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Volume 16, Number 21,
Issue of November 1, 1996
pp. 6634-6647
Copyright ©1996 Society for Neuroscience
AMPA Receptor Flip/Flop Mutants Affecting Deactivation,
Desensitization, and Modulation by Cyclothiazide, Aniracetam, and
Thiocyanate
Kathryn M. Partina,
Mark W. Flecka, and
Mark L. Mayer
Laboratory of Cellular and Molecular Neurophysiology, National
Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, Maryland 20892-4495
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
AMPA receptor GluRA subunits with mutations at position 750, a
residue shown previously to control allosteric regulation by
cyclothiazide, were analyzed for modulation of deactivation and
desensitization by cyclothiazide, aniracetam, and thiocyanate. Point
mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val,
and Tyr were constructed in GluRAflip. The last four of
these mutants were not functional; S750D was active only in
the presence of cyclothiazide, and the remaining mutants exhibited
altered rates of deactivation and desensitization for control responses
to glutamate, and showed differential modulation by cyclothiazide and
aniracetam. Results from kinetic analysis are consistent with
aniracetam and cyclothiazide acting via distinct mechanisms. Our
experiments demonstrate for the first time the functional importance of
residue 750 in regulating intrinsic channel-gating kinetics and
emphasize the biological significance of alternative splicing in the
M3-M4 extracellular loop.
Key words:
glutamate receptors;
cyclothiazide;
aniracetam;
thiocyanate;
mutagenesis;
AMPA;
desensitization;
deactivation;
alternative splicing;
flip and flop
INTRODUCTION
AMPA subtype ionotropic glutamate receptors are
assembled from subunits encoded by multiple gene families that are
differentially expressed in subpopulations of neurons and glia (Boulter
et al., 1990
; Keinänen et al., 1990; Hollmann and Heinemann,
1994). The wide variety of functionally distinct native AMPA receptor
responses recorded in CNS tissue (Burnashev et al., 1992; Jonas and
Sakmann, 1992; Raman and Trussell, 1992; Livsey et al., 1993) is likely
to result from assembly of different combinations of subunits. The
results of single-cell PCR analyses provide strong support for this
hypothesis (Lambolez et al., 1992; Bochet et al., 1994; Geiger et al.,
1995) and suggest that the kinetics of excitatory postsynaptic currents
in individual cells, which are determined by a combination of receptor
deactivation, desensitization, and the rate of recovery from the
desensitized state (Edmonds et al., 1995; Trussell and Otis, 1996),
must also be controlled by subunit composition.
Recent studies on memory- and cognition-enhancing drugs implicate AMPA
receptors as their underlying target of action (Staubli et al.,
1994a,b; Zivkovic et al., 1995). Cognition-enhancing drugs that are
thought to work by potentiating glutamatergic synaptic efficacy fall
into two categories, the pyrrolidinones aniracetam, piracetam, and the
related compound 1-BCP (Isaacson and Nicoll, 1991; Tang et al., 1991;
Vyklicky et al., 1991; Hestrin, 1992; Gouliaev and Senning, 1994;
Staubli et al., 1994b) and the benzothiadiazines cyclothiazide,
diazoxide, and IDRA21 (Yamada and Rothman, 1992; Bertolino et al.,
1993; Patneau et al., 1993; Yamada and Tang, 1993). Both categories of
drug slow the rate of AMPA receptor deactivation and desensitization
(Isaacson and Nicoll, 1991; Vyklicky et al., 1991; Hestrin, 1992;
Patneau et al., 1993; Barbour et al., 1994). Previous work has
demonstrated that for AMPA receptors an alternatively spliced exon
termed the flip/flop domain (Sommer et al., 1990), which forms part of
the extracellular M3-M4 loop (Hollmann et al., 1994; Stern-Bach et
al., 1994; Bennett and Dingledine, 1995), regulates the kinetics of the
onset of and recovery from desensitization as well as AMPA receptor
sensitivity to cyclothiazide and aniracetam (Sommer et al., 1990;
Lomeli et al., 1994; Mosbacher et al., 1994; Partin et al., 1994;
Johansen et al., 1995). The findings that RNA splicing and RNA editing
of this region are regulated in a cell-specific and developmentally
programmed manner (Monyer et al., 1991; Lomeli et al., 1994) suggest
that this region plays a critical role for AMPA receptor synaptic
function.
The localization of molecular determinants of desensitization to this
small (38 amino acid) region of the protein makes the flip/flop domain
a good target for mutational analysis. We showed previously that
exchange of a single residue at position 750 in GluRA (flip Ser and
flop Asn) underlies differential modulation of desensitization by
cyclothiazide for AMPA receptor splice variants (Partin et al., 1995).
The role of this residue in controlling sensitivity to other allosteric
modulators of AMPA receptors has not been addressed. Also unknown is
whether the effects of cyclothiazide and aniracetam on deactivation are
regulated by alternative splicing. To address these issues, we
constructed a series of position 750 mutations substituting amino acids
with different side chain moieties. These were compared to wild-type
receptors for differences in deactivation, desensitization, and
modulation by cyclothiazide, aniracetam, and thiocyanate. Using kinetic
modeling to simulate experimental findings, we suggest that
cyclothiazide and aniracetam act via different mechanisms: that
aniracetam modulates desensitization as a consequence of slowing
channel closing, whereas cyclothiazide modulates desensitization by
stabilizing a nondesensitized agonist-bound closed state.
MATERIALS AND METHODS
Plasmids and mutagenesis. cDNAs for wild-type
GluRAi (flip) and GluRAo (flop) in CMV
expression vectors were generous gifts of Dr. Peter Seeburg
(Heidelberg, Germany). Point mutations were made in pBS/GluRAi by
dut-ung- oligonucleotide missense mutagenesis (Bio-Rad
Muta-Gene Phagemid In Vitro Mutagenesis Kit, Version 2; Bio-Rad,
Hercules, CA) and then shuttled into the CMV expression clone using the
BspE1
BspE1 (New England Biolabs, Beverly, MA) fragment of GluRA (nt
1777-2320), after which the sequence of the mutation was confirmed.
S750N, S750Q, GluRAi[io], and
GluRAo[oi] were described previously (Partin et al.,
1995). All cDNAs were purified through two cesium chloride gradients
before transfection.
Cells and transfections. Human embryonic kidney 293 fibroblasts (ATCC CRL 1573) were cultured in DMEM supplemented with
10% fetal bovine serum and 2 mM glutamine (Life
Technologies, Bethesda, MD). Cells were kept at a low density and
passaged only up to passage number 15 (P15). Transfections were
performed according to the CaCl2 method of Chen and Okayama
(1987), with 10-20 µg of total DNA per 35 mm dish. Transfections
with glutamate receptor cDNAs also included 10-20% CMV-GFP, a
plasmid encoding the cDNA for green fluorescent protein (Chalfie et
al., 1994) (which was a gift from Dr. Peter Seeburg) into which we
introduced a point mutation of S65T (Cubitt et al., 1995)
for identification of successfully transfected cells.
Whole-cell recording. Transfected 293 cells were
voltage-clamped at a holding potential of 
60 mV using an Axopatch-1C
amplifier (Axon Instruments, Foster City, CA), and solutions were
applied using a 9-barrel flowpipe, stepper-motor-based perfusion system
as described previously (Vyklicky et al., 1990). Thin-walled
borosilicilate glass microelectrodes (TW150F, World Precision
Instruments, Sarasota, FL) had resistances of 2-5 M
when filled
with (in mM): 135 CsCl, 10 CsF, 10 HEPES, 5 Cs-BAPTA, 1 MgCl2, and 0.5 CaCl2, pH 7.2, 295 mOsm. The
series resistance was <10 M and compensated by at least 80%;
voltage-clamp errors were <5 mV for typical AMPA receptor responses.
Responses were filtered at 2 kHz with an 8-pole Bessel filter
(Frequency Devices, Haverhill, MA), digitized at 0.25-10 msec/point as
required, and stored on a Macintosh IIfx computer using an ITC-16
interface (Instrutech, Great Neck, NY) under control of the data
acquisition and analysis program Synapse (Synergistic Research, Silver
Spring, MD). Extracellular recording solution contained (in
mM): 145 NaCl, 5.4 KCl, 5 HEPES, 1 MgCl2, 1.8 CaCl2, and 0.1 mg/ml phenol red, pH 7.3, 295 mOsm.
Cyclothiazide and aniracetam were dissolved in DMSO before dilution
with extracellular solution (final DMSO concentration, 0.5 or 1%) with
DMSO added at equal concentrations to other solutions. For experiments
with thiocyanate, 20 mM NaCl was replaced with 20 mM NaSCN. Salts, biochemicals, and amino acids were
purchased from Aldrich (Milwaukee, WI), Molecular Probes (Eugene, OR),
Sigma (St. Louis, MO), and Tocris Cookson (Bristol, UK).
Outside-out patch recording. Outside-out membrane patches
from transfected 293 cells were voltage-clamped at a holding potential
of 60 mV using an Axopatch 200A amplifier (Axon Instruments). Agonist
responses were filtered at 10 kHz and digitized at 20-100
µsec/point. Solutions were applied with an infusion pump (World
Precision Instruments), using a flowpipe constructed from 4-barrel
square glass tubing with 0.16 mm bores and 0.4 mm outer width (Vitro
Dynamics, Rockaway, NJ). Movement was driven by a Piezo translator
(P245.30, Physik Instrumente, Waldbronn, Germany), which typically
permitted solution exchange times in <200 µsec as determined by
measurements of open-tip junction potentials recorded from the upper
and lower interfaces after disruption of the patch at the end of every
experiment; data were excluded for junction potentials with 20-80%
rise times > 500 µsec. Normal extracellular solution with or
without 10 mM L-glutamate was driven through
the lower bores, and the same solutions plus 100 µM
cyclothiazide, 5 mM aniracetam, or 20 mM NaSCN
were driven through the upper bores. Movement between the upper and
lower bores within an experiment was achieved using a vernier
micrometer (SM-13, Newport Instruments, Irvine, CA).
Data analysis. The rate of onset of desensitization was
estimated in both whole-cell and outside-out patch experiments by
fitting the decay of the response in the continuous presence of agonist
from 95% of peak to steady state with a single-exponential function
(
des); where noted we used the sum of two exponentials
(f and s). The rate of deactivation was
estimated by fitting a single exponential (deact) to
responses to 1 msec applications of agonist. When there was substantial
desensitization during the initial component of responses to 1 msec
applications of agonist, the rate of deactivation was estimated from
fits to the subsequent fast component of decay that developed after
removal of agonist and that varied in amplitude between 90 and 20% of
the peak response. In all cases, deactivation was faster than
desensitization, such that there was a clear increase in rate of decay
after removal of glutamate, even for responses for which there was
substantial (>50%) desensitization in response to 1 msec applications
of glutamate. Sensitivity to cyclothiazide, aniracetam, and thiocyanate
was estimated by comparing desensitization and deactivation time
constants in the absence and presence of modulator. Whole-cell
responses are shown individually; patch responses are the average of
5-20 successive agonist applications. Data are presented as mean ± SEM except as noted. When statistical analysis was performed,
Student's unpaired one- and two-tailed t tests were
used.
Kinetic modeling was performed using FastFlow, a previously
described program written by Dr. John Clements (Benveniste et al.,
1990). The initial rate constants used for modeling were taken from the
three-binding-site model of Raman and Trussell (1995) but were
modified to reproduce the properties of recombinant GluRA as
determined experimentally. This entailed (1) removing the third
agonist-binding step (C3) and two open states (O2slow
and O3), yielding decays that were well fit by single-
rather than double-exponential functions, (2) slowing the rates of
channel opening and closing by half, and (3) slowing the microscopic
rates for onset of desensitization and recovery from desensitization
such that the modeled responses desensitized and recovered with time
constants similar to those found experimentally for recombinant GluRA.
The affinity of glutamate for the desensitized agonist-bound states was
adjusted to satisfy microscopic reversibility. The rates for binding of
cyclothiazide and aniracetam were modeled based on experimental
measurements of the affinities/efficacies of the respective
modulator.
Analysis of the structure of GluRA was made using model coordinates for
GluR3 provided by Dr. Patrick J. O'Hara (ZymoGenetics, Seattle, WA).
The model was visualized using either QUANTA V4.0 (Molecular
Simulations, Burlington, MA) or LOOK V2.0 (Molecular Applications
Group, Palo Alto, CA). The predicted effects of point mutations were
calculated and visualized using an algorithm of LOOK that allows point
mutations at a single residue to be generated, identifies the nearest
neighbors (within a 5 Å radius) of that residue, and then calculates
the predicted conformation of the side chains of the selected residue
and the nearest neighbors as a consequence of the mutation. The mutated
structure was then overlaid with the nonmutated structure for
comparison.
RESULTS
Position 750 mutations differentially regulate responses to
cyclothiazide and aniracetam
To determine the constraints at position 750 that influence
receptor gating and allosteric modulation by cyclothiazide and
aniracetam, we mutated the Ser residue at this site in
GluRAi to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val,
and Tyr (Fig. 1A). The resulting cDNAs
were expressed as homo-oligomers by transient transfection of 293 cells
and assayed by rapid perfusion in the whole-cell configuration. The
mutants S750T, S750L, S750Y, and
S750V did not generate functional responses;
S750C and S750M expressed poorly;
S750D gave responses only in the presence of cyclothiazide;
and the remaining mutants generated robust responses to glutamate in
both the presence and the absence of modulators. Figure
1B shows examples of whole-cell currents evoked by 1 mM glutamate in the presence of 100 µM
cyclothiazide for wild-type GluRAi, GluRAo, and
some of the position 750 substitutions that were found to be
functional. The only receptor for which desensitization was essentially
blocked was wild-type GluRAi (Table 1); all
of the functional mutants studied continued to show desensitization in
the presence of cyclothiazide. The rate of onset of desensitization was
slowed most for S750A (des 1690 ± 149 msec; n = 11). An intermediate response similar
to that for wild-type GluRAo (des 301 ± 14 msec; n = 9) was observed for S750G
(des 332 ± 45 msec; n = 6) and
S750N (des 336 ± 20 msec;
n = 12). For S750M, S750C, and
S750Q, the rate of onset of desensitization was rapid
(des 3-6 msec; n = 2-7) and not
significantly different from control (Table 1). S750D was
unusual in that it expressed no current under control conditions, but
large currents in the presence of cyclothiazide; the decay in the
presence of cyclothiazide was best fit by a double exponential
[f 58 ± 5.0 msec (Af 38 ± 3%);
s 2857 ± 328 msec; n = 9].
Because of the limited solubility of cyclothiazide in physiological
solutions, we were unable to examine the effects of higher
concentrations on S750D or the other mutants.
Fig. 1.
Mutations at GluRAflip
Ser750 and their modulation by cyclothiazide.
A, Schematic diagram of an AMPA receptor subunit;
gray boxes represent membrane domains; a black
box represents the flip/flop domain (residues 739-777 of the
mature peptide for GluRA). Below is the amino acid
sequence of this domain for GluRAi (top) and
GluRAo (bottom). The site for RNA editing
(R/G) that occurs in GluRB, -C, and -D is shown by +; amino acids for
which there are conserved differences between the flip and the flop
variants of AMPA receptors are marked by *; the point at which the
structural homology with LAOBP ends is marked with 
. Amino acids
listed between the GluRAi and GluRAo
sequences were introduced in GluRAi at position
750 and are grouped according to functional criteria. B,
Whole-cell currents evoked from transiently transfected 293 cells
expressing wild-type or mutant receptors. Currents were evoked by a 2 sec application of 1 mM glutamate after preincubation for
>30 sec with 100 µM cyclothiazide. Decays were well fit
with a single exponential (superimposed on traces), with the exception
of wild-type GluRAi, which desensitized too slowly to be
fit, and S750D, which was best fit by a double exponential.
Every point mutation studied altered the ability of cyclothiazide to
slow the onset of desensitization, as compared to wild-type
GluRAi, with S750M and S750Q
permitting little and no modulation, respectively. Calibration bars
represent 500 msec and 500 pA, except for S750M, for which
the current bar represents 50 pA.
[View Larger Version of this Image (15K GIF file)]
As reported previously, aniracetam also shows splice-variant
specificity for modulation of desensitization but, in contrast to
cyclothiazide, the effect is greater for GluRAo than
GluRAi (Johansen et al., 1995). To determine the role of
position 750 in differential modulation by aniracetam, the
desensitization kinetics of the same mutants assayed for modulation by
cyclothiazide were analyzed for sensitivity to 5 mM
aniracetam (Fig. 2), a concentration chosen on the basis
of previous work on native AMPA receptors and which is at the
solubility limit of aniracetam in physiological saline with 1% DMSO
(Isaacson and Nicoll, 1991; Vyklicky et al., 1991). As seen from the
overlay of representative traces in Figure 2B,
aniracetam slowed the onset of desensitization of S750N
(des 31 ± 3.2 msec; n = 7) to a
greater extent than for GluRAi (des 20 ± 1.4 msec; n = 5); the extent of modulation for
S750N was similar to that for GluRAo
(des 32 ± 9 msec; n = 6),
suggesting that an Asn residue at position 750 underlies the higher
sensitivity of flop splice variants to aniracetam (Johansen et al.,
1995). However, the equilibrium current in the presence of aniracetam
for S750N was substantially greater (15- ± 5-fold
potentiation of steady-state current) than for GluRAo (2.8- ± 0.6-fold) or the other mutations (5- to 10-fold range). A similar
observation was made for potentiation of equilibrium currents by
cyclothiazide; after a 15 sec application of glutamate in
Xenopus oocytes, S750N equilibrium current was
potentiated by cyclothiazide 2.5-fold more than either
S750A or S750G (data not shown), even though in
the present experiments des in the presence of
cyclothiazide was much slower for S750A than for
S750N or S750G (Table 1).
Fig. 2.
Aniracetam modulation of GluRAo is
directed by the ASN residue at position 750. A,
Superimposed whole-cell responses to 0.5 sec applications of 1 mM glutamate recorded before and after preincubation for
>30 sec with 5 mM aniracetam. Single-exponential fits are
superimposed on responses recorded in the presence of aniracetam, which
slowed the onset of desensitization for GluRAo to a greater
extent than GluRAi. The point mutation S750N
was sufficient to change flip-like modulation to flop-like modulation.
Neither a control current nor a current in the presence of aniracetam
could be recorded for S750D. B,
Superimposition of the traces shown above and recorded in the presence
of aniracetam, normalized to their peak amplitude, demonstrate that no
other point mutation permits better modulation by aniracetam than
S750N. Traces for wild-type GluRAi and
GluRAo are shown in bold for
comparison.
[View Larger Version of this Image (16K GIF file)]
Figure 3 plots the change in time
constant of onset of desensitization for mutations that were modulated
by cyclothiazide and aniracetam versus increasing size of the amino
acid side chain at position 750. For both drugs, one residue is clearly
preferred; however, the amino acid permitting the most efficacious
modulation for cyclothiazide is different than for aniracetam. For both
drugs, side chains that are either larger or smaller result in less
efficacious modulation. In the case of cyclothiazide, this effect is
extreme, and although cyclothiazide is much more efficacious than
aniracetam it is only Ser750 for which cyclothiazide
essentially blocks desensitization. For aniracetam, mutants with a
broader range of side chains show modulation of desensitization
(compare modulation by aniracetam but not by cyclothiazide of
S750M and S750Q), yet the difference in
efficacy of aniracetam between the most and least sensitive mutants is
less than fourfold. An interesting exception to this is
S750D, which is strongly modulated by cyclothiazide yet
insensitive to aniracetam. S750D also shows no control
response in the absence of modulators, although when expressed in
oocytes there was a small (1-2 nA) equilibrium current in response to
glutamate (data not shown). The relative differences in side chain
sensitivity and efficacy for modulation by aniracetam and cyclothiazide
for position 750 mutants could arise through a number of mechanisms
including changes in the structure of a single binding pocket that
differentially affects the two drugs, or modulation via different
mechanisms, one of which limits the efficacy of modulation by
aniracetam but not cyclothiazide.
Fig. 3.
Comparison of the ability of cyclothiazide and
aniracetam to modulate desensitization for GluRAi and
position 750 point mutants. A, Bar plot showing the
effect of position 750 point mutants on modulation by cyclothiazide
(des cyclothiazide/des control); mutants
are displayed in increasing order of size with the structures of
corresponding amino acid side chains shown above the
bars. The asterisk denotes that the decay
for wild-type GluRAi (Ser) was estimated
from studies on the dissociation kinetics of cyclothiazide in the
presence of glutamate (see Fig. 8). The plus sign (+)
indicates that the value for S750D is an average of the
fast and slow components weighted by their relative amplitudes.
B, Bar plot showing the effect of position 750 point
mutants on modulation by aniracetam (des
aniracetam/des control). S750D expressed
neither a control response nor any response in the presence of
aniracetam. Comparison of A and B reveals
no correlation between modulation by cyclothiazide and aniracetam,
suggesting that the molecular determinants governing allosteric
modulation of desensitization differs for these ligands. The plots
summarize data from whole-cell experiments performed on transfected 293 cells (see Figs. 1, 2).
[View Larger Version of this Image (24K GIF file)]
The mutants S750T, S750L, S750Y,
and S750V generated nonfunctional receptors. This could
arise from a variety of causes: gross misfolding of the protein,
inappropriate intermolecular contacts such that the subunits cannot
associate to form homo-oligomers and subsequently fail to reach the
plasma membrane, or inappropriate intramolecular packing resulting in
localized deformation of the helix-turn-helix motif that is thought
to exist in this region of the flip/flop module (O'Hara et al., 1993;
Stern-Bach et al., 1994). To test for appropriate folding and assembly,
we performed immunofluorescence experiments, staining both live and
permeabilized transfected 293 cells with anti-peptide antisera raised
against the N-terminal domain of GluR1 (R. Wenthold, unpublished
observations). We found that the level of immunoreactivity for the
nonfunctional mutant receptors S750L and S750V
and their cellular localization in the plasma membrane of live cells
were indistinguishable from that for wild-type GluRAi (data
not shown), suggesting that these mutant proteins were correctly
folded, assembled, and inserted in the plasma membrane. In contrast,
although the intracellular level of S750T immunoreactivity
was similar to wild-type, little or no immunoreactivity could be
detected on the plasma membrane of live cells.
To predict the effect of the mutations on intramolecular packing, we
analyzed the predicted structure of the mutations using the LOOK
modeling program (see Materials and Methods). Substitutions at position
750 were introduced into a model of GluR3 developed by O'Hara and
colleagues (Stern-Bach et al., 1994) based on homology of glutamate
receptors with bacterial periplasmic amino acid-binding proteins
(O'Hara et al., 1993; Kuryatov et al., 1994; Stern-Bach et al., 1994).
The predicted perturbations of local structure as a consequence of
mutations that disrupted modulation by cyclothiazide
(S750Q) or resulted in a nonfunctional receptor
(S750L) were both minimal. This suggests that position 750 mutations that result in nonfunctional receptors either disrupt
contacts in the flip/flop region that are beyond the region of homology
of AMPA receptor subunits with bacterial periplasmic amino acid-binding
proteins or disrupt intermolecular contacts with a neighboring subunit.
Mutations at Ser750 alter control kinetics of
deactivation and desensitization
Whole-cell recordings with position 750 mutants suggest that
S750Q desensitized slightly faster and that
S750A desensitized slower than the other receptors tested
(Table 1). Observations of altered desensitization kinetics raised the
possibility that deactivation kinetics could also differ, but the
whole-cell recording technique lacks sufficient resolution for this
determination. Therefore, deactivation kinetics were measured for
GluRAi, GluRAo, S750N,
S750Q, and S750A with 1 msec applications of 10 mM glutamate to outside-out patches. The subunits that were
chosen for further study represent naturally occurring isoforms
(GluRAi and GluRAo), express a critical
determinant of differential modulation by aniracetam and cyclothiazide
(S750N), or exhibit altered desensitization kinetics
(S750A and S750Q). Outside-out patch responses
of wild-type and mutant GluRA subunits revealed rapid kinetics, with
onset of desensitization even during a 1 msec application of glutamate,
such that for different subunits the start of deactivation was fit well
only for the later phase of the response, beginning from between 95 and
20% of the peak response. The deactivation kinetics of
S750A (deact 0.97 ± 0.06 msec;
n = 21) were slower (p < 0.01 vs GluRAi), and those for S750Q
(deact 0.68 ± 0.03 msec; n = 17)
were faster (p < 0.05 vs GluRAi)
than other subunits tested (Fig.
4A,B). Similarly,
S750A (des 3.95 ± 0.17 msec;
n = 22) desensitized significantly more slowly
(p < 0.001) and S750Q
(des 1.59 ± 0.04 msec; n = 16)
desensitized more rapidly (p < 0.001) than
wild-type GluRAi. There were no significant differences
between GluRAi and GluRAo in deactivation
kinetics (Fig. 4C), although there was a statistically
significant difference (p < 0.001) in
desensitization kinetics: GluRAi des
2.45 ± 0.1 msec (n = 28); GluRAo
des 3.23 ± 0.1 msec (n = 32; Fig.
4D). The deactivation kinetics of wild-type
GluRAi (deact 0.79 ± 0.04;
n = 28) are much faster than those of AMPA receptors in
hippocampal neurons (Vyklicky et al., 1991; Colquhoun et al., 1992),
are reminiscent of the fast kinetics found for AMPA receptors in nMAG
neurons (Raman and Trussell, 1995), and are only slightly slower than
reported for homomeric GluRD (Mosbacher et al., 1994).
Fig. 4.
Deactivation and desensitization kinetics
for control responses to glutamate in outside-out patches.
A, Responses for S750A, evoked by 1 msec
(deactivation) or 100 msec (desensitization) applications of 10 mM glutamate. B, Similar responses for
S750Q. Shown above the current traces in
A and B are open tip junction currents
recorded after the patch was blown off. Time constants for deactivation
were estimated from single-exponential fits starting from ~90 to 60%
of the peak amplitude (arrows); time constants for
desensitization were estimated from a single-exponential fit from 95%
of peak. C, Bar plot of the time constants of
deactivation (mean ± SEM) as determined from 1 msec application
of 10 mM glutamate. Asterisks above the
bars indicate that S750A deactivated
significantly more slowly (p < 0.01) and
S750Q deactivated more rapidly
(p < 0.05) than wild-type
GluRAi. Values in parentheses represent the
number of patches analyzed. D, Bar plot of the time
constants of desensitization (mean ± SEM) as determined from
20-100 msec applications of 10 mM glutamate.
Asterisks above the bars indicate that
S750A desensitized significantly more slowly
(p < 0.001) and S750Q
desensitized more rapidly (p < 0.001) than
GluRAi. E, Traces from a paired-pulse
protocol used to determine the kinetics of recovery from
desensitization for wild-type GluRAi. Shown are 12 overlaid
traces, each consisting of a 10 msec conditioning pulse of 10 mM glutamate followed by test pulses at intervals of
10-670 msec. Traces are normalized to correct for rundown (<6% in
the example shown). F, The ratio of the peak amplitude
of the test response to the conditioning response
(I2/I1, mean ± SEM) plotted versus interpulse interval for GluRAi,
GluRAo, and three point mutants were well fit with an
exponential function, 1 [exp(t/rec)], of
time constant 147 msec. There was no significant difference for
rec among the five receptors studied.
[View Larger Version of this Image (24K GIF file)]
It had been reported that RNA editing of the Arg/Gly site in the
N terminus of the flip/flop domain results in a change in the time
constant (rec) for recovery from desensitization (Lomeli
et al., 1994). However, the intronic sequences necessary for RNA
editing at this site are not found in the GluRA gene and, therefore,
RNA editing of GluRA is not found in vivo. To verify that
point mutations at position 750 did not affect recovery from
desensitization, we measured rec for GluRAi,
GluRAo, and three of the point mutations (Fig.
4E,F) and found that they had
indistinguishable time constants for recovery from desensitization
(mean rec 147 ± 3 msec; n = 5-8).
Surprisingly, this value is substantially slower than reported
previously for homo-oligomeric AMPA receptors assembled from GluRC or
GluRD (Lomeli et al., 1994). Technical reasons for this difference were
eliminated by measuring rec for hetero-oligomeric AMPA
receptors generated by assembly of GluRAi with
GluRBi, which gave rec 71 ± 5 msec
(n = 5), similar to previously published values (Lomeli
et al., 1994).
Cyclothiazide and aniracetam selectively modulate deactivation of
AMPA receptor splice isoforms
Both cyclothiazide and aniracetam modulate deactivation of native
AMPA receptors (Hestrin, 1992; Patneau et al., 1993; Barbour et al.,
1994). Because AMPA receptor splice variants show different sensitivity
to these drugs, we examined whether modulation of deactivation is also
sensitive to alternative splicing and whether there is a correlation
between modulation of deactivation and modulation of desensitization.
We looked first at the effects of cyclothiazide and aniracetam on
deactivation of wild-type GluRAi and GluRAo
(Fig. 5). Cyclothiazide slowed the rate of deactivation
of GluRAi by 2.40- ± 0.27-fold (n = 8),
but did not modulate deactivation of GluRAo
(n = 11). Surprisingly, in view of the much weaker
effects of aniracetam on desensitization, we found that deactivation
for GluRAo was slowed 4.09- ± 0.24-fold (n = 5) by aniracetam even though cyclothiazide failed to modulate
deactivation for this splice variant. For GluRAi aniracetam
produced a 2.23- ± 0.19-fold (n = 6) slowing of
deactivation similar to the effect of cyclothiazide. We concluded that
both compounds differentially modulate deactivation as well as
desensitization; for cyclothiazide the effects were greater for
GluRAi than for GluRAo, whereas the opposite
was true for aniracetam.
Fig. 5.
Differential modulation of deactivation by
cyclothiazide and aniracetam for wild-type GluRAi and
GluRAo. A, Responses of outside-out patches
to 1 msec applications of 10 mM glutamate, in the absence
or presence of 100 µM cyclothiazide; responses are
normalized to their peak amplitudes to compare their time courses. Open
tip junction potentials are shown above each set of
responses. Arrows denote the start of single-exponential
fits for analysis of deactivation. Cyclothiazide slowed deactivation of
GluRAi but failed to modulate deactivation of
GluRAo. B, Aniracetam (5 mM)
slowed deactivation more effectively for GluRAo than
GluRAi.
[View Larger Version of this Image (21K GIF file)]
We then examined modulation of deactivation for three of the point
mutants that showed altered desensitization in the presence of
cyclothiazide or aniracetam, S750N, S750A, and
S750Q (Fig. 6). Modulation of deactivation
by cyclothiazide followed the same trend as modulation of
desensitization in that clearly a Ser residue at position 750 permits
the most efficacious slowing of deactivation (2.40- ± 0.27-fold;
n = 8); deactivation of S750A was slowed by
1.51- ± 0.11-fold (n = 11), whereas deactivation of
S750N and S750Q was not affected (Fig.
6B, Table 2). Aniracetam slowed
deactivation for S750N (3.41- ± 0.22-fold;
n = 9) to a greater extent than the other mutants
examined, similar to its greater effect on desensitization for
S750N versus S750A and S750Q (Fig.
3). In general, although there was a clear correlation between the
effect of these modulators on desensitization and deactivation kinetics
(Fig. 6B,C), this is true qualitatively
rather than quantitatively because for S750A
desensitization was efficaciously modulated by cyclothiazide (145-fold
slowing of the onset of desensitization), whereas deactivation was not
(1.52-fold slowing). The kinetics of desensitization measured in
outside-out patches were consistently faster (1.1- to 2.9-fold,
depending on the subunit and modulator) than those observed by
whole-cell recording (Tables 1, 2). The reasons for this difference are
unclear, but most likely reflect slow application of agonist for
whole-cell recording and possibly altered channel properties in
outside-out patches (Tong and Jahr, 1994).
Fig. 6.
Modulation of deactivation and desensitization for
position 750 mutants. A, Outside-out patch responses to
1 msec applications of 10 mM glutamate, in either the
absence or the presence of 100 µM cyclothiazide; open tip
junction potentials are shown above each set of current
traces. Arrows denote the start of single-exponential
fits for analysis of deactivation. Cyclothiazide slowed deactivation
for S750A, but not for S750N or
S750Q. Responses to 1 msec applications of 10 mM glutamate in the absence or presence of 5 mM
aniracetam revealed that modulation of deactivation was most
efficacious for S750N but also occurred for
S750Q for which cyclothiazide was inactive.
B, Bar plots of the time constants for deactivation
(deact, mean ± SEM) as determined for controls
(open bars), in the presence of 100 µM
cyclothiazide (solid bars), or in the presence of 5 mM aniracetam (hatched bars) for
GluRAi, GluRAo, and the three point mutants
examined in the outside-out patch configuration. C, Bar
plots of time constants for desensitization (des,
mean ± SEM) for the same receptors. Note that there are three
y-axes: one for control responses to 10 mM
glutamate; one for desensitization in the presence of cyclothiazide;
and one for desensitization in the presence of aniracetam.
Asterisk denotes that, for GluRAi, the value
represents the time constant of dissociation of cyclothiazide
(15.7 ± 0.7 sec) estimated from experiments shown in Figure
8D. Plus signs (+) denote values
that include the weighted averages of data fit with two exponentials
(see Table 2).
[View Larger Version of this Image (25K GIF file)]
Thiocyanate modulation of AMPA receptor splice isoforms
Recent experiments on native and reconstituted AMPA receptors
(Bowie and Smart, 1993; Arai et al., 1995; Kessler et al., 1996) have
shown that thiocyanate is a modulator of desensitization, but with the
opposite effect from that produced by cyclothiazide and aniracetam,
because thiocyanate increases the extent and rate of onset of
desensitization. To test for differential sensitivity of
AMPA receptor splice isoforms to thiocyanate, we
first compared whole-cell responses to 500 µM AMPA in the
presence and absence of 20 mM thiocyanate (Fig.
7A) using protocols similar to those for
experiments on native receptors expressed in hippocampal slices (Arai
et al., 1995), for which the effect of thiocyanate was shown to be
greater with AMPA rather than glutamate as agonist. The effect of
thiocyanate on GluRAi was similar to that found for native
AMPA receptors in hippocampal neurons in that the peak was reduced
17 ± 3% and des accelerated from 7.2 ± 0.5 to 5.1 ± 0.3 msec (n = 21). The results were
different for GluRAo, however, in that the peak was
consistently increased by 38 ± 14%, and des
slowed from 7.4 ± 0.6 to 8.3 ± 0.5 msec (n = 6). We also tested S750Q (resistant to cyclothiazide) and
S750N (increased sensitivity to aniracetam) for modulation
of desensitization by thiocyanate (Fig. 7A) and found that
responses for S750N were similar to those for
GluRAo, with des essentially unchanged
(7.6 ± 0.9 to 7.9 ± 1.0 msec; n = 15) but
with the peak amplitude potentiated (44 ± 6%), whereas for
S750Q des accelerated from 6.1 ± 0.6 to 4.4 ± 0.5 msec (n = 12) without a significant
change in the amplitude of the current (98 ± 14%). Although
S750A desensitized almost twice as slowly
(des 11.4 ± 1.3; n = 13) as
S750Q (6.1 ± 0.6 msec; n = 12),
thiocyanate accelerated the rate of onset of desensitization (~33%)
to a similar extent for both mutants (Fig. 7A, Table 1).
Fig. 7.
Thiocyanate differentially modulates
desensitization of GluRA flip/flop isoforms. A,
Whole-cell responses to 500 µM AMPA in the absence
(thin trace) or presence (bold trace) of
20 mM thiocyanate; single-exponential fits of the indicated
time constant are superimposed on the traces. Thiocyanate decreased the
peak response and increased the rate of onset of desensitization for
GluRAi and S750Q. In contrast, thiocyanate
consistently increased the peak amplitude and slowed the onset of
desensitization for both GluRAo and S750N. Bar
plots summarize the kinetics of onset of desensitization
(left) and peak current amplitudes
(right) in the presence of thiocyanate plotted relative
to control for GluRAi, GluRAo, and the three
point mutants studied (S750X, where
X represents N, A, or Q). B, Outside-out
patch responses to 10 mM glutamate in the presence of 20 mM thiocyanate. Arrows denote the start of
single-exponential fits. Open tip junction potentials are shown
above the current traces. Deactivation was essentially
unchanged for GluRAi but slowed for GluRAo in
the presence of thiocyanate. The onset of desensitization of
GluRAi was faster in the presence of thiocyanate, whereas
thiocyanate slowed the onset of desensitization for GluRAo,
similar to whole-cell responses recorded using AMPA as agonist.
[View Larger Version of this Image (24K GIF file)]
To better resolve modulation of desensitization by thiocyanate, as well
as to determine whether thiocyanate modulates deactivation for
wild-type GluRAi and GluRAo, we tested the
effects in outside-out patches of 20 mM thiocyanate on
responses evoked by 10 mM glutamate, which was chosen to
allow comparison with the effects of other modulators in outside-out
patches (Fig. 7B). We found a 1.6-fold slowing of
deact for GluRAo from 0.69 ± 0.05 to
1.12 ± 0.08 msec (n = 8, p > 0.0001), whereas for GluRAi deact was not
significantly changed (0.65 ± 0.04 to 0.73 ± 0.04 msec;
n = 8). Thiocyanate had a differential effect on
desensitization, accelerating des for GluRAi
(2.17 ± 0.11 to 1.58 ± 0.09 msec), but slowing
des for GluRAo (from 3.21 ± 0.27 to
3.77 ± 0.29 msec). Therefore, modulation of deactivation and
desensitization by thiocyanate is distinct from modulation by
cyclothiazide and aniracetam, but is sensitive to amino acid changes in
the flip/flop domain and, specifically, to changes at position 750.
Kinetic modeling of differential modulation by aniracetam
and cyclothiazide
A variety of kinetic models for ligand-gated ion channels
have been developed to describe activation, deactivation, and
desensitization. Figure 8A presents a
kinetic model for AMPA receptor modulation by aniracetam and
cyclothiazide, derived primarily from the work of Vyklicky et al.
(1991), Raman and Trussell (1995), and Kessler et al. (1996). The rate
constants for the model are given in Table 3 (see also
Materials and Methods). The model describes binding of two molecules of
agonist leading to a conformational transition to a single open state.
In the continued presence of agonist, there is a high probability that
the receptor will undergo further transitions to nonconducting,
agonist-bound desensitized states (RdA and
RdA2). After channel opening by brief pulses of
agonist, a significant fraction of receptors return to the unliganded
state without entering the desensitized states RdA and
RdA2. The model assumes binding of one molecule
of modulator and does not address whether aniracetam and cyclothiazide
bind to the same site. The model allows desensitization to occur only
through closed states, although this assumption has not been proven.
Although these simplifications facilitate analysis of modulation,
native AMPA receptors are known to have subconductance states (Howe et
al., 1991) and, in addition, as modeled by Raman and Trussell (1995),
there is evidence for multiple and kinetically distinct open states.
Figure 8B shows simulated responses to 10 mM glutamate generated by the models developed here. These
are similar in many ways to experimental responses for GluRA (Figs. 4,
5): there is marked desensitization with a 1 msec pulse of agonist;
deact (0.85 msec) is 3.6-fold faster than
des (3.1 msec); responses to prolonged application of
glutamate desensitize to <1% of peak. Modulation by aniracetam
similar to that observed for GluRAo was achieved by slowing
the closing rate constant sixfold. This reproduced slowing of
deactivation (deact 5.0 msec) and desensitization
(des 16.6 msec); for GluRAo the
experimentally recorded mean time constants were 3.31 and 26 msec,
respectively. As would be expected from a cyclic model, slowing the
closing rate constant changes accessibility to ligand-bound
desensitized states, which results in a slowing of the rate of onset of
desensitization (Vyklicky et al., 1991). A similar stabilization of the
open state could occur alternatively if modulation by aniracetam
resulted from an accelerated rate of channel opening. A kinetic model
increasing the opening rate constant 30-fold did slow deactivation
(deact 3.4 msec), but it also slowed desensitization
(des 95 msec) to a much greater extent than observed
experimentally, leading us to conclude that modulation of the channel
closing rate rather than channel opening was a more probable
explanation for the effects of aniracetam. In the model developed here,
the channel opening rate constant, 30,000 sec
1 in the
absence of modulators, would have to be changed to 900,000 sec1 to slow deactivation to the extent seen
experimentally. This high rate seems improbable and, indeed, is
substantially faster than the maximum opening rate estimated for
nicotinic (12,000 sec1; Liu and Dilger, 1991) and
GABAA (3500 sec1; Jones and Westbrook, 1995)
receptor channels. Additional evidence consistent with modulation of
the closing rate constant by aniracetam comes from single-channel
studies on native receptors for which modulation by aniracetam is
observed as an increased mean open time and burst length (Vyklicky et
al., 1991). A final prediction of our model is a linear relationship
between modulation of deactivation and modulation of desensitization by
aniracetam, a correlation confirmed experimentally (see Discussion;
Fig. 9).
Fig. 8.
Kinetic modeling suggests different mechanisms for
modulation by aniracetam and cyclothiazide. A, Cyclic
model describing kinetic reactions between states of an AMPA receptor
in the presence of either aniracetam (left) or
cyclothiazide (right). Bold lines
indicate altered rate constants in the presence of aniracetam or
cyclothiazide. R, Free receptor; M,
modulator; A, agonist (glutamate);
Rd, desensitized state. B,
Simulated currents representing modulation of deactivation and
desensitization by aniracetam (left) or cyclothiazide
(right); modulators (open bar) were
assumed to be pre-equilibrated with the receptor before application of
10 mM glutamate (closed bar). Responses are
normalized to their peak amplitudes to compare their time courses.
Modulation of GluRAo by 5 mM aniracetam was
simulated by slowing only the rate of channel closing sixfold versus
control. Modulation of GluRAi by 100 µM
cyclothiazide was simulated by slowing the rate of onset of
desensitization 10,000-fold and increasing the agonist affinity of the
cyclothiazide-bound state RMA2 20-fold; single-exponential
fits are superimposed on the simulated traces. C,
Simulated recovery from desensitization in the absence of modulator.
The data were fit with a single exponential, rec = 125 msec, similar to experimental values. D, Decays of a
simulated response (thin trace) and an experimentally
recorded response of GluRAi to 1 mM glutamate
in the absence of modulator after preincubation with 100 µM cyclothiazide. The rate of decay is assumed to reflect
the unbinding of cyclothiazide. For GluRAi the mean
dissociation rate was 15.7 ± 0.7 sec; for the simulation it was
11.3 sec.
[View Larger Version of this Image (25K GIF file)]
Fig. 9.
Deactivation and desensitization are linked
processes. A, Correlation between the time constants for
deactivation and desensitization combining previously published values
for native and recombinant AMPA receptors (
) with data from the
present study (
). HMC, Hilar mossy cells;
CA3, hippocampal CA3 pyramidal cells;
Cx-pyramidal, neocortical layer V
pyramidal cells; Cx-interneuron,
neocortical layer IV nonpyramidal cells;
DG-basket, dentate gyrus basket cells;
MNTB, medial nucleus of the trapezoid body (auditory
nucleus). Values are taken from Geiger et al. (1995), except for nMAG
(chick nucleus magnocellularis), which was taken from Raman and
Trussell (1995) and recombinant GluRAi, -Ao,
-Di, and -Do (Lomeli et al., 1994). Data from
the present study include GluRAi, S750A,
S750N, and S750Q. Note that the time constants
for outside-out patches presented in this study were obtained using 10 mM glutamate, whereas most previous studies used 1 mM glutamate. A linear fit through the data yields a slope
of 5.3 (r = 0.96). A similar finding was reported
recently by Trussell and Otis (1996). B, Comparison of
the relationship between control deactivation and desensitization rates
for mutations at position 750 or amino acids in the C terminus of the
flip/flop domain (GluRAi[io] and
GluRAo[oi]). A linear fit through GluRAi,
S750N, S750Q, and S750A yields a
slope of 8.1 (r = 0.99). The
asterisk indicates that data for GluRD are taken from
Mosbacher et al. (1994). C, Comparison of the linear
relationship between deact and des after
making incremental changes in the closing rate constant for simulated
data (solid line), and between deact and
des in the presence of aniracetam () or cyclothiazide
(
) for mutations described in this study. The data for aniracetam
lie along the line, consistent with the hypothesis that aniracetam acts
via slowing the closing rate constant. D, Schematic
representation of AMPA receptor domains (O'Hara et al., 1993;
Stern-Bach et al., 1994; Kuryatov et al., 1994). The N-terminal domain
shares homology with LIVBP (bacterial periplasmic
eucine-
soleucine-
aline-
inding
rotein; hatched region). The
agonist-binding pocket is formed by two lobes (derived from N-terminal
residues and residues from the M3-M4 extracellular loop) sharing
homology with LAOBP
(ysine-
rginine-
rnithine-inding
rotein; gray stippled region); S1/S2 refers
to the chimeric constructs (Stern-Bach et al., 1994; Kuusinen et al.,
1995), confirming that this forms the agonist-binding pocket. Putative
membrane domains shown in black are embedded within the
lipid bilayer (Hollmann and Heinemann, 1994; Bennett and Dingledine,
1995). The cytoplasmic domain is shown with wavy
hatching; P represents the site for
phosphorylation by CaM-kinase II (McGlade-McCulloh et al., 1993; Yakel
et al., 1995). The flip/flop region is shown as a helix-turn-helix
motif that connects the agonist-binding region to M4.
[View Larger Version of this Image (35K GIF file)]
A kinetic scheme reproducing modulation by cyclothiazide for
GluRAi is shown in Figure 8A
(right), but in this case modulation occurs through
``stabilization'' of an agonist-bound nondesensitized closed state
(RMA2). This is achieved by increasing agonist affinity for
the RMA2 state 20-fold and slowing the rate of onset of
desensitization from the RMA2 state 10,000-fold. Modulation
of the onset of desensitization would not account for the slowing of
deactivation that is seen experimentally, whereas increasing agonist
affinity slows the rate of deactivation by slowing agonist
dissociation, but only when the rate of entry into desensitized states
is reduced. The model predicts very efficacious modulation of
desensitization with weaker modulation of deactivation, an effect
observed experimentally for cyclothiazide modulation of all GluRA
constructs studied. The model also predicts that, different from
modulation by aniracetam, there should not be a linear correlation
between modulation of deactivation and desensitization by
cyclothiazide. The simulated data traces reproduce these features, with
deact (4.8 msec) slowed only 5.6-fold but
desensitization essentially abolished. This is similar to data
collected for GluRAi (deact 2.1 msec, 2.4-fold slower
than control) but with <6% desensitization within 100 msec. Figure
8C shows that for the models developed the kinetics of
recovery from the desensitized state are slow (rec 125 msec) with kinetics similar to those recorded experimentally for GluRA
(rec 147 msec; Fig. 4E). Because
GluRAi desensitizes very little in the presence of
cyclothiazide, it is difficult to assign a value for the ``rate of
onset of desensitization'' as a measure of the efficacy of
cyclothiazide. We therefore performed experiments to measure the
dissociation rate of cyclothiazide in the presence of glutamate for
GluRAi (off 15.7 ± 0.7 sec) and for
the model (off 11.3 sec; Fig. 8D) and
found the rates of dissociation to be similar. Simulated dose-response
relationships with 1 mM glutamate as agonist yielded an
EC50 for cyclothiazide of 1.0 µM and a shift
in the EC50 of the glutamate peak response from 936 to 26 µM in the presence of 100 µM cyclothiazide.
This substantially reproduces the results of Yamada and Tang (1993),
for which the cyclothiazide EC50 was 12 µM
with quisqualate as agonist, and the EC50 of the
quisqualate peak response was shifted from 280 to 3 µM in
the presence of 10 µM cyclothiazide. The apparent
stabilization of RMA2 as the underlying mechanism of
modulation by cyclothiazide has been discussed previously for native
AMPA receptors (Patneau et al., 1993; Kessler et al., 1996). It is
supported by work demonstrating that there is no change in
single-channel conductance in the presence of cyclothiazide (Yamada and
Tang, 1993), by data that show a decrease in [3H]AMPA
binding in the presence of cyclothiazide attributable to the higher
affinity of AMPA for the desensitized state (Hall et al., 1993), and by
the finding that cyclothiazide causes a leftward shift of kainate
dose-response curves (Patneau et al., 1993; Partin et al., 1994).
However, it is not possible with the present experiments to distinguish
whether this occurs through a cyclothiazide-induced destabilization of
the desensitized state, stabilization of the closed nondesensitized
state (RMA2), or a combination of both.
DISCUSSION
We report here a detailed analysis of a critical site in the
alternatively spliced flip/flop domain of AMPA receptors (position 750 in GluRA) that affects intrinsic channel gating kinetics and the
ability of three different compounds to modulate both desensitization
and deactivation. These effects could reflect (1) coupling of
deactivation and desensitization, (2) that structural determinants
directing these conformational transitions share a set of amino acids
(some of which are encoded within the flip/flop domain), and/or (3)
that the modulators bind at or near this site. In the next section, we
discuss our interpretation of the functional contributions of the
flip/flop domain to AMPA receptor properties.
In the kinetic model presented in Figure 8, slowing the closing rate
constant results in a concomitant slowing of the macroscopic rate of
onset of desensitization (Vyklicky et al., 1991). As recently noted by
Trussell and Otis (1996), experimental observations on AMPA receptors
in different neuronal populations show a similar covariance between the
time constants of deactivation and desensitization. Further support for
this is shown in Figure 9A, which combines data from our
study with published values for a large number of neuronal and
recombinant AMPA receptors. Of the position 750 point mutations studied
in detail, S750Q and S750A varied from
GluRAi in both des and deact,
whereas for S750N neither process was altered (Fig.
9B). This suggests that determinants at position 750 can
directly influence the kinetics of deactivation, resulting in a
secondary effect on the kinetics of desensitization. Although for
native receptors heterogeneity in the rate of onset of desensitization
would be expected to result from heterogeneity in the closing rate
constant, the two kinetic processes do vary independently to some
extent, perhaps according to subunit composition and assembly (Geiger
et al., 1995). Evidence for variation in the rate of onset of
desensitization, independent from the closing rat
