The Journal of Neuroscience, September 3, 2003, 23(22):8051-8059
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Glycine Receptor Knock-In Mice and Hyperekplexia-Like Phenotypes: Comparisons with the Null Mutant
Geoffrey S. Findlay,1
Rachel Phelan,1
Michael T. Roberts,1
Gregg E. Homanics,2
Susan E. Bergeson,1
Gregory F. Lopreato,1
S. John Mihic,1
Yuri A. Blednov,1 and
R. Adron Harris1
1Waggoner Center for Alcohol and Addiction
Research, Section of Neurobiology, University of Texas at Austin, Austin,
Texas 78712, and 2Departments of Anesthesiology and
Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261
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Abstract
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Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in
the spinal cord and brainstem. To better define the function of this receptor
in vivo, we constructed a point mutation that impairs receptor
function in the 
1-subunit and compared these knock-in mice
to oscillator (spdot) mice lacking
functional GlyR 1-subunits. Mutation of the serine residue
at amino acid 267 to glutamine (1S267Q) results in a GlyR
with normal glycine potency but decreased maximal currents, as shown by
electrophysiological recordings using Xenopus oocytes. In addition,
single-channel recordings using human embryonic kidney 293 cells indicated
profoundly altered properties of the mutated GlyR. We produced knock-in mice
bearing the GlyR 1 S267Q mutation to assess the in
vivo consequences of selectively decreasing GlyR efficacy. Chloride
uptake into brain synaptoneurosomes from knock-in mice revealed decreased
responses to maximally effective glycine concentrations, although wild-type
levels of GlyR expression were observed using 3H-strychnine binding
and immunoblotting. A profound increase in the acoustic startle response was
observed in knock-in mice as well as a "limb clenching" phenotype.
In contrast, no changes in coordination or pain perception were observed using
the rotarod or hot-plate tests, and there was no change in
GABAA-receptor-mediated chloride uptake. Homozygous S267Q knock-in
mice, like homozygous spdot mice, exhibited
seizures and died within 3 weeks of birth. In heterozygous
spdot mice, both decreased
3H-strychnine binding and chloride flux were observed; however,
neither enhanced acoustic startle responses nor limb clenching were seen.
These data demonstrate that a dominant-negative point mutation in GlyR
disrupting normal function can produce a more dramatic phenotype than the
corresponding recessive null mutation, and provides a new animal model to
evaluate GlyR function in vivo.
Key words: GlyR; knock-in; oscillator; spd-ot; mice; channel gating; glycine; hyperekplexia; strychnine; chloride flux; Xenopus oocytes; acoustic startle response
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Introduction
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Glycine receptors (GlyRs) are the primary inhibitory neurotransmitter
receptors in the brainstem and spinal cord. GlyRs are pentameric ligand-gated
ion channels that conduct chloride in response to the neurotransmitter glycine
and are inhibited by the competitive antagonist strychnine. Four GlyR
-subunits and one 
-subunit have been identified. GlyRs are
typically composed of three -subunits and two -subunits. Although
the 2-subunit is found throughout the prenatal brain and is
important for synaptogenesis, the 1-subunit predominates in
the adult spinal cord and brainstem, whereas the -subunit is widely
distributed in the brain (Laube et al.,
2002
). The -subunit localizes GlyR at the synapse by binding
gephyrin, which in turn binds to microtubules
(Kirsch and Betz, 1995;
Meyer et al., 1995). Although
the 1-subunit can form functional GlyRs in vitro
that exhibit properties similar to those of native GlyRs, the -subunit
is important for GlyR function in vivo
(Becker et al., 1992) and
coassembles with the 1-subunit
(Kuhse et al., 1995). Specific
point mutations in GlyR 1 can cause dominant hyperekplexia
("startle disease"), in which an exaggerated response to acoustic
stimuli can result in uncontrolled falling
(Shiang et al., 1993).
Oscillator (spdot) mice possess a 7 bp
deletion in the GlyR 1-subunit gene that results in a
complete loss of 1-subunits in mice homozygous for the
spdot mutation and is lethal in mice homozygous for this
deletion (Buckwalter et al.,
1994). In contrast, loss of 50% of 1-subunits in
heterozygous spdot mice leads to a relatively mild
behavioral phenotype (Kling et al.,
1997). We hypothesized that mice harboring a mutation leading to
GlyR hypofunction by disrupting the function at each individual GlyR
(dominant-negative effect), rather than decreasing the number of GlyR
(haplotype insufficiency), would result in a different mode of receptor
function reduction. This in turn might preclude the compensation or subunit
substitution that appears to occur in mice with reduced levels of GlyR protein
(Kling et al., 1997). We
hypothesized that this might result in a more dramatic phenotype in
vivo. To test this hypothesis, we generated knock-in mice possessing the
GlyR 1 serine to glutamine (S267Q) mutation that decreases
glycine efficacy and compared them with the corresponding GlyR
1 null mutant mice. The knock-in approach represents a
powerful and proven technique for evaluating the significance of receptor
function in vivo (Low et al.,
2000; McKernan et al.,
2000; Crestani et al.,
2001).
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Materials and Methods
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Oocyte electrophysiology. Two-electrode voltage-clamp
electrophysiological recordings were performed on Xenopus laevis
oocytes as described previously (Mihic et
al., 1997; Findlay et al.,
2001). The effect of the S267Q mutation of the
1-subunit was compared in homomeric 1 and
heteromeric 1 GlyR. Oocytes were injected with either
wild-type or mutated human GlyR 1 cDNAs individually or in
combination with the wild-type GlyR -subunit at a ratio of 30 :1
to reach a final concentration of 1.5 ng/30 nl. Wild-type and
cDNAs were subcloned into the pBKCMV vector that had been modified
previously by removal of the lac promoter and the lacZ ATG
(Mihic et al., 1997). The
S267Q GlyR 1 cDNA was subcloned into the pCIS vector.
Electrophysiological measurements were made 4-14 d after the cDNA injection in
a counter-balanced design, such that each group contained an equal number of
oocytes from each frog, and oocytes in each group were tested at equal times
after cDNA injection. All oocytes received an initial 15 sec application of
200 µM glycine to test for GlyR expression. If oocytes did
express GlyR, experiments were started after an 8 min wash-out period. All
experimental glycine applications were 30 sec in duration, with wash-out
periods (5-18 min) long enough to ensure complete resensitization between drug
applications. Oocytes were constantly perfused with modified Barth's saline
(Beckstead et al., 2000) buffer
(with or without glycine) at a rate of 2 ml/min. Peak currents were measured
and used in data analysis.
Single-channel recordings. Single-channel recordings were made
from outside-out patches pulled from human embryonic kidney 293 (HEK 293)
cells transiently transfected with cDNAs encoding either wild-type or S267Q
1 glycine receptor subunits. Briefly, HEK 293 cells obtained
from ATCC (Manassas, VA) were cultured in a 5% CO2 atmosphere at
37°C in DMEM + L-glutamine + sodium pyruvate + 10% fetal bovine
serum (Invitrogen, Carlsbad, CA). Cells were split every 2-3 d using 0.25%
trypsin and 1 mM EDTA in HBSS (Invitrogen) and were used through
passage 20. The day before transfection, cells were split onto 15 mm Thermanox
coverslips (Nunc, Rochester, NY). Transfections were carried out using a
GlyR-optimized calcium phosphate transfection protocol
(Groot-Kormelink et al.,
2002). Transfected cells were used for experiments 1-2 d
later.
Outside-out patch recordings were made according to standard methods
(Hamill et al., 1981). Patch
pipettes were pulled from thick-walled borosilicate glass (Sutter Instruments,
Novato, CA), coated with Sylgard 184 (Dow Corning, Midlands, MI), and fire
polished to tip resistances of 8-15 M
. The pipette solution was
composed of the following (in mM): 145 CsCl, 2 CaCl2, 2
MgCl2, 10 HEPES, and 10 EGTA, pH 7.3, with CsOH. The bath was
perfused with, and glycine solutions were prepared in, an external solution
containing the following (in mM): 140 NaCl, 5 KCl, 2
CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4, with
NaOH. Glycine applications were made using the SF-77B Perfusion Fast-Step
apparatus (Warner Instruments, Hamden, CT). Data were acquired using an
Axopatch 200B amplifier (Axon Instruments, Union City, CA) interfaced with a
Pentium class computer running pClamp 8.0 software (Axon Instruments).
Single-channel data were low-pass-filtered at 3-5 kHz (-3 dB, four pole
Bessel), digitized at 30 kHz, stored on a hard drive, and analyzed using
Clampfit (Axon Instruments).
Mouse production. A mouse strain 129/SvJ BAC genomic DNA library
(Genome Systems, St. Louis, MO) was screened with an 
700 bp ApaI
fragment of the human glycine 1-receptor cDNA. This fragment
contained the putative transmembrane region 2 (TM2) region. A 10 kb
HindIII-PstI subclone was used to create the targeting
construct illustrated in Figure
3. The Quickchange Site Directed Mutagenesis kit (Stratagene, La
Jolla, CA) was used to change three nucleotides in exon 7 so that the codon
for serine (AGC) at position 267 now encoded glutamine (CAG). In addition to
changing the codon, this mutation also destroyed an SstI restriction
site. A neomycin resistance cassette that was flanked by FLP recombinase
target sites (Meyers et al.,
1998) was inserted into intron 7 in a NheI site and a
HSV-TK (herpes simplex virus-tyrosine kinase) cassette was inserted at the
5' end of the targeting construct. The vector was linearized with
NotI and introduced into strain 129/SvJ "Go Germline"
mouse embryonic stem (ES) cells (Genome Systems) as described previously
(Homanics et al., 1997). ES
cells that survived G418 selection were screened for targeting by Southern
blot analysis of HindIII-digested DNA and hybridization with a
3' probe that is external to the targeting construct. This probe was an
350 bp PstI-HindIII genomic subclone that spanned exon
8. Correctly targeted clones were also analyzed with additional enzymes,
including SstI, and probes (data not shown).
Correctly targeted ES cell clones were microinjected into C57BL/6J
blastocysts to produce chimeric animals. Highly chimeric mice were mated to
C57BL/6J mice to produce the first filial (F1) generation. Mice
heterozygous for the neo-containing knock-in (KI) allele (S/Qneo)
were mated to enhanced FLP (FLPe) general deleter mice
(Rodriguez et al., 2000)
(C57BL/6 background) to remove the neo marker cassette. The FLPe transgene was
detected on Southern blot analysis with an 500 bp
EcoRI/EcoRV fragment of the FLPe coding sequence. For the
experiments described here, all knock-in mice were of a mixed genetic
background of (C57BL/6J x 129/SvJ) of the F3-4 generations.
Littermate mice bearing wild-type 1 GlyR subunits served as
controls in behavioral and biochemical experiments.
PCR genotyping for GlyKI mice was performed by using a forward primer
(sequence: CCTGCTCATCGTCATCCT) and a reverse primer (sequence:
TGCAGCTCTCTCCTCCTC) with an annealing temperature of 55°C to amplify a
fragment of the genomic GlyR 1-subunit. These primers flank
the GlyR 1 S267Q mutation. The fragment was subsequently
digested with SacI and run on a 4% MetaPhor/TBE gel. The wild-type
gene produced two bands: 185 and 110 bp; homozygous knock-in mice were
observed because of a single band: 295 bp; heterozygous mice were identified
by three bands: 295, 185, and 110 bp. The point mutation S267Q in heterozygous
or homozygous mice was observed because of the elimination of a SacI
cut site by the mutation. This genotyping protocol was confirmed using
Southern blotting.
Heterozygous +/spdot mice carrying the oscillator
mutation on a C57BL/6J background were purchased from Jackson Laboratories
(Bar Harbor, ME) to establish a breeding colony in our facility. PCR
genotyping for spdot mice was performed as described
previously (Kling et al.,
1997). In all cases, wild-type littermates were used as controls
for comparison with mutant mice.
Cloning of GlyR 1 cDNA. RNA
was extracted from spinal cord and brainstem tissue using a modified
phenol-chloroform extraction protocol. Brainstem and spinal cord tissues were
isolated and immediately frozen in liquid nitrogen before being stored at
-70°C overnight. Frozen tissue samples were placed into 2 ml of Trizol
reagent (Invitrogen, Carlsbad, CA) and immediately homogenized using a
Powergen 700 (Fisher Scientific, Pittsburgh, PA) for 2 min. The tissue was
incubated at room temperature for 5 min, followed by centrifugation at 12,000
x g for 10 min at 4°C. Chloroform (0.2 ml/1 ml reagent) was
added to the supernatant, and the solution was shaken vigorously for 30 sec.
After 2 min of incubation at room temperature, the solution was centrifuged at
12,000 x g for 15 min at 4°C. The top aqueous phase was
removed and isopropanol (0.5 ml/1 ml reagent) was added and mixed. After 10
min of incubation at room temperature, the solution was centrifuged at 12,000
x g for 10 min at 4°C. The RNA pellets were recovered by
aspiration of the supernatant and then washed with 70% ethanol (1 ml/ml
reagent) and then centrifuged at 7500 x g for 10 min at
4°C. The pellet was air-dried and resuspended in diethylpyrocarbonate
(DEPC)-double distilled H2O and stored at -70°C. RNA quality
was analyzed using a RNA Nano Chip with an Aligent 2100 Bioanalyzer (Aligent
Technologies, Waldbrom, Germany). Reverse transcription of the RNA was
performed using Superscript II (Invitrogen, Carlsbad, CA). GlyR
1 cDNA was amplified with PCR using a primer upstream
(sequence: CGTGGACTTTACAGCACT) and downstream (sequence: CCTCCCACCACCCTCTCC)
from the coding region of the GlyR 1 cDNA, with an annealing
temperature of 54°C. The GlyR 1 cDNA was then cloned
using the TA cloning kit (Promega, Madison, WI) and sequenced. Primers for
both complementary strands were used.
Immunoblot analysis. Immunoblot analysis of combined spinal cord
and brainstem tissues was performed as described previously
(Findlay et al., 2002), using
a polyclonal antibody to the N-terminal of the 48 kDa GlyR
-subunit (rabbit anti-glycine receptor antibody; Chemicon
International, Temecula, CA; Wick et al.,
1999). After testing a range (1.25-30 µg of protein) of tissue
amounts, a moderate (10 µg of protein) amount of tissue was used for each
sample in the immunoblots.
Glycine-stimulated chloride (36Cl-)
uptake of synaptoneurosomes. Isolated brainstem and spinal cord membrane
vesicles (synaptoneurosomes) were prepared as described previously
(Blednov et al., 1996), with
modifications. The tissue was homogenized in 4.5 ml of assay buffer containing
the following (in mM): 145 NaCl, 5 KCl, 1 MgCl2, 10
glucose, 1 CaCl2, 10 HEPES, pH 7.5, using Tris-base) using a hand
homogenizer (Thomas Scientific, Swedesboro, NJ), and the tissue was then
centrifuged at 900 x g for 15 min. Combined brainstem and
spinal cord samples were resuspended in 10 ml of assay buffer, and the
suspensions were then filtered through one layer of 100 µm nylon
microfilament cloth (PGC Scientific, Frederick, MD) to remove myelin. Samples
were then centrifuged again at 900 x g for 15 min. The pellet
was suspended in ice-cold assay buffer and tissue aliquots (0.8-1.2 µg of
protein) were incubated at 34°C for 15 min in the presence or absence of
500 µM strychnine. Uptake was initiated by adding 200 µl of
36Cl - solution (2 µCi/ml of assay buffer) containing
glycine (50-500 µM final concentration). The influx reaction was
terminated 9 sec later by the addition of 4 ml of ice-cold quench buffer
(assay buffer containing 0.1 µM strychnine) and rapid filtration
through a GB100R filter (Advantec MFS, Dublin, CA). The filter was then washed
once with 8 ml of quench buffer. Filters were incubated in 4 ml of Biosafe II
scintillation liquid (Research Products International, Mount Prospect, IL)
before analysis in a Beckman LS 6500 scintillation counter (Beckman Coulter,
Fullerton, CA). The amount of 36Cl - that was bound to
the filter in the absence of membrane (no-tissue blank) was subtracted from
all values. Glycine-dependent chloride uptake was defined as the amount of
chloride taken up while agonist was present in the medium (total intake) minus
the amount taken up in the absence of agonist (glycine-independent or basal
uptake). Strychnine-sensitivity was determined by comparing the basal chloride
uptake with the glycine-stimulated chloride uptake in the presence of
strychnine.
[3H]strychnine binding. [3H]strychnine
binding was performed as reported previously
(Findlay et al., 2002).
Specific binding was calculated by subtracting nonspecific binding (in the
presence of glycine) from total binding. Kd and
Bmax values were calculated for each individual saturation
curve using the Prism 2.0 program (GraphPad Software, San Diego, CA).
Behavioral experiments and phenotype analysis. Acoustic startle
responses were measured using SR-LAB test stations and software (San Diego
Instruments, San Diego, CA). Test stations were both standardized and
calibrated, and startle responses are expressed in milliNewtons. Startle
responses were recorded as described previously
(Bullock et al., 1997). After a
3 min acclimation period, trials of single 40 msec bursts of white noise
ranging from 95 to 125 dB were presented in a pseudorandom order with a 10 sec
intertrial interval. The background noise was 70 dB. Eight no-stimulus
recordings were recorded at pseudorandom intervals to measure spontaneous
activity. Each stimulus (with the exception of the no-stimulus trial) was
presented 15 times. The entire session comprised 113 trials and took 22 min.
The highest and lowest responses to each stimulus intensity and the
no-stimulus recordings were excluded. The average response to a stimulus trial
at a given sound (decibel) intensity was defined as the average startle
response minus the average of the background no-stimulus trials.
Locomotor activity was performed as described previously
(Findlay et al., 2002). Total
beam breaks were measured in 30 min increments over a 24 hr period for each
mouse tested.
Hot-plate latency was determined by placing a mouse on a Hotplate Analgesia
Meter (Columbus Instruments, Columbus, OH) set at a constant temperature of
52.5°C. Latency was defined as the time that elapsed between the mouse
being placed on the hotplate and when it engaged in stereotypical behaviors
such as paw licking, rearing on hindlegs, or leg lifting.
Rotarod coordination was determined by first training mice on a rotarod
(Economex; Columbus Instruments, Columbus, OH) at 2.5 rpm until they were able
to walk on it for 1 min. Successively increasing speeds were then tested: 5,
7.5, 10, 12.5, and 15 rpm. Success was defined as the mouse remaining on the
moving rotarod for 1 min. Failure was defined as the mouse falling from the
rotarod four times. If the mouse failed at a given speed, higher speeds were
not tested. The highest speed of success for each mouse was recorded and
analyzed statistically. Adult mice, age 2-4 months, were tested in all
behavioral experiments.
Statistical analysis. Student t tests, one-way and
two-way ANOVAs, and curve-fitting were performed using the Prism 3.0 program
(Graph-Pad Software) or Origin 7 (Microcal Software, Northampton, MA). All
graphs are represented as means ± SEM with levels of significance
indicated as *p < 0.05, **p <
0.01, and ***p < 0.001.
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Results
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The effects of the GlyR 1 S267Q mutation were
investigated in Xenopus oocytes by expressing wild-type or mutant
S267Q GlyR 1 cDNA and measuring currents produced by
glycine. If results were expressed as the percent of the maximal current
observed in each oocyte, no differences were observed between homomeric GlyR
1 S267Q and the wild-type receptors, indicating that the
mutation did not alter the potency of glycine
(Fig. 1A). However,
when these same data were expressed as absolute currents, a decreased maximal
response was seen in the S267Q mutant (Fig.
1B). The S267Q mutation also produced a large decrease in
the maximal response to glycine in 1 heteromeric
receptors (Fig. 1D).
Results obtained from multiple batches of oocytes tested showed a consistent
decrease in the maximal glycine-stimulated currents as a result of the S267Q
mutation when the 1-subunit was expressed homomerically
(30% reduction), or heteromerically with the -subunit (60%
reduction) (Fig. 2).
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Figure 1. Glycine concentration-response relationships of wild-type (WT) or mutant
S267Q 1 GlyR expressed in Xenopus laevis oocytes.
A, Electrophysiological responses of WT (filled circles) or S267Q
(open circles) GlyR to varying concentrations of glycine (0.01-10
mM) were measured (n = 7-8 per point). The responses are
expressed as a percentage of the maximal response for each oocyte. WT and
S267Q concentration-response relationships were statistically
indistinguishable using a two-way ANOVA followed by Tukey's multiple
comparisons. Glycine EC50 values were 0.2 ± 0.08 and 0.2
± 0.04 µM, and Hill coefficients were 2.3 ± 0.5
and 1.9 ± 0.3 for WT and S267Q GlyR, respectively. B, The data
from A expressed as absolute currents. A two-way ANOVA followed by
Tukey's multiple comparisons reveal that the concentration-response curves for
WT and S267Q are significantly different (p < 0.01, n =
7-8 per point). Emax values were 24 ± 5 and 10
± 2 µA in WT and S267Q GlyR (p < 0.05 using a two-tailed
t test, n = 7-8 per point). EC50 or Hill values
were the same as calculated in A using normalized data. C,
Electrophysiological responses to glycine (0.01-10 mM) of either
heteromeric WT (filled triangles) or S267Q (open triangles)
1-subunit coexpressed with the -subunit. WT and mutant
concentration-response relationships were statistically different as measured
by a two-way ANOVA followed by Tukey's multiple comparisons (p <
0.05, n = 5-8 per point). Glycine EC50 values were 0.2
± 0.04 and 1.0 ± 0.3 mM (p > 0.05), and
Hill coefficients were 1.8 ± 0.4 and 1.1 ± 0.2 (p >
0.05) for WT and 1(S267Q) GlyR. D, The data
from C are presented as the absolute currents. A two-way ANOVA
followed by Tukey's multiple comparisons revealed that the
concentration-response curves were significantly different (p <
0.01, n = 5-8 per point). Emax values were 11
± 2 and 4.7 ± 1 µA in WT and 1(S267Q)
GlyR (p < 0.05 using two-tailed t test, n = 5-8
per point). EC50 or Hill values were the same as calculated in
C using normalized data. In all panels, EC50,
Emax, and Hill values were calculated from
concentration-response curves obtained from individual oocytes.
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To evaluate the in vivo consequences of the GlyR
1 S267Q mutation, knock-in mice bearing this mutation were
generated (Fig. 3). To ensure
that the knock-in mice carried only the desired mutation, knock-in and
wild-type cDNAs for the GlyR 1-subunit gene were cloned and
sequenced. Two splice variants exist for the GlyR 1-subunit
(Malosio et al., 1991) and
both were observed for wild-type and mutant cDNA. Wild-type cDNA was identical
to the GlyR 1 sequence that we characterized previously
(GenBank accession number AY129229
[GenBank]
). The knock-in cDNA was identical to the
wild-type cDNA except for the intended AGC
CAG mutation that produced the
S267Q mutation.
Mice homozygous for the S267Q mutation exhibited seizures and died by 3
weeks after birth. As observed previously
(Kling et al., 1997;
Findlay et al., 2002),
homozygous oscillator (spdot/spdot)
mice also die within 3 weeks because of a mutation in the GlyR
1 subunit gene that results in a loss of functional
1 polypeptide. Therefore, heterozygous
spdot mice were simultaneously tested with heterozygous
S267Q 1 knock-in mice to allow direct comparisons to be
made. Hindfeet clenching or limb clenching was consistently observed in
heterozygous S267Q 1 knock-in mice when they were lifted by
their tails (Fig. 4). Hindfeet
clenching is associated with dominant hyperekplexia
(Becker et al., 2002), but this
phenotype was not observed in wild-type or heterozygous
spdot mice. No spontaneous convulsions were observed in
heterozygous S267Q 1 knock-in mice, and these mice were
easily able to right themselves quickly when placed on their backs.
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Figure 4. Hindfeet clenching or limb clenching phenotype in adult knock-in mice.
Wild-type (A), heterozygous knock-in (B), and heterozygous
spdot (C) mice were evaluated. When lifted by the
tail, heterozygous knock-in mice displayed an obvious hindfeet clenching or
limb clenching phenotype. Neither wild-type nor heterozygous
spdot mice displayed this phenotype.
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A dramatic increase in the acoustic startle responses was observed in S267Q
1 heterozygous knock-in mice
(Fig. 5A). In
contrast, no differences in the acoustic startle response were observed as a
result of heterozygosity for the spdot mutation
(Fig. 5B). Wild-type
mice for both groups displayed similar acoustic startle responses. The large
increase in the acoustic startle response observed in S267Q knock-in mice did
not extend to a more general disruption of coordination as measured using the
rotarod test. All mice were successfully trained at 2.5 rpm on the rotarod.
The highest speed of success was 11.0 ± 1 rpm (n = 18) and
12.4 ± 1 rpm (n = 17) for wild-type and knock-in mice,
respectively. In addition, no differences were observed in spontaneous
locomotor activity as a result of the knock-in mutation (data not shown). To
measure pain perception, the hot-plate test was used. No differences between
knock-in and wild-type mice were observed using this test; the hot-plate
latency was 9.1 ± 0.7 sec (n = 11) and 9.9 ± 0.7 sec
(n = 17) in wild-type and knock-in mice, respectively.
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Figure 5. Acoustic startle responses. A, A profound increase in startle
responses was observed for heterozygous knock-in mice (+/KI) compared with
corresponding wild-type (+/+) mice (n = 17-24 per group). B,
No differences in acoustic startle responses were observed between
heterozygous spdot (+/spdot) and corresponding
wild-type (+/+) mice (n = 14-15 per group). Both groups of wild-type
mice displayed similar acoustic startle responses. Note that there is a large
difference in the y-axis between A and B.
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The S267Q knock-in mutation produced no differences in GlyR expression as
assessed by immunoblotting and 3H-strychnine binding
(Fig. 6). In contrast, the
spdot mutation did produce a decrease in
3H-strychnine binding (Fig.
6B), confirming earlier work
(Kling et al., 1997). Unlike
transgenic mice with mutant GlyR (Becker et
al., 2002; Findlay et al.,
2002), no ectopic expression of GlyR was observed in the cortex of
GlyR 1 S267Q knock-in mice using immunoblotting
(Fig. 6A).
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Figure 6. GlyR expression as measured by immunoblotting and 3H-strychnine
binding. A, Immunoblotting was used to determine GlyR -subunit
protein levels in wild-type (+/+) and knock-in (+/KI) mice. Results from a
typical immunoblot are shown. GlyR levels in the spinal cord and brainstem
(SpBr) were indistinguishable between heterozygous knock-in and wild-type
tissue (n = 3 per group). In addition, using this method, SpBr tissue
taken from homozygous knock-in mice at postnatal day 18 displayed
indistinguishable GlyR levels compared with wild-type controls (n = 3
per group). GlyR was not detected in the cortex (Ctx) of knock-in or wild-type
mice. B, 3H-strychnine binding was measured in the
combined spinal cord and brainstem of heterozygous spdot
(+/spdot) and heterozygous knock-in (+/KI) mice compared with
corresponding wild-types (+/+, filled squares) and (+/+, filled circles),
respectively. Bmax values were 599 ± 27, 588
± 19, 588 ± 24, and 347 ± 38; Kd
values were 11.6 ± 1.5, 10.2 ± 0.8,9 ± 0.5, and 8.5
± 0.6 for +/+ (filled circles), +/KI (open circles), +/+ (filled
squares), and +/spdot (open squares) mice, respectively (n
= 6-8 per point per group). +/+ (filled circles), +/+ (filled squares), and
+/KI Bmax values were statistically indistinguishable.
3H-strychnine Bmax values were reduced in
+/spdot mice (p < 0.001 using two-tailed t
test, n = 6-8 per point per group).
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To evaluate GlyR function in knock-in and spdot mice,
glycine-stimulated chloride (36Cl-) uptake was
determined for brainstem and spinal cord synaptoneurosomes. Glycine-stimulated
36Cl- uptake was inhibited by strychnine (data not
shown). Decreased maximal responses to glycine were observed as a result of
the S267Q knock-in or spdot mutations
(Fig. 7A,B). A measure
of ion flux per receptor (chloride uptake/Bmax) was
decreased for the knock-in, but not the spdot mice
(Table 1). These data suggest
that the S267Q mutation disrupts channel function, unlike in wild-type and
spdot mice. In contrast to the changes in glycine receptor
function, maximal responses to muscimol, a GABA agonist, were not altered by
the knock-in mutation (Fig.
7C).
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Figure 7. Glycine-stimulated strychnine-sensitive chloride uptake in
synaptoneurosomes of spinal cord + brainstem. A, Maximal glycine
responses were decreased in heterozygous knock-in mice (+/KI) (p <
0.0001 for genotype interaction using two-way ANOVA; p < 0.01
using Bonferroni post tests for 50 and 100 µM glycine;
n = 9-10 per group). B, Maximal glycine responses were also
decreased in heterozygous spdot (+/spdot) mice
(p < 0.05 for genotype interaction using two-way ANOVA; p
< 0.05 using Bonferroni post tests for 100 µM glycine;
n = 9 per group). C, No changes in maximal GABA responses of
heterozygous knock-in mice (+/KI) (n = 6). Corresponding wild-type
mice are labeled (+/+).
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To examine the possibility that the knock-in mutation disrupts GlyR gating,
single-channel recordings were made using outside-out patches pulled from HEK
293 cells transiently expressing GlyR. Consistent differences were observed
between wild-type and S267Q 1 receptors. Wild-type
1 GlyR displayed bursts of channel openings to stable
conductance levels, similar to those shown previously
(Beato et al., 2002). However,
recordings made from outside-out patches of S267Q 1 GlyR
displayed brief openings that rarely attained a stable amplitude and had no
resolvable bursts of openings (Fig.
8). Nonetheless, these data qualitatively support the idea that
the GlyR 1 S267Q mutation severely disrupts channel
function.
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Figure 8. Single-channel tracings of glycine-activated currents from wild-type
(A, B) and S267Q (C-F) 1 glycine
receptors. Outside-out patches pulled from transiently transfected HEK 293
cells were voltage clamped at -60 mV and perfused with 10 µM
(A-D) or 1 mM (E, F) glycine. A portion
of each of the tracings in the left-hand column (marked with a line above it)
is expanded in the right-hand column to provide a detailed view of individual
channel opening events. In E, the first 1.5 sec and the final 1.5 sec
of a 60 sec glycine application are shown with the preceding and following 0.5
sec of baseline (included to indicate the quality of the patch). The downward-
and upward-pointing arrows indicate the onset and offset, respectively, of the
glycine application. Perfusion artifacts were subtracted. Whereas wild-type
receptors exhibited bursts of openings to stable amplitude levels, S267Q
receptors tended to open very briefly and exhibited little or no burst
structure. Each panel is from a single patch but is representative of multiple
(n = 5) patches tested.
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Discussion
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The S267Q 1 GlyR knock-in mouse model demonstrates that
heterozygous mice with a point mutation altering GlyR function exhibit a
phenotype similar to forms of dominant hyperekplexia. This phenotype is more
dramatic than that seen in heterozygous mice for the recessive null mutation
(spdot), which are by comparison behaviorally
asymptomatic. Unlike mutations that cause hyperekplexia by decreasing the
glycine affinity of GlyR (Laube et al.,
2002), the GlyR 1 S267Q mutation appears instead
to decrease the efficacy of glycine. As noted previously
(Findlay et al., 2002), no
differences were observed in the glycine concentration-response relationships
for wild-type and S267Q homomeric GlyR 1 receptors expressed
in Xenopus laevis oocytes, and minimal changes in glycine sensitivity
were observed in heteromeric 1 and
1(S267Q) GlyR, when results were expressed as the
percentage of the maximal response. However, when the results are expressed in
absolute currents, large differences emerged. In agreement with these data,
maximal glycine-stimulated, strychnine-sensitive 36Cl-
uptake into synaptoneurosomes of the spinal cord and brainstem were reduced
50% in heterozygous knock-in mice.
Oscillator (spdot) mice have a 7 bp microdeletion in
the gene for the GlyR 1-subunit that results in the complete
lack of GlyR 1 polypeptide in homozygous
(spdot/spdot) mice; heterozygous mice had an 50%
decrease in the GlyR 1-subunit and an 30% reduction in
total GlyR levels (Kling et al.,
1997). These results suggest that some compensation by GlyR
subunit substitution may occur in heterozygous spdot mice.
Consistent with these results, we observed an 40% reduction in GlyR
levels using 3H-strychnine binding and chloride uptake. In
contrast, GlyR levels were unaltered in heterozygous knock-in mice. The lack
of ectopic GlyR expression in the cortex and identical GlyR levels in the
spinal cord and brainstem demonstrate a substantial advantage of expression of
the mutant GlyR 1 S267Q via the endogenous GlyR
1 promoter over transgenic expression of mutant GlyR
(Becker et al., 2002;
Findlay et al., 2002).
Unlike wild-type and heterozygous spdot mice, knock-in
mice exhibited a hindfeet clenching or limb clenching phenotype when lifted by
the tail. This phenotype is similar to that observed in homozygous
spastic (spa) (Kingsmore
et al., 1994; Mulhardt et al.,
1994) or spasmodic (spd) mice
(Ryan et al., 1994;
Saul et al., 1994), which have
mutations in GlyR 1- and -subunits, respectively. This
may be a phenotype common to mutations leading to GlyR hypofunction. Unlike
the spa and spd phenotypes, which are most pronounced during
postnatal week 2-3 but progressively lessen with age
(Kingsmore et al., 1994;
Mulhardt et al., 1994;
Ryan et al., 1994;
Saul et al., 1994), the limb
clenching phenotype is observed in adult GlyR 1 S267Q
knock-in mice, suggesting that the S267Q mutation may produce a larger
disruption of GlyR function or reduce the possibility of compensation compared
with spa or spd mutations. Homozygous
spdot mice also have a similar phenotype before death
(Buckwalter et al., 1994).
However, unlike these other mutants, heterozygous knock-in mice did not
display episodes of myoclonus, and the righting abilities of these mice were
not impaired.
The acoustic startle response is very sensitive to changes in GlyR
function. Disruption of GlyR function by hyperekplexia mutations or by
sublethal concentrations of strychnine will enhance startle responses.
Previously, a modest increase in the startle responses for heterozygous
spdot mice was observed
(Kling et al., 1997). In the
current study, no alteration in acoustic startle responses was observed in
heterozygous spdot mice. It is possible that differences
in experimental procedure can account for these differences or that subsequent
generations of spdot mice have become more resistant to
this phenotype. However, S267Q 1 GlyR knock-in mice display
a tremendous increase in acoustic startle responses. Although different
genetic backgrounds in mice can produce different behavioral responses
(Crawley, 2000), the wild-type
controls for spdot mice (C57BL/6J) and wild-type controls
for knock-in mice (C57BL/6J x 129/SvJ) have similar startle responses.
This phenotype of the knock-in mice demonstrates that a point mutation
altering receptor function can produce a larger impact on behavior than a
corresponding null mutation for the same gene. Considering that the transgenic
expression of a -subunit that provided 50% of the wild-type
3H-strychnine binding was sufficient to rescue the phenotype of
homozygous spastic (spa) mice
(Hartenstein et al., 1996), it
is likely that the mutant GlyR 1 S267Q subunit coassembles
with other wild-type subunits to affect function adversely to a degree greater
than that produced by a loss of half of the receptors. This idea is consistent
with the decrease in chloride flux per receptor in 1 S267Q
knock-in mice (Table 1), as
well as the observation that 1 S267Q mutation decreases
currents in heteromeric 1 receptors
(Fig. 2). Thus, the formation
of mixed wild-type/mutant channel complexes may result in the dominant
negative effect observed in S267Q knock-in mice. The S267Q 1
knock-in mutation may also decrease the potential for compensation that is
seen in heterozygous spdot mice, perhaps because there is
no shortage of 1-subunits seen in the knock-in animals;
i.e., perhaps receptor number rather than receptor function per se determines
whether compensation occurs.
In contrast to previous work showing that transgenic expression of a
wild-type GlyR 1-subunit produced a progressive limb
incoordination (Becker et al.,
2002), the performance of heterozygous knock-in mice using the
rotarod test was indistin-guishable from the wild-type. Thus, although
knock-in mice display profound increases in acoustic startle responses, this
phenotype does not appear to extend to a more general ability of coordination.
Although it has been postulated that GlyR function may be important for pain
perception (Keller et al.,
2001), no differences in pain perception of the knock-in mice were
observed using the hot-plate latency test. However, it is possible that
differences could still emerge using a different behavioral test.
GABAA receptor function was measured in the knock-in mice
because previous data suggested that alterations in GlyR can affect
GABAA receptor function. GABAA receptor protein
expression levels are increased in spa mice
(White and Heller, 1982;
Biscoe et al., 1984). In
addition, GABAA receptor function was decreased, although protein
expression was unaltered, because of the transgenic expression of a mutant
GlyR (Becker et al., 2002). In
contrast, no alteration in GABAA receptor function was observed as
a result of the S267Q 1 knock-in mutation, measured by
muscimol-stimulated 36Cl- uptake in spinal cord and
brainstem synaptoneurosomes.
Our findings raise the question of how a point mutation in TM2 can produce
such marked physiological changes in receptor function. Previous in
vitro studies demonstrated that point mutations in the extracellular
region linking TM2 and TM3 of GlyR are critical for coupling glycine binding
to channel opening (Rajendra et al.,
1995; Lynch et al.,
1997; Lewis et al.,
1998). Near the extracellular edge of TM2, the GlyR
1 Q266H mutation can cause dominant hyperekplexia
(Milani et al., 1996). The
Q266H mutation reduces the open channel time (similar to S267Q) and decreases
the potency of glycine and taurine (unlike S267Q) without altering
single-channel conductance, the ion/cation selectivity ratio, strychnine
binding, or the effects of several modulators (Zn2+, pH, and DEPC)
(Moorhouse et al., 1999). It
has been postulated that Q266H stabilizes the closed state of the receptor
(Tang et al., 2002), and S267Q
may function via a similar mechanism. The S267 site is located near this
region and is of interest because it is important for the effects of alcohols
and volatile anesthetics and seems to be part of an alcohol and anesthetic
binding site (Mihic et al.,
1997; Mascia et al.,
2000b), and the S267Q mutation can affect the allosteric
modulation of GlyR function by alcohol and anesthetics
(Mascia et al., 2000a;
Findlay et al., 2002). Ethanol
is changed from a positive to a negative allosteric modulator by the S267Q
mutation (Findlay et al.,
2002), strongly suggesting that S267 is involved with channel
gating. Mutations at the homologous amino acid in GABAA receptors
can also alter channel gating by drastically reducing allosteric modulation,
increasing GABA sensitivity, and producing spontaneous channel openings
(Findlay et al., 2001;
Nishikawa et al., 2002). The
results of GlyR TM2 NMR structure analyses suggest that a mere 10° of
rotation of the TM2 regions could be sufficient to induce channel gating; this
work also demonstrates that single amino acid mutations at S267 are likely to
alter channel gating (Tang et al.,
2002), although the S267 residue appears to face away from the
channel (Trudell and Bertaccini,
2002). Because strychnine and glycine share overlapping binding
sites, the similar affinity of strychnine binding to wild-type and S267Q
mutant receptors provide evidence that the mutation has not altered the
binding site. Furthermore, the S267Q mutation reduced maximal glycine
responses in knock-in mouse synaptoneurosomes, as measured using chloride
uptake. Finally, briefer channel openings in cell-detached patches containing
GlyR 1 S267Q suggest changes in channel gating, although
changes in conductance or in vivo receptor trafficking cannot be
ruled out. These changes are consistent with the idea that S267 is involved
with allosteric transitions required for channel gating and may also explain
the lethality of this mutation in vivo.
We propose that construction of knock-in mice bearing defective receptors
may provide an additional avenue to assess the roles of a particular receptor,
complementing studies involving the deletion (knock-out) of genes. Deletion of
a protein often allows for the substitution of related proteins, especially
for receptors such as the GABAA receptor, in which there are many
related subunits (Kralic et al.,
2002; Peng et al.,
2002; Ramadan et al.,
2003). In the case of the glycine 1-subunit, it
is likely that the marked differences seen in heterozygous mice are caused by
sufficient substitution or sufficient function of the normal allele in
spdot mice, but little or no substitution and more
impaired GlyR function in the S267Q mouse. In homozygous mice, sufficient
substitution is not possible, and both mutations are lethal. In conclusion,
these studies demonstrate that a point mutation in the 1
GlyR can produce a more dramatic phenotype than the corresponding null
mutation and demonstrate that the S267 residue plays a critical role in
allosteric transitions required for channel function in vivo.
|
Footnotes
|
|---|
Received Feb 13, 2003;
revised July 8, 2003;
accepted July 17, 2003.
This work was supported by National Institute on Alcohol Abuse and
Alcoholism Grants AA11525 and AA13520. We thank Marilee Wick and Carolyn
Ferguson for molecular expertise and Virginia Bleck for assistance in
genotyping mice.
Correspondence should be addressed to Dr. Adron Harris, Waggoner Center for
Alcohol and Addiction Research (A4800), The University of Texas at Austin,
Austin, TX 78712. E-mail:
harris{at}mail.utexas.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/238051-09$15.00/0
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Top
Abstract
Introduction
). An SstI site was eliminated by the S267Q mutation. D, Knock-in locus after FLPe-mediated deletion of the neomycin marker cassette by site-specific recombination.
