1Department of Neuroscience and 2Department of Pharmacology and Therapeutics, University of Florida Medical College, J. H. Miller Health Center, Gainesville, Florida 32610-0267
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ABSTRACT |
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Karkanias, Nikolas B. and
Roger L. Papke.
Subtype-specific effects of lithium on glutamate receptor function.
We report that substitution of sodium with lithium
(Li+) in the extracellular solution causes subtype-specific
changes in the inward and outward currents of glutamate receptors
(GluRs), without a shift in reversal potential. Li+
produces an increase of inward and outward currents of
-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and
decreases in the currents of kainate (KA) and
N-methyl-D-aspartate receptors. The greatest
effect of Li+ was observed with GluR3. A
concentration-response curve for GluR3 reveals that the potentiation
caused by Li+ is greatest at saturating agonist
concentrations. GluR1, which shows no potentiation by Li+
at 100 µM KA, shows a small but significant potentiation at
saturating KA and glutamate concentrations. The effects of
Li+ on outward current, where Li+ is not the
primary charge carrier, and the lack of reversal potential shift argue
for a mechanism of potentiation not associated with Li+
permeation. This potentiation of current is specific for
Li+ because rubidium, although causing an increase of
inward current, shifted the reversal potential and did not increase
outward current. The effects of Li+ are different for KA, a
weak desensitizing agonist, and glutamate, a strong desensitizing
agonist, suggesting that Li+ might interact with a
mechanism of desensitization. By using cyclothiazide (CTZ) to reduce
desensitization of GluR3, we find that for low concentrations of KA and
glutamate potentiation of the response by a combination of CTZ and
Li+ is no greater than by CTZ or Li+ alone.
However, at high concentrations of agonist, the potentiation of the
response by a combination of CTZ and Li+ is significantly
greater than by CTZ or Li+ alone. This potentiation was
additive for glutamate but not for KA. At high agonist concentration in
the presence of CTZ, the intrinsically lower desensitization produced
with KA-evoked responses may preclude Li+ from potentiating
the current to the same degree as it can potentiate glutamate-evoked
responses. The additive effects of CTZ and Li+ were unique
to the flop variant of GluR3.
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INTRODUCTION |
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Ionotropic glutamate receptors (GluRs) are
responsible for most of the fast excitatory neurotransmission in the
mammalian brain, which includes activity-dependent synaptic
modifications such as long-term potentiation and long-term depression
(Bliss and Gardner-Medwin 1973; Bliss and Lynch
1988
; Collingridge and Bliss 1987
). A
differential expression of GluR subunit genes gives rise to the
functional diversity of GluRs among brain regions with unique
permeability and kinetic properties for specific receptor subtypes.
Characteristics of GluRs such as ionic selectivity and kinetics are
vital to the understanding of fast excitatory synaptic transmission and
how synaptic activity and neuronal plasticity may be coupled in various
parts of the brain.
Several subtypes of GluRs contribute to fast excitatory transmission,
and they can be pharmacologically distinguished into two major classes,
non-N-methyl-D-aspartate (NMDA) and NMDA
sensitive. The non-NMDA-sensitive channels contain the receptor
subunits GluR1-GluR7. The channels composed of subunits GluR5-GluR7
can assemble with accessory subunits KA1 or KA2 to form receptors that
are activated by kainate (KA). Channels composed of GluR1-GluR4 are
activated by -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)
as well as KA. The NMDA-sensitive channels are composed of NMDAR1 and
NMDAR2a-NMDAR2d. Functional properties of these GluRs, such as their
permeability to sodium, potassium, and calcium and their kinetics, are
influenced by the specific subunit composition of the channel
(Hollmann et al. 1991
; Monyer et al.
1992
) .
Receptor desensitization is another property of GluRs that may regulate
synaptic function. When the glutamate transient time course is slow,
because of the nature of the synaptic morphology, the duration of
synaptic current may be determined primarily by desensitization
kinetics (Barbour et al. 1994). Desensitization is
promoted by agonist exposure, and experimental agonists can vary in
their relative desensitizing effect. For example, KA produces less
desensitization in AMPA-selective receptors than either AMPA or
glutamate. The desensitization kinetics vary among the specific AMPA
receptor subtypes as a result of RNA editing and alternative splicing.
The flip/flop domain is a 38-amino acid cassette located extracellularly and N-terminal to the final transmembrane domain of
AMPA receptors. Alternative splicing of this cassette yields mature
flip or flop AMPA receptors that vary in their desensitization kinetics
(Mosbacher et al. 1994
; Sommer et al.
1990
). Immediately before the flip/flop domain is the R/G site
(Lomeli et al. 1994
). RNA editing at the R/G site can
also influence AMPA receptor desensitization kinetics with edited
channels (G) recovering from desensitization faster.
Although pharmacological modulators such as cyclothiazide (CTZ) are
thought to reduce desensitization of AMPA receptors thus providing
pharmacological tools with which to study this property (Partin
et al. 1993; Vyklicky et al. 1991
; Wong
and Mayer 1993
), desensitization was largely assumed to be
independent of the charge-carrying ion. In a previous study we reported
the preliminary observation that Li+ produced
subtype-specific alterations of macroscopic current (Karkanias
et al. 1998
). Further investigation of Li+ effects
on GluR leads us to propose that modulation of receptor desensitization
is a mechanism that causes the flop variant of GluR3 to display a
modified conductance in the presence of Li+.
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METHODS |
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Oocyte preparation
Female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI) and kept in tanks at 17°C. Frogs were anesthetized for 30 min on ice in 2.2 g/1.5 l of 3-aminobenzoic acid ethyl ester purchased from Sigma (St. Louis, MO). Oocytes were obtained in lobes through a small abdominal incision made just above the leg and near the midline on the ventral surface of the frog. Two to three lobes were pulled from the frog, cut, and placed in collagenase from Worthington Biochemical (Freehold, NJ) (1 mg/ml in calcium-free Barth's solution containing 88 mM NaCl, 1 mM KCl, 15 mM HEPES, 0.33 mM MgSO4, and 0.1 mg/ml gentamicin sulfate, pH 7.6) for 2 h to enzymatically remove the native follicular cell layer. After the follicular cell layer was removed, oocytes were washed several times with calcium-free Barth's and then washed several times with Barth's solution containing 88 mM NaCl, 1 mM KCl, 15 mM HEPES, 0.33 mM CaNO3, 0.41 mM CaCl2, 0.33 mM MgSO4, and 0.1 mg/ml gentamicin sulfate (pH 7.6) and stored at 17°C. Mature oocytes were injected the same or following day with the appropriate RNA transcripts (20 ng/oocyte).
mRNA transcription and injections
cDNA clones containing the appropriate gene and T3/T7 bacterial
promoters were isolated from bacteria and purified with a kit from
Qiagen (Santa Clarita, CA). Purified cDNA clones were linearized with
the necessary restriction enzyme and then purified to serve as template
for in vitro transcription. Briefly, in vitro cRNA transcripts were
prepared with the appropriate mMessage mMachine kit from Ambion
(Austin, TX). Transcription reactions were performed with 1 µg cDNA
as template, an RNA polymerase (T3 or T7, depending on clone),
DTT, RNase inhibitor, dNTPs, and 32P.
Nucleotide incorporation was evaluated by DEAE81 filter binding assays
and a liquid scintillation counter. RNA was stored in DEPC water stocks
at 80°C, and aliquots were used for injection into the oocytes. The
accession numbers for the clones used in this study were GluR1
(X17184), GluR2 (M85035), GluR3 (M85036), GluR6 (Z11548),
NMDAR1-1a (L19708), NMDAR2a (AF001423), and NMDAR2b (U11419).
Unless otherwise noted, we used the flop variants of AMPA receptors in
our experiments.
Two-electrode voltage clamp
For the conventional two-electrode, voltage-clamp experiments,
oocytes were placed in a Warner Instruments (Hamden, CT) recording chamber and perfused with frog Ringer solution [containing (in mM) 115 NaCl, 2.5 KCl, 1.8 BaCl2, and 10 HEPES, pH 7.3]. Ringer solutions containing lithium (Li+) or rubidium were made by
substituting the ion for sodium. Osmolarity of different Ringer
solutions was checked with a Precision Systems (Natick, MA) Osmette A
osmometer. A Warner Instruments Oocyte Clamp OC-725B and Frequency
Devices model 902 filter were interfaced with National Instruments
(Austin, TX) LabVIEW software and a Macintosh computer for data
acquisition. Electrodes were fabricated from glass capillary tubes
(KG-33) from Garner Glass (Claremont, CA) with a DKI (Tujunga, CA)
model 750 needle/pipette puller. Voltage electrodes were filled with 3 M KCl and had resistances on the order of 1-5 M, whereas current
electrodes were filled with 0.25 M CsCl, 0.25 M CsF, and 100 mM EGTA
(pH 7.3) and had resistances of 0.5-3 M
. Experiments were performed
at room temperature, and the oocyte membrane was clamped at
50 mV.
Currents were measured to the nearest nanoampere. At least three and
usually four or more oocytes were used for each measurement. Drugs were
dissolved in Ringer and applied by filling a 2.0-ml length of tubing at the end of the perfusion line. A discrete volume of agonist was thereby
administered over a 10-s period. Some drug stocks were dissolved in
DMSO and then diluted in Ringer to <1% DMSO. No effect on control
response was observed when the agonist was dissolved in DMSO. In most
experiments, barium was used instead of calcium in the Ringer to
minimize contributions of endogenous calcium-activated chloride
current. However, similar results were obtained in the presence of
calcium. For experiments with GluR6(Q/R), a 2.0-ml, 10-s pulse of
concanavalin A (Sigma; St. Louis, MO, type IV; 1.2 mg/ml) was applied 5 min before applying agonist.
Current-voltage relationships were performed by delivering a voltage
ramp, 50.0 mV to +50.0 mV, during the plateau phase of the response
to agonist (pClamp 5.5, Axon Instruments; Foster City, CA). The passive
current-voltage response of the cell membrane in the absence of agonist
was subtracted from the current-voltage response in the presence of
agonist. Permeability ratios for Li+ and rubidium with
respect to sodium were calculated with Eq. 1
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(1) |
Concentration-response relationships
The responses of GluR3 expressing oocytes to various test
concentrations of KA were normalized by the response to the
EC50 KA concentration (100 µM) immediately preceding the
test concentration. First, a response was recorded to 100 µM KA, and
after a 5-min washout a response was recorded to a test dose of KA.
After 5 min, 100 µM KA was applied again to determine any residual
effects from the test dose of KA. If the response to 100 µM KA after
the test dose was 75% of the response to 100 µM KA before the test dose, the oocyte was tested further at other concentrations of KA. The
resulting concentration-response relationship was fitted with Eq. 2 (Luetje and Patrick 1991
)
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(2) |
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RESULTS |
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Effect of Li+ on neuronal GluR function
Specific GluR subtypes were evaluated for their potential modulation by Li+. Effects on GluR function were noted in the range of 5-115 mM Li+. For our standard agonist applications we used 100 µM KA for AMPA receptors, 100 µM glutamate for KA receptors, and 100 µM glutamate + 10 µM glycine for NMDA receptors. Under these conditions, KA/NMDA-receptor currents appeared to be reduced in Li+ solutions, whereas AMPA receptor currents were potentiated in Li+ solutions. Subtype-selective differences in potentiation were observed within the AMPA receptor class and were investigated further with ionic variation in extracellular solutions, different agonists at multiple concentrations, and chemical modulators.
Permeability and conductance of neuronal GluRs to Li+
Compared with their respective sodium controls, the amplitude of the KA-evoked current in Li+ varied across subtype of GluR. However, when extracellular sodium was replaced with Li+, no shift in reversal potential was detected for any of the subtypes that were tested. For the AMPA-selective flop variants of GluRs, both inward and outward currents in Li+ Ringer were equal to or greater than the current in Na+ Ringer. A representative I-V relationship for GluR3 + R2 is shown in Fig. 1A. The increase of both inward and outward currents without a shift in the reversal potential suggests an effect on the Popen rather than an increase in single channel conductance with Li+. In general, the inward and outward currents of various AMPA receptors were potentiated by Li+, ranging from 40 to 200% above control (Fig. 1B). However, the current evoked by 100 µM KA in Li+ Ringer through GluR1 was not significantly increased. In contrast, under these conditions the current in Li+ Ringer was decreased by 40-60% for KA and NMDA receptors (Fig. 1, C and D). Because GluR1 + R2 receptor currents were potentiated but GluR1 currents were not (Fig. 1B), we investigated the role of the GluR2 subunit in Li+ potentiation. Wild-type GluR2 alone does not function well in oocytes. Therefore for these experiments we expressed wild-type GluR2 with a Q/R site mutant, GluR2(R586Q), as well as the mutant alone. The inward and outward currents of heteromeric GluR2 + GluR2(R586Q) receptors were increased in Li+ by 74 ± 7.9% (n = 6) and 72 ± 10% (n = 6), respectively (Fig. 2A). The conductance of the mutant homomer was increased 134 ± 26% above control (n = 3) in the presence of Li+ (Fig. 2B). These results indicate that GluR2 is intrinsically capable of Li+ modulation. However, further experiments focused on the GluR3 subtype because it was most sensitive to Li+ potentiation and easily formed homomeric receptors.
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Specificity of the effect of Li+ on GluR currents
To test the specificity of Li+ effect on GluR currents, we performed similar experiments in rubidium-based Ringer. Raw waveforms recorded from a GluR3-expressing oocyte in Na+, Rb+, and Li+ Ringer are shown in Fig. 3A. Rubidium produced a mean increase in current of 56 ± 4.6% (n = 4) but correspondingly shifted the reversal potential 6.60 ± 1.03 mV (n = 7) for GluR3 (Fig. 3A, inset) and 4.05 ± 0.46 mV (n = 10) for GluR3 + R2 (Fig. 4, A and B). There is no potentiation of outward current by Rb+ for GluR3 (Fig. 3A, inset) or for GluR3 + R2 (Fig. 4B). By using the shift in reversal potentials and solving for the ratio PRb/PNa (see METHODS), we calculated a PRb/PNa of 1.30 for GluR3 and 1.17 for GluR3 + R2.
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Concentration dependence of extracellular Li+
We wished to determine if the magnitude of Li+
potentiation of GluR3 response varies linearly with external
Li+ concentration. We noted that when 100 µM KA is used
as the agonist an increase of the extracellular Li+
concentration causes a current potentiation that could be fit by
Eq. 2 with an EC50 of 14.85 ± 1.71 mM
(n 4) (Fig. 5).
However, when saturating concentrations (1 mM) of KA were used we saw a further anomalous increase in potentiation at the highest extracellular Li+ concentration. To test if this peculiar increase with 1 mM KA in 100% Li+ (115 mM Li+, 0 mM
Na+) could be associated with the total absence of sodium
we performed experiments at the same Li+ concentration but
with added Na+ (115 mM Li+, 10 mM
Na+) as well as an osmotic control (115 mM Li+,
20 mM sucrose). The presence of 10 mM Na+ in 115 mM
Li+ Ringer did not significantly reduce the response to 1 mM KA (data not shown). These results indicate that the extreme
potentiation reported for 1 mM KA responses obtained in the presence of
115 mM Li+ was not due to a specific effect associated with
the removal of sodium. Note that when 1 mM Glu was used as the agonist
the GluR3 current displayed an increased threshold for potentiation by
Li+ that did not appear to saturate and reached a maximum
potentiation of 357% with 100% mole fraction of Li+ (Fig.
7B).
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It is interesting to note the receptor sensitivity to potentiation by concentrations of Li+ that approach levels used for treatment of bipolar disorder. We observed an increase of 21 ± 3% (n = 10, P < 0.001) for GluR3 and 26 ± 3% (n = 6, P < 0.05) for GluR3 + R2 in response to 100 µM KA at 5 mM extracellular Li+. If the potentiation was linearly dependent on extracellular Li+ concentration, at 5 mM extracellular Li+ (1/23 of 115 mM) one would expect that the potentiation would be ~1/23 of the maximum potentiation observed with 100 µM KA and 115 mM Li+ (or 7-8%). This observation further supports the hypothesis that the effects of Li+ are most likely due to effects on Popen rather than channel conductance. The response waveforms recorded in 115 mM Na+, 115 mM Li+, and 5 mM Li+ from GluR3-expressing oocytes are shown in Fig. 6, A-C. Rubidium has no effect on GluR3 current at 5 mM extracellular concentration in contrast to Li+ (Figs. 3B and 6). Potentiation by Li+ is readily reversible at both high and low concentrations on washout of the Li+ Ringer. In Fig. 6D, an I-V relationship of a GluR3 + R2-expressing oocyte recorded in 5 mM Li+ showed an increase of both inward and outward currents analogous to that shown in Fig. 1A.
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Li+ effects on GluR agonist potency
We examined the potency and efficacy of KA in the presence and
absence of Li+. The concentration-response relationship
determined for GluR3 in Na+ Ringer yielded a Hill slope of
1.14 ± 0.07 and an EC50 of 125 ± 8 µM (Fig.
7A, ). In Li+
Ringer, the concentration-response relationship yielded a Hill slope of
2.19 ± 0.56 and an EC50 of 211 ± 28 µM (Fig.
7A,
). Li+ increased the maximal attainable
response by >500% and increased the EC50 by 60% compared
with sodium. The total percent increase caused by Li+ for
glutamate and KA at low and high concentrations relative to the same
agonist and concentration in Na+ is presented in Fig.
7B. GluR1 does not show the same magnitude of increase
compared with GluR3 with either KA or glutamate at saturating
concentrations.
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Li+ effects and desensitization
We examined the effect of Li+ when desensitization of GluR3 channels was reduced with the compound CTZ (Fig. 8). With 100 µM KA or glutamate as the agonist, both Li+ and 100 µM CTZ potentiate GluR3 responses to the same extent. There was no further increase when the treatments were combined (Fig. 8, A and B). With 1 mM KA as the agonist, the combination of 100 µM CTZ and Li+ substitution produced a current increase that was 30% larger than the increase CTZ produced in Na+ Ringer (Fig. 8C, P < 0.05, unpaired t-test). At a high glutamate concentration (1 mM), the combination of 100 µM CTZ and Li+ substitution produced a current increase that was 353 ± 54% larger than the increase CTZ produced in Na+ Ringer (Fig. 8D, P < 0.001, unpaired t-test). Although CTZ potentiated GluR1 currents in sodium, there was no apparent interaction between the combination of CTZ with Li+ at either agonist concentration (data not shown).
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Because flip/flop variants were reported to vary in their intrinsic desensitization, we set out to determine the interaction of the flip/flop domain with Li+ potentiation. We investigated GluR3 flip currents for their capacity to be modulated by Li+ at saturating agonist concentrations in the presence and absence of CTZ and Li+ (Fig. 8, E and F). We found that Li+ potentiated the current evoked from GluR3 flip channels less than from GluR3 flop. Potentiation of responses to 1 mM KA and 1 mM Glu was only 19 and 67% of the potentiation obtained with GluR3 flop. We also examined the effect of Li+ when desensitization of GluR3 flip channels was reduced with the compound CTZ (Fig. 8, E and F). In contrast to the results obtained with GluR3 flop, Li+ actually decreased 1 mM KA + 100 µM CTZ-evoked GluR3 flip currents by 19% (n = 6) compared with the 1 mM KA + 100 µM CTZ-evoked currents in Na+ Ringer (n = 6) (Fig. 8E, P < 0.05, unpaired t-test). Similarly, Li+ decreased 1 mM Glu + 100 µM CTZ-evoked GluR3 flip currents by 35% (n = 11) compared with the 1 mM Glu + 100 µM CTZ-evoked currents in Na+ Ringer (n = 11) (Fig. 8F, P < 0.05, unpaired t-test). This reduction in GluR3 flip current when CTZ and Li+ are combined was also seen when lower agonist concentrations were used (100 µM Glu), indicating that this effect is not specific to high agonist concentrations (n = 3, P < 0.05, data not shown).
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DISCUSSION |
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We characterized the subtype-selective potentiation of GluR3 by Li+ through an evaluation of Li+ effects with agonist concentration, mole fraction of extracellular Li+, and compounds affecting receptor desensitization. Our results permit us to propose that the effect of Li+ is to modify desensitization in a manner that depends on the flop domain.
Li+, rubidium, and sodium are all monovalent cations in
group I of the periodic table, but only Li+ is used for the
treatment of the mental illness bipolar disorder. Studies involving
Li+ inhibition of second-messenger systems often use
rubidium to confirm the specificity of the inhibition by
Li+ (Ebstein et al. 1980). We therefore
compared GluR3 currents in Li+-, Na+-, and
Rb+-based Ringer. Although we observed a potentiation of
the responses in both Rb+ and Li+ Ringer
compared with Na+, the potentiation in Rb+ was
qualitatively different than in Li+ because it coincided
with a shift of the reversal potential in the positive direction and
was not observed on outward currents. These data suggest that the
effects of Rb+ were largely due to an increased
permeability of Rb+ through the channel.
The magnitude of Li+ potentiation increased with the concentration of extracellular Li+ independently of agonist concentration except at saturating agonist concentration in the presence of 115 mM Li+. One potential explanation for this observation is that there are two processes that contribute to Li+ potentiation of GluR current. One process, which is independent of agonist concentration, may predominate at lower Li+ concentrations. At very high Li+ concentrations, a second form of potentiation may manifest that selectively enhances responses to high, potentially desensitizing concentrations of agonists.
The results of our concentration-response experiments further suggest
that Li+ caused a dramatic change in the apparent efficacy
of the agonist. A raised maximal response suggests an increase in the
probability of a channel being open (Po) or an
increase in the single-channel conductance (). However, the effects
of Li+ on outward current, where it is not the primary
charge carrier, favors the interpretation that there is an alteration
in the percentage of time that channels are open.
CTZ was reported to potentiate AMPA receptor currents by reducing
receptor desensitization (Partin et al. 1996). Because
Li+ effects were greatest under desensitizing conditions,
we hypothesized that the effect of Li+ might also be to
reduce desensitization, and we sought to determine if CTZ and
Li+ acted through similar or different mechanisms. If
Li+ and CTZ work through distinct mechanisms, the
potentiation that each causes individually might be additive when they
are applied in combination. Our results indeed suggest that, at high
concentrations of KA, Li+ and CTZ do not act entirely
through the same mechanism. Moreover, when a high concentration of
glutamate (1 mM) was used, the effects of Li+ and CTZ
appeared to be completely additive for GluR3. These observations are
consistent with the idea that Li+ produces potentiation of
current in GluR3 by modulating the amount of receptor desensitization
and that desensitization of the channel can be influenced by specific
agonists. Desensitization is also controlled by the presence of
specific protein domains, as in the alternative splice variants, flip
and flop, in AMPA receptors. Flop receptors are thought to desensitize
more than flip variants. Indeed, it appears that flop receptor currents
are also potentiated to a greater degree by Li+ than are
flip receptor currents. The specificity of the Li+ effect
for the flop domain is further supported by the observation that, under
conditions of the additive CTZ effect for flop variants, there was an
antagonism of the CTZ effect in flip variants.
The inconsistent interaction of Li+ with CTZ suggests a novel mechanism for this effect, although with a similar requirement for the flop domain. The modulation of desensitization could involve alterations of rate constants into or out of the desensitized state as well as alterations of rate constants leading toward the desensitized state. Our data suggest that further detailed studies of desensitization may exploit the use of Li+ as a tool to dissect the mechanisms of desensitization.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Michael Hollmann, Jane Sullivan, Steve Heinemann, and Jim Boulter for the glutamate receptor (GluR) subunit genes and Dr. R. Dingledine for providing the mutant GluR2 channel cDNA. We thank Dr. Robert Lenox for helpful discussion.
This work was supported by predoctoral fellowships to N. B. Karkanias from the University of Florida Center for Neurobiology and Behavior and the University of Florida Division of Sponsored Research. Technical assistance was provided by C. Stokes.
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FOOTNOTES |
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Address for reprint requests: R. L. Papke, Depts. Of Pharmacology & Therapeutics and Neuroscience, University of Florida Medical College, J. H. Miller Health Center, Box 100267, Gainesville, FL 32610-0267.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 September 1998; accepted in final form 15 December 1998.
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REFERENCES |
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