB>)<UP>exp</UP>(<UP>−</UP>([B]k<SUB><UP>sum</UP></SUB>t))+I<SUB>∞</SUB>}](/content/vol18/issue20/fulltext/8175/img005.gif)
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(4)
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where ksum is the sum of all rate
constants (Eqs. 1-3) and t is time. Membrane current before
blocker entry (I0) was defined as:
|
(5)
|
where Vrev is the reversal potential,
G0 is conductance at 0 mV,
Gmin is the minimal conductance (normalized
value of 1), and Vc is a constant. Averaged
values for Vrev,
G0, and Vc were 1.06 ± 0.57, 1.10 ± 0.01, and 51.1 ± 0.10 mV
(n = 57 patches; mean ± SEM), respectively, and
did not vary with blocker concentration. Membrane current at
equilibrium block (I
) was defined as:
![I<SUB>∞</SUB>=<FR><NU>I<SUB>0</SUB></NU><DE>1+<FR><NU>[<IT>Blocker</IT>]</NU><DE>K<SUB><UP>d</UP></SUB></DE></FR></DE></FR>](/content/vol18/issue20/fulltext/8175/img007.gif)
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(6)
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To obtain unique fits to Equation 4, the equilibrium
dissociation constant (Kd) calculated
from Equations 1-3 as:
|
(7)
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was redefined to
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(8)
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where
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(9)
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and
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(10)
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Thus, koff and
kperm can also be redefined accordingly:
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(11)
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(12)
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Definitions of koff and
kperm (Eqs. 11 and 12) were substituted into
ksum of Equation 4, and this form of the
function with the redefined Kd (Eq. 8) were used
to determine rate constants from fits to experimental data. Unless
indicated otherwise, all values are presented as the mean ± SEM
in the text and figure legends.
Kinetic modeling of GluR6(Q) channel activity. The basic
framework for the construction of a gating model for GluR6 was aided by
previous studies investigating the kinetic behavior of this receptor
(Heckmann et al., 1996; Traynelis and Wahl, 1997). Simulations were
performed using code written in Mathematica assuming 1000 channels of
conductance at 16 pS (Traynelis and Wahl, 1997). Occupancy of each
state was calculated from Q-matrices using the method of Colquhoun and
Hawkes (1977) and Mathematica code provided by Dr. A. Roth (Max Planck
Institute, Heidelberg, Germany), available at MathSource
Electronic Library (http://www.mathsource.com). Numerical values
assigned to rate constants for binding and gating steps gave a maximum
open probability consistent with dose-response analysis (Traynelis and
Wahl, 1997). Close attention was given to choosing values for channel
closure (
) and the rate of entry (d1)
into the double-liganded, desensitized state to match the decay rates
of responses observed experimentally after 1 and 50 msec pulses of 10 mM Glu at a range of membrane potentials (100 to +125
mV). Although single-channel analysis reveals substates for GluR6(Q)
(Swanson et al., 1996), deactivation of responses to 1 msec pulses of
Glu was well fit by a single exponential function, justifying the use
of a single open state. Experimentally observed decay rates were weakly
voltage-dependent in both cases and could be adequately modeled when
was assumed to be voltage-dependent (see Fig.
6B). The state diagram for the simple sequential
open-channel-blocking scheme in Model 1 is shown
below:
with rate constants as follows: k1 = 2 × 107 mol
1
sec1; k1 = 300 sec-1; k2 = k3 = 1 × 107
mol1 sec1;
k2 = 105
sec1; d1 = 100 sec1; d1 = 1 sec-1; d2 = 1.39 × 104 sec1;
d2 = 0.2 sec1; 
= 5000 sec1; = 324 exp(V/305)
sec1. To satisfy the law of microscopic
reversibility, k3 was set to 144 sec1. The numerical values for Spm block rate
constants are summarized in Table 1. To
simulate the gating of homomeric GluR-D (see Fig. 6), the channel
closing rate was increased fivefold ( = 1500 exp(V/305)
sec1); in this case, current relaxations generated
by 1 msec applications of 10 mM L-Glu decayed
with first order kinetics in the absence of polyamines (tau, 0.6 msec
at 60 mV), similar to previously published experimental values
(Lomeli et al., 1994). Simulations of trains were performed with
desensitized states omitted from the model.
The state diagram of Model 2 for a mechanism with closed- and
open-channel block is shown below:
We assumed that Spm does not affect agonist binding or
desensitization rates. Therefore, the numerical values assigned to these rate constants are the same as for Model 1. The closing rate
constant (') was twofold faster for open blocked channels to account
for the experimentally observed faster rate of deactivation in the
presence of Spm. In addition, before the first agonist application, all
channels were assumed to be in the closed blocked state of the
channel (RB). This satisfies the slow voltage-dependent rise times
observed experimentally in the presence of Spm, because channels first
have to unblock before reaching the open state. We have no quantitative
information describing the kinetics of closed-channel block and,
therefore, simulations were performed omitting reactions between closed
and closed blocked states. However, recent experiments indicate that
the rate of polyamine block of closed AMPA receptors occurs on a time
scale that would not significantly influence the simulations performed
in the present study (Rozov et al., 1998).
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RESULTS |
Rapid onset of open-channel block by polyamines
Although it is known that the development and recovery of GluR
block by polyamines is very rapid, previous studies with voltage jumps
had failed to resolve the relaxations expected for open-channel block
by Spm (Koh et al., 1995). To study the kinetics of block by
polyamines, the membrane potential of outside-out patches containing GluR6(Q) channels was stepped in 15 mV increments from 100 mV to +125
mV in the presence of the weakly desensitizing agonist domoate (50 µM). Membrane currents after depolarizing voltage steps
showed well resolved relaxations, which decayed with first order
kinetics when 15-100 µM PhTX (n = 15)
(Fig. 1A), 5-30
µM Spm (n = 19) (Fig.
1B), or 15-100 µM Spd
(n = 23; data not shown) were added to the internal
solution. I-V relationships constructed for peak responses
exhibited weak outward rectification (Fig. 1C,D),
similar to that observed in the absence of polyamines (Figs. 1E,F,
2C). In contrast,
I-V plots constructed 5 msec after the onset of
depolarization were strongly rectifying, consistent with previous
analysis of equilibrium polyamine block (Bowie and Mayer, 1995;
Bähring et al., 1997; Bähring and Mayer, 1998). In contrast to Spm and Spd, which showed biphasic rectification attributable to
relief of block with strong depolarization, outward currents remained
fully blocked in the presence of 100 µM PhTX, consistent with previous findings that the larger cross-sectional width of PhTX
greatly slows progress through the ion-permeation pathway (Bähring et al., 1997; Bähring and Mayer, 1998). Because
current relaxations were observed only when polyamines were added to
the internal solution (Fig. 1E), our observations
suggest that after voltage steps from 100 mV, open channels begin to
accumulate in a voltage-dependent blocked state and that the current
decay observed in each case reflects the time course of polyamine
block.
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Figure 1.
Time course of open-channel block by polyamines.
A, GluR6(Q) responses recorded from an outside-out patch
after voltage steps from 100 to +125 mV in 15 mV increments with 50 µM domoate and 100 µM internal PhTX.
B, Similar experiment on another patch with 30 µM internal Spm. C, D,
I-V relationship for the responses shown in
A and B measured at peak (open
circles) and 5 msec after depolarization (closed
circles). E, In the absence of internal
polyamines, GluR6(Q) responses to domoate did not show relaxations
after voltage steps from 100 to +125 mV. Dashed lines
in A, B, and E indicate
zero current. F, G-V plot for
seven experiments similar to that shown in E (mean ± SD) normalized to the conductance at 100 mV. The continuous
line through the data points predicts the voltage dependence of
domoate responses in the absence of polyamines and was generated using
Equation 5, with mean values for G0
(1.10 ± 0.01) and Vc (51.1 ± 0.10 mV) obtained from kinetic analysis of responses in the presence of
Spm, Spd, and PhTX (n = 57 patches; see Table 1),
as shown in Figure 2A.
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Figure 2.
Fits of a sequential open-channel block model to
polyamine responses. A, Current relaxations recorded
with 50 µM domoate and 20 µM internal Spm
after voltage steps from 100 to +95 mV in 15 mV increments.
Lines through the data points are fit by a single
binding site reaction scheme for a permeant blocker (Eq. 4).
B, Rate of onset of block observed at different voltages
with 20 µM Spm (n = 5; mean ± SD). Data points indicate the reciprocal of the time constant of single
exponential fits to responses like those shown in A;
solid lines through the data points are the sum of mean
values for kon,
koff, and
kperm (n = 57 patches;
see Table 1) estimated by fitting Equation 4 to responses like those
shown in A. C, I-V plots
recorded either in the absence of blocker (n = 7)
or with 20 µM Spm (n = 5); in the
latter case, responses were measured 100 µsec and 5 msec after the
onset of depolarization. Lines through data points show
the current predicted by Equation 4 using mean values from Table 1 at
time 0, before the onset of block, at 100 µsec after depolarization
and at equilibrium (t =  ). D,
Relaxations observed with different Spm concentrations (5, 10, and 30 µM) after voltage steps from 100 to +35 mV. Responses
are from different patches normalized to the amplitude of the fully
unblocked response at 100 mV; the rate of onset of block
(solid line) was fit by a single exponential.
E, Rate of onset of block at +35 mV for different
concentrations of Spm reveals a linear relationship, consistent with an
open-channel-blocking scheme. The solid line was fit by
linear regression; the slope yields an estimate for
kon at +35 mV of 7.4 × 107 mol1
sec1, and the intercept was 132 sec1. Both values are in good agreement with
predictions of 6.9 × 107
mol1 sec1 and 83 sec1, respectively, calculated from values in
Table 1. F, Relaxations after voltage steps from +40 mV
to potentials ranging from 100 to +110 mV with 30 µM
internal Spm. Simulations (smooth line) using mean
values for the rate constants for Spm block (Table 1) accurately
predict the time course of the relaxations, except at 85 and 100
mV, for which the reaction scheme predicts slightly faster
reequilibration than observed experimentally
(arrow).
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Kinetic analysis of rate constants for polyamine block
The kinetics of onset of block at each membrane potential were
used to determine rate constants for binding, unbinding, and permeation
of polyamines by fitting current relaxations with a single binding site
reaction scheme for a permeant blocker (see experimental procedures in
Materials and Methods). Figure 2A shows a
typical fit to experimental data, in this case, with 20 µM internal Spm. Fits of similar quality in which the
data points are fit well throughout the time course of the onset of
block were also obtained with PhTX and Spd (data not shown). Estimates
of rate constants were independent of blocker concentration; for
example, fits for 5, 10, 20, and 30 µM Spm gave estimates
for Kd(0 mV) of 3.3 ± 0.6 (n = 4), 4.1 ± 1.7 (n = 4),
2.8 ± 0.2 (n = 5), and 3.3 ± 0.7 (n = 6) µM, respectively, where
Kd was calculated from (koff + kperm)/kon. The
sum of the rate constants for binding, dissociation, and permeation
predicted well the experimentally observed kinetics of block at all
membrane potentials tested when relaxations were fit by single
exponential functions. Figure 2B shows an example for
20 µM Spm. As an additional test of the adequacy of the
estimated rate constants for predicting polyamine block, we simulated
I-V relationships at different time points to permit comparison with experimental observations. I-V plots in
Figure 2C show data from experiments performed in the
absence of polyamines (open circles) or with 20 µM Spm at 100 µsec and 5 msec after a depolarizing step
from 100 mV. Lines in Figure 2C through
the points are simulated I-V plots generated using the rate
constants for block by Spm (Table 1) and illustrate that the scheme
accurately predicts current before entry of polyamines into the
channel, as well as during the development of block. The voltage
dependence of binding (kon,
e-fold per 98.8 mV), unbinding
(koff, e-fold per 18.1 mV),
and permeation (kperm, e-fold
per 19.5 mV) for Spm suggests that at membrane potentials below the
threshold for initiation of action potentials the voltage dependence of
block is almost entirely attributable to the unbinding rate, a feature also possessed by our previously published Eyring rate theory model,
which contains an asymmetrical inner barrier (Bähring et al.,
1997). Rate constants of binding and dissociation for both Spd and PhTX
exhibited similar voltage dependencies to those for Spm, suggesting
that the location of the binding site was probably the same for each
blocker (Table 1).
Because the proposed mechanism of block displays first order kinetics,
we would expect the binding rates for polyamines to be linearly
dependent on concentration. To examine the effect of polyamine
concentration on block rates, current relaxations with different Spm
concentrations were compared at +35 mV, because the magnitude of
kon with respect to unbinding rates is greatest at this voltage (Fig. 2B). Figure
2D shows that current relaxations decayed more
rapidly with increasing Spm concentration, consistent with the
prediction. Block rates determined from single exponential fits of
similar data from multiple patches displayed a linear relationship with
Spm concentration, yielding an estimate for kon
at +35 mV of 7.4 × 107
mol1 sec1 (Fig.
2E), in excellent agreement with the value of
6.9 × 107 mol1
sec1 predicted from values in Table 1. These
observations are all consistent with an open-channel-blocking scheme.
The values of the rate constants estimated from fits to the data from
voltage-jump experiments are summarized in Table 1. Note that the rate
of binding (kon) of Spm was more than
threefold faster than for PhTX (Table 1). PhTX, which is a bulky analog
of Spd with a total charge of +3, most likely needs to assume a
particular orientation to enter the channel, reducing its effective
binding rate. Similar to results obtained with Spm, the rate constants
for kon,
koff, and kperm
for PhTX were independent of concentration of 15-100 µM.
In agreement with recent experimental observations (Bähring and
Mayer, 1998), simulations of equilibrium block by low concentrations of
PhTX showed saturation at strongly depolarized potentials because of
the voltage dependence of kperm (Table 1),
indicating that despite its large size PhTX is weakly permeant.
As a final evaluation of the adequacy of the fit parameters, responses
evoked by voltage steps from +40 mV were used to compare predictions
and experimental observations during reequilibration from blocked to
unblocked states; this is in contrast to the previous analysis of
responses to voltage steps from 100 mV (Fig. 2A), which reflect reequilibration from unblocked to blocked states. Figure
2F shows relaxations observed with 30 µM Spm after voltage steps from +40 mV to both
hyperpolarized and depolarized potentials for which recovery from block
occurs via unbinding and permeation of Spm, respectively. The
smooth line in Figure 2F
shows predictions of the reaction scheme, which matches well
experimental observations at all membrane potentials, except at 85 mV
and 100 mV (Fig 2F, arrow) where
reequilibration rates for the reaction scheme are slightly faster than
observed experimentally. This could indicate the existence of a closed
blocked state, the exit from which is slower than for open blocked
channels at strongly negative potentials. Indeed, although the results
shown in Figure 2 can be well explained by a simple model for
open-channel block, as described below, clear evidence for closed
blocked states was obtained from subsequent experiments indicating that
the action of polyamines is more complex than previously realized.
Analysis of responses to Glu reveals additional blocked states
To determine whether polyamine block might influence activity at
central synapses with unedited AMPA or kainate receptors (McBain and
Dingledine, 1993; Isa et al., 1996), we developed a kinetic model for
GluR6(Q) responses that matched well experimentally observed responses
to brief applications of Glu in the absence of polyamines. The rate
constants for polyamine block given in Table 1 were then used to
simulate responses with 20 µM internal Spm and 1 msec
pulses of a saturating concentration (10 mM) of the
neurotransmitter L-Glu, conditions typical for prototypic excitatory synapses (Clements et al., 1992). These simulated responses were then compared with experimental observations recorded using the
same concentrations of Glu and Spm. Figure
3A shows a typical control
experiment in which Glu was applied for 1 msec at different membrane
potentials (range, 100 to +125 mV) in the absence of internal
polyamines. Because of the brief duration of the agonist pulse, the
current decay reflects primarily the rate of channel closure. The rate
of decay after removal of agonist was weakly voltage-dependent,
decreasing e-fold per 300 mV depolarization (Fig.
3E). A six-state cyclic gating model with reaction rates chosen to simulate GluR6 responses described by Heckmann et al. (1996)
was modified to match additional properties revealed by our
experimental observations (Model 1) (see experimental procedures in
Materials and Methods) and gave simulated responses that
predicted well responses to Glu over a wide range of membrane
potentials (Fig. 3B).
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Figure 3.
Sequential block fails to predict the kinetics of
responses to Glu. A, Membrane currents evoked by 1 msec
applications of 10 mM Glu at various holding potentials
(100 to +125 mV, 15 mV increments) in the absence of internal
polyamines; lines fit through the data points are single
exponential functions. Top trace shows the junction
current (1 msec) recorded with an open patch electrode at the end of
the experiment. B, Simulation of GluR6(Q) responses by
Model 1 with the time course of agonist application adjusted to give
exchange rates similar to those achieved in experiments (top
trace). C, Simulation of GluR6(Q) responses by
Model 1 but with 20 µM internal Spm; note that at
potentials producing strong block, the outward currents decay
biexponentially. D, Experimentally recorded membrane
currents evoked by 1 msec applications of 10 mM Glu in the
presence of 20 µM internal Spm. Responses to Glu decayed
with first order kinetics at all voltages; lines fit
through the data points are single exponential functions.
E, Deactivation time constants as a function of voltage
for control responses (open circles)
(n = 7; mean ± SEM) and with 20 µM Spm (filled circles)
(n = 5; mean ± SEM). Deactivation was
accelerated by Spm at membrane potentials more positive than 50 mV.
The line fit through control responses indicates an
e-fold decrease in the time constant of deactivation per
303 mV depolarization; filled triangles indicate the
time constant of the slow component of deactivation
(y-axis scale is  of measured values)
estimated from double exponential fits to the simulated responses shown
in C. F, G-V plots for
peak Glu responses observed in the presence (n = 11 patches) and absence (n = 8 patches) of 20 µM Spm. Lines through the data points for
20 µM Spm show fits based on Equation 8, corrected for
the weak outward rectification of Glu responses observed in the absence
of polyamines (Vc = 56 mV; Eq. 5).
Filled triangles indicate peak conductance values
determined from measurement of simulated responses with the same
programs used for analysis of experimental data.
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When block by Spm was included in simulations with Model 1, the
current, particularly at positive potentials, decayed biexponentially with fast and slow components, reflecting the classical properties of a
sequential open-channel-blocking scheme (Neher, 1983), similar to
results obtained, for example, at the endplate (Colquhoun and Sheridan,
1981). The fast component of decay reflects the rate of open-channel
block, whereas the slow component reflects the channel burst length,
which is prolonged in the presence of open-channel blockers (Neher,
1983). In marked contrast to the predictions of Model 1, experimental
responses evoked by 1 msec pulses of 10 mM Glu in the
presence of 20 µM Spm decayed with first order kinetics
without fast and slow components (Fig. 3D). Indeed, the rate
of decay was faster than observed in the absence of blocker with no
evidence of the slow voltage-dependent component predicted by
simulations with 20 µM Spm (Fig. 3E).
Consistent with the notion that an open-channel block mechanism alone
is insufficient to account for the results shown in Figure
3D, conductance-voltage (G-V) plots for
peak responses determined from simulations with Model 1 for 20 µM Spm showed only weak attenuation of the peak response
to Glu in contrast to the strong and voltage-dependent block observed
experimentally (Fig. 3F). The model predicts weak attenuation of the peak response, because the rate of onset of block by
20 µM Spm (Fig. 2) is slow compared with the rate of activation of GluR6(Q) by 10 mM Glu in the absence of
polyamines. Together, these findings indicate that block had developed
before activation of channel gating by Glu, suggesting that polyamines can bind to closed channels.
Polyamine block slows the rise time of responses to Glu
If channels were already blocked before activation by agonist and
unblock after channels open, we would predict that the rising phase of
responses to Glu would show biphasic voltage dependence, determined by
the rate constants for binding and dissociation of polyamines to the
open state (Fig. 2). To investigate this possibility, the rise times of
Glu responses were analyzed in the presence or absence of blocker and
compared as functions of membrane potential. We predicted that if Glu
receptor channels were already blocked in the closed state, then the
rise time after activation by agonist would be slowed and that this
effect would be strongest at potentials in which polyamines bind with
highest affinity. Thus, we would expect the rise times to become faster with either hyperpolarization or depolarization away from the potential
for maximum block. Our experimental results fully confirmed this
prediction.
In the absence of Spm, the 10-90% rise times for responses to 10 mM Glu at 100 mV and +50 mV were similar, at 0.37 ± 0.05 and 0.44 ± 0.05 msec, respectively (n = 9 patches; mean ± SEM), and were essentially voltage-independent at
all membrane potentials tested (Fig.
4A,C).
When compared with open tip junction potentials recorded at the end of
experiments, responses to Glu developed with a delay of 62 ± 13 µsec, close to the limit of resolution determined by the solution
exchange rate and the sample period for data acquisition
(n = 9). In contrast, as shown in Figure 4,
B and C, Glu responses recorded with 20 µM internal Spm showed voltage-dependent rise times that
were much slower than control (1.44 ± 0.06 msec at +50 mV and
0.53 ± 0.04 msec at 100 mV) and a delay in channel opening
after Glu application (147 ± 21 µsec at 100 mV;
n = 9). The rise times exhibited a bell-shaped voltage dependence (Fig. 4C), with the slowest responses recorded at
+20 mV (rise time, 1.67 ± 0.18 msec).
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Figure 4.
GluR6 activation kinetics are slowed in the
presence of Spm. A, Control responses evoked by 50 msec
applications of 10 mM Glu at 100 and +50 mV and the open
tip junction current for this experiment (top trace).
The 10-90% rise times of the Glu responses (240 and 300 µsec) were
similar at 100 and +50 mV. B, In the presence of 20 µM Spm, responses to 10 mM Glu exhibited much
slower and voltage-dependent 10-90% rise times of 420 and 940 µsec
at 100 and +50 mV, respectively. The responses shown are an average
of 11 trials and are scaled to match the amplitude of the control
responses shown in A (dotted lines).
Scale bar: 175 pA at 100; 44 pA at +50 mV.
Arrows indicate the time of the peak response, which at
+50 mV was estimated by fitting a seventh order polynomial to the
rising and falling phases (solid line); in
A and B, dashed lines
indicate zero current. C, In the absence of Spm,
10-90% rise times for control responses to Glu were
voltage-insensitive for both symmetrical 150 mM Na
(open circles) and symmetrical 405 mM Na (data not shown). With 20 µM Spm, rise
times showed biphasic voltage dependence for 150 mM Na
(filled circles) with weaker voltage dependence
for 405 mM Na (filled diamonds).
D, G-V plots for peak Glu responses in
symmetrical 150 mM Na (filled
circles) and symmetrical 405 mM Na (open
circles). Fits of G-V plots with a single
binding site reaction scheme (Eq. 4) reveals that Spm affinity is
fourfold higher in 150 mM Na than in 405 mM Na.
G-V plots were corrected for the weak outward
rectification observed in the absence of polyamines (Fig.
3F).
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The slow voltage-dependent rise time of responses to Glu is consistent
with our proposal that Spm can access its binding site when channels
are closed, because the activation kinetics would then reflect
reequilibration of the binding of blocker with the open state after
channel activation by agonist (Fig. 2F). To further test this hypothesis, we examined the effect on Glu response rise times
of lowering the affinity of the blocker for the open state, because in
this scenario, the blocker would reside for a shorter period on its
binding site and thus rise times should become faster. To achieve this,
responses to 10 mM Glu in the presence of 20 µM Spm were recorded in high permeant ion concentrations
(symmetrical 405 mM Na). We have shown previously that
polyamine affinity is strongly influenced by asymmetrical changes in
either internal or external permeant ion concentration (Bähring
et al., 1997). However, the shifts in Vrev
observed in asymmetrical ion gradients did not permit a distinction to
be made between changes in ion flux or the membrane potential as the
mechanism underlying shifts in the voltage dependence of polyamine
block. Consistent with our previous finding that equilibrium block of
responses to domoate was reduced on increasing
[Na]o, block of Glu responses by Spm was reduced
in symmetrical solutions of 405 mM Na (Kd(0
mV) = 31.6 ± 3.5 µM; n = 3)
when compared with results for symmetrical 150 mM Na
(Kd(0 mV) = 7.9 ± 1.27 µM;
n = 8), suggesting that block is governed primarily by
ion flux via the channel and not membrane potential per se (Fig.
4D). Fits of the sum of two Boltzman functions showed
that the voltage dependence of onset (150 mM Na,
e-fold per 18.3 ± 1.3 mV; 405 mM Na,
e-fold per 16.8 ± 0.8 mV) and relief (150 mM Na, e-fold per 22.4 ± 2.3 mV; 405 mM Na, e-fold per 21.3 ± 7.6 mV) from
block was almost identical in each case, suggesting that although the
affinity of the blocker has changed, the location of the binding site
within the membrane electric field is unaltered (see Eq. 8) (Fig.
4D). Glu response rise times in symmetrical 405 mM Na with 20 µM Spm (0.83 ± 0.17 msec;
n = 3 at +20 mV) were, as predicted, faster than
observed with 150 mM Na and 20 µM Spm
(1.67 ± 0.18 msec), suggesting that the slow kinetics of the
rising phase of the Glu response with Spm was attributable to
reequilibration of the binding of blocker with the open state of the
channel (Fig. 4C). In contrast, the delay in channel opening
observed with 20 µM Spm in symmetrical 405 mM
Na (290 ± 34 µsec; n = 4) remained slower than
in the absence of polyamines (56 ± 29 µsec; n = 4), suggesting that the molecular mechanisms responsible for the delay
in channel activation are probably separate from those affecting the
rise times of responses to Glu. In view of the difficulty in accurately
resolving small differences in response latency, we have not
investigated further the underlying mechanisms of this effect.
Ion flux-dependent transitions between open and closed
blocked states
To explore further the conditions that influence transitions
between open-channel block by Spm on the one hand (Figs. 1, 2) and
closed-channel block on the other (Figs. 3, 4), we examined the effect
of changing the membrane potential on responses to 10 mM
Glu (50 msec duration) when channels were either open or closed (Fig.
5). Glu responses with 20 µM Spm were recorded at +50 mV, at which strong block
occurs, and at 100 mV, at which blocker affinity is low and thus
relief of block is fast (Fig. 2F). The membrane
potential was then stepped from 100 to +50 mV at various times
preceding and during the rising and falling phase of the response to
Glu. Figure 5, A and B, illustrates typical examples of this experiment in the presence and absence of 20 µM Spm, respectively. The response profile in the
presence of 20 µM Spm was strongly influenced by the time
at which the step from 100 mV to +50 mV occurred (Fig.
5A). When the step to +50 mV preceded the application of
agonist by 250 or 500 µsec, the peak response to Glu, when corrected
for the change in driving force, was greatly reduced in amplitude
(7.2 ± 1.2% of responses at 100 mV; n = 10)
and showed a slow rise time (10-90%, 1.34 ± 0.19 msec at +50
mV); these features are anticipated from the closed-channel block
mechanism suggested by the experiments shown in Figures 3 and 4. In
contrast, when the step from 100 to +50 mV occurred when channels
were open, during either the rising or falling phase of the response to
Glu, the instantaneous response at +50 mV showed almost no sign of
block. For example, when corrected for the change in driving force at
the peak of the Glu response, the amplitude at +50 mV was 98.5 ± 4.1% of that at 100 mV (n = 7).
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Figure 5.
Voltage jumps reveal transitions between open and
closed blocked states. A, Depolarizing steps to +50 mV
were applied before or during the rising and falling phase (time
increment, 500 µsec) of responses evoked by 50 msec applications of
10 mM Glu at 100 mV. When the depolarizing step preceded
the application of Glu, the response at +50 mV showed strong block. In
contrast, the envelope of the instantaneous currents evoked by
depolarizing steps during the response to Glu matched well the
amplitude of the response predicted for fully unblocked channels
(open circles); this was estimated by averaging
responses at 100 mV and correcting for the change in driving force
and the weak outward rectification observed in the absence of
polyamines (Fig. 3F). Note that the instantaneous
currents evoked by steps from 100 to +50 mV at first overshoot the
response observed when Glu was applied at +50 mV but then decay with
double-exponential kinetics faster than the response at 100 mV, as
expected for onset of open-channel block (Fig. 3C).
B, Same experimental paradigm as in A but
in the absence of polyamines. Note that the amplitude of the response
recorded when the depolarizing step to +50 mV preceded the application
of Glu matched well both the envelope of the instantaneous currents
recorded on depolarization from 100 to +50 mV, as well as the
amplitude and time course of the response at 100 mV scaled for the
change in driving force (open circles).
C, Responses to 10 mM Glu (50 msec duration)
at +50 and 100 mV were recorded immediately after steps to prepulse
potentials between 100 to +50 mV (15 mV increments, 200 msec
duration); the amplitude of individual responses did not vary with
prepulse potential. Solid lines show the average of the
responses at 100 and +50 mV. In A-C, open tip
junction potentials indicate the time of application of Glu.
D, Similar analysis of results from four and eight
patches, as indicated, confirm that the prepulse potential did
not affect the amplitude of responses to Glu.
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In the presence of Spm, the large instantaneous outward currents evoked
by steps from 100 to +50 mV decayed much faster than could be
accounted for by the rate of onset of desensitization at +50 mV
observed in the absence of polyamines (Fig. 5) and most likely reflects
onset of open-channel block by Spm. Because the rate of onset of
open-channel block is predicted to depend on Spm concentration, we
compared the kinetics of such responses with 20 and 60 µM
Spm. In both cases, the current observed at +50 mV after voltage steps
from 100 mV at the peak of the Glu response decayed biexponentially
but with faster kinetics for 60 versus 20 µM Spm. The
fast component of block was threefold more rapid for 60 µM Spm (tau, 0.14 ± 0.07 msec; n = 4) compared with measurements with 20 µM Spm (tau,
0.38 ± 0.04 msec; n = 7), as anticipated from an
open-channel block mechanism. The slower component of decay was also
faster with 60 µM Spm (tau, 0.84 ± 0.59 msec;
38.7 ± 8.9%) compared with responses with 20 µM
Spm (tau, 3.2 ± 0.35 msec; 41.7 ± 4.6%), and in both
cases, the onset of block was faster than the rate of onset of
desensitization of control responses with no polyamines (tau, 4.58 ± 0.65 msec; n = 5) (Fig. 5B). The slower
component of decay with polyamines observed in these experiments most
likely reflects a combination of the burst length of open blocked
channels and the onset of desensitization. Our observation that the
slow component of decay recorded with polyamines occurs faster than the
onset of desensitization and is much faster than predicted for a
sequential open-channel block mechanism (Fig. 3C) most
likely reflects truncation of the burst length attributable to entry of
open blocked channels into closed blocked states. Together, our
observations suggest that block of GluR6(Q) channels by internal
polyamines is strongly activity-dependent.
To test whether occupancy of the closed blocked state by Spm is
sensitive to the membrane potential, responses to 10 mM Glu were examined at 100 (n = 4) and +50 mV
(n = 8) after prepulses over a range of membrane
potentials previously shown to produce large changes in polyamine block
when channels are open. In contrast, we found that when channels were
closed the response amplitude was unaffected by the prepulse potential,
with steps of 200 msec duration (Fig. 5C,D); in
preliminary experiments, similar results were obtained with prepulses
of 2 sec duration (n = 2).
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DISCUSSION |
A kinetic model for bimodal block by polyamines
Based on our experimental observations, we developed a kinetic
model with open and closed blocked states (Model 2) (see experimental procedures in Materials and Methods). The model was based on
simulations that predicted well control responses to Glu in the absence
of polyamines (Model 1) but included additional closed blocked states. The model was tested by its ability to predict both the voltage dependence of the peak conductance and accelerated rate of decay of
responses to 1 msec applications of Glu (Fig. 3), as well as the
biphasic voltage dependence of the rise time of Glu responses (Fig. 4).
These key predictions of the model are summarized in Figure
6. In the presence of Spm, the
voltage-dependent rise time of responses to 10 mM Glu
observed experimentally was well achieved by Model 2, confirming that
equilibration of the binding of Spm with the open state during the
rising phase of the response to Glu was sufficient to account for this
effect (Fig. 6A). The model, however, did not predict
fully the delay in channel activation observed experimentally (Fig.
4B), implying that Spm most likely stabilizes some of
the closed states of the receptor; without additional information that
would be difficult to obtain experimentally, there is no justification
for incorporating additional closed states into the model or for
altering the rate constants for binding of agonist and polyamines to
closed states to increase their occupancy. The accelerated rate of
deactivation observed in the presence of Spm (Fig. 3E) was
achieved in the model by increasing the channel closing rate twofold
for Spm-blocked channels (Fig. 6B). Although the
molecular mechanism responsible for destabilization of the open state
is not yet understood, such effects are typical of the action of many
trapped channel blockers (Blanpied et al., 1997). One attractive
possibility would be that because of their large size, polyamine
molecules may exclude permeant cations from the pore, with consequent
effects on channel-closing kinetics similar to the effect of
use-dependent blockers on K+-channels (Baukrowitz
and Yellen, 1996).
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Figure 6.
Modulation of responses to Glu by a bimodal
blocking scheme. A, Simulation of responses to 1 msec
applications of 10 mM Glu, as shown in Figure
3D but with Model 2. Consistent with experimental
observations (Fig. 4), the peak response shows strong rectification,
and the rise times show a bell-shaped voltage dependence; the time of
occurrence of the peak response is indicated by filled
circles. B, Simulation of the voltage dependence
of deactivation of responses to 1 msec applications of Glu
(filled triangles) agrees well with experimental
observations (filled circles) when the rate of
channel closure is increased twofold for Spm-blocked channels.
C, Schematic diagram for Model 2 indicating cycling
between open and closed blocked states. D,
E, Simulations showing facilitation of responses to 50 Hz trains of 1 msec applications of 10 mM Glu with 20 µM Spm using rate constants adjusted to produce control
responses like those for GluR-D (Lomeli et al., 1994).
Traces in E show occupancy of the closed
(R) and closed blocked (RB) states of
Model 2 during facilitation of responses to L-Glu; note the
progressive decrease in occupancy of closed blocked states during
successive responses to Glu.
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Binding of polyamines to closed GluR channels
The recent elucidation of K+-channel structure
from Streptomyces lividans (Doyle et al., 1998) offers a
possible explanation to account for our observation that the occupancy
of closed blocked states by polyamines was not detectably altered by
membrane potential within the limit of resolution of our experimental
protocols, whereas ion flux through the pore rapidly relieves polyamine
block (Fig. 5). A diagram illustrating the key features of the proposed model is shown in Figure 6C and requires that polyamines
bind to a water-filled internal vestibule accessible in the closed state. This avoids the difficulty that the local potential around a
charged molecule, such as Spm, would be large in a region of low
dielectric constant, such as a narrow channel pore in a membrane. However, if the narrow pore is replaced by a larger cavity that accommodates polarizable water molecules, as proposed from
K+-channel structure (Doyle et al., 1998),
polyamines will be shielded and experience less of the potential drop
across the membrane. The structure of the pore region in Glu receptors
is unknown, but data from accessibility experiments using
Cys-substituted residues on NMDA (Kuner et al., 1996) and AMPA
receptors (Kuner et al., 1997) indicate that the M2 segment
forms a channel-lining hairpin loop accessible from the cytoplasmic
side of the channel. The inverted orientation of the pore loop in Glu
receptors relative to that in potassium channels would generate a
tepee, with an external gate and internal selectivity filter
surrounding a central hydrophobic cavity. Although it is premature to
speculate on the precise orientation of the structural elements in Glu
receptor pores, it is likely that the binding site with which
polyamines interact during permeation would become fully accessible
only after channel activation and that movement of polyamines into the
blocking site after channel opening would precede and thus prevent
measurable ion flux.
The blocking mechanisms that we propose underlie the action of Spm are
summarized schematically in Figure 6C, which outlines the
cyclic nature of the proposed scheme. Before receptor activation, cytoplasmic polyamines reside in a water-filled cavity that is accessible to the cytoplasm in the closed conformation and represents the closed blocked state. When the receptor binds Glu, the
conformational steps associated with channel activation expose a
polyamine binding site within the membrane electric field. The rate of
binding and dissociation of Spm from the open state of the channel
underlies the voltage dependence of the rise time of responses to Glu.
Our results suggest that this process is governed to a large extent by
ion flux via the channel rather than membrane potential per se. Such a
model helps to explain the strong flux dependence of polyamine unblock
(Fig. 5) and our previous observation that at equilibrium the voltage
dependence of GluR6(Q) responses to domoate shows strong coupling to
permeant ion concentrations (Bähring et al., 1997).
Physiological consequences of polyamine block
The rate of decay of the response to 1 msec applications of
L-Glu and presumably the rate of decay of EPSCs at synapses
with polyamine-sensitive Glu receptors found in some interneurons
(Geiger et al., 1995; Koh et al., 1995) will be determined primarily by the closing rate of open and open blocked channels as they relax into
adjacent closed and closed blocked states, respectively, as shown in
Model 2. At the modestly depolarized membrane potentials required for
initiation of action potential discharge, the rate of open-channel
block by polyamines is quite modest (Fig. 3E) and would not
be expected to greatly accelerate the rate of decay of EPSCs; however,
the relative occupancy of closed and closed blocked states would be
transiently altered immediately after an EPSC. This is because the
occupancy of these closed states reflects voltage-dependent block by
polyamines of the adjacent open states. Although, because of the slow
kinetics of recovery from desensitization of GluR6 (Heckmann et al.,
1996; Traynelis and Wahl, 1997), we have no information on the kinetics
of rebinding of Spm to closed channels, the presence of polyamines in
the cytoplasm requires that closed channels will eventually relax back
into the closed blocked state. One consequence of this is that our model for polyamine block shows short-term plasticity similar to that
recently observed for polyamine block of both native and recombinant
AMPA receptors (Rozov et al., 1998). Although our experiments were
performed using GluR6, the high expression of which facilitated
analysis of macroscopic GluR responses in outside-out patches, we
believe that the mechanism we propose is similar for both kainate and
AMPA receptors.
Simulations with Model 2 show that the peak amplitude of successive
responses to Glu occurring before the reaccumulation of channels in the
closed blocked state will progressively increase during train
applications of agonist or presumably with repeated activation of
synapses, the explanation being that after each successive release of
L-Glu, fewer and fewer channels remain in the closed
blocked state. A direct test of this prediction is not possible with
GluR6(Q) channels, which recover slowly from desensitization. However,
when rate constants are adjusted to simulate responses for GluR-D, an
AMPA receptor with rapid kinetics of recovery from desensitization
(Lomeli et al., 1994) that is expressed at high levels in hippocampal
interneurons (Geiger et al., 1995), our model predicts facilitation for
responses evoked by a train of pulses of Glu (Fig. 6). Such
polyamine-dependent potentiation of peak Glu responses has been
recently observed for both recombinant and native AMPA rec
Top
Abstract
Introduction
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