Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating.

Novel GLRA1 Missense Mutation (P250T) in Dominant
Hyperekplexia Defines an Intracellular Determinant of Glycine
Receptor Channel Gating
Brigitta Saul,1 Thomas Kuner,2 Diana Sobetzko,1,4 Wolfram Brune,3 Folker Hanefeld,4 Hans-Michael Meinck3
and Cord-Michael Becker1
1Institut fu¨r Biochemie, Universita¨t Erlangen-Nu¨rnberg, D-91054 Erlangen, Germany, 2Max-Planck-Institut fu¨r Medizinische
Forschung, D-69120 Heidelberg, Germany, 3Neurologische Klinik, Universita¨t Heidelberg, D-69120 Heidelberg, Germany,
and 4Zentrum fu¨r Kinderheilkunde, Schwerpunkt Neuropa¨diatrie, Universita¨t Go¨ttingen, D-37075 Go¨ttingen, Germany
Missense mutations as well as a null allele of the human glycine
receptor a1 subunit gene GLRA1 result in the neurological
disorder hyperekplexia [startle disease, stiff baby syndrome,
Mendelian Inheritance in Man (MIM) #149400]. In a pedigree
showing dominant transmission of hyperekplexia, we identified
a novel point mutation C1128A of GLRA1. This mutation en-
codes an amino acid substitution (P250T) in the cytoplasmic
loop linking transmembrane regions M1 and M2 of the mature
a1 polypeptide. After recombinant expression, homomeric
a1P250T subunit channels showed a strong reduction of maxi-
mum whole-cell chloride currents and an altered desensitiza-
tion, consistent with a prolonged recovery from desensitization.
Apparent glycine binding was less affected, yielding an approx-
imately fivefold increase in Ki values. Topological analysis pre-
dicts that the substitution of proline 250 leads to the loss of an
angular polypeptide structure, thereby destabilizing open chan-
nel conformations. Thus, the novel GLRA1 mutant allele P250T
defines an intracellular determinant of glycine receptor channel
gating.
Key words: glycine; hyperekplexia; inhibition; receptor; startle
disease; stiff baby syndrome
Strychnine-sensitive glycine receptors (GlyRs) represent a family
of ligand-gated chloride channels that exist as pentameric protein
complexes. The GlyR isoform prevailing in brainstem and spinal
cord of adult mammals is an assembly of ligand-binding a1 and
structural b subunits (Betz, 1992; Becker, 1995; Becker and Lan-
gosch, 1998). In addition, a2, a3, and a4 subunit genes have been
identified in the human and rodents (Grenningloh et al., 1990;
Kuhse et al., 1990; Kingsmore et al., 1994; Matzenbach et al.,
1994; Nikolic et al., 1998). Mature GlyR subunit polypeptides are
thought to cross the postsynaptic membrane four times, with
transmembrane segment M2 delineating the inner wall of the
anion pore. Determinants of ligand binding have been assigned to
the large extracellular N-terminal domain of the a subunit vari-
ants (Betz, 1992; Breitinger and Becker, 1998). Glycinergic ago-
nist responses also depend on amino acid residues situated within
the extracellular loop linking segments M2 and M3 (Becker and
Langosch, 1998). The human genes encoding the a1 (GLRA1),
a2 (GLRA2), a3 (GLRA3), and b subunits (GLRB) have been
localized to the chromosomal regions 5q32, Xp21.2-p22.1, 4q33-
q34, and 4q31.3, respectively (Grenningloh et al., 1990; Shiang et
al., 1993; Baker et al., 1994; Shiang et al., 1995; Handford et al.,
1996; Milani et al., 1998; Nikolic et al., 1998).
Glycine binding is efficiently antagonized by the plant alkaloid
strychnine, which produces both increases in muscle tone and
exaggerated startle responses to external stimuli (Becker, 1995).
Symptoms of the human neurological disorder hyperekplexia
[startle disease, stiff baby syndrome, STHE, Mendelian Inheri-
tance in Man (MIM) #14940] are reminiscent of strychnine-
induced GlyR dysfunction (Tijssen et al., 1995). Affected infants
display exaggerated startle responses and severe muscle stiffness,
which may result in fatal apnea. During the first year of life,
muscle tone returns to normal whereas excessive startling, which
may culminate in immediate, unprotected falling, persists into
adulthood (Ryan et al., 1994; Tijssen et al., 1995; Brune et al.,
1996). Dominant traits of hyperekplexia were found to correlate
to GLRA1 missense mutations affecting segment M2 and the
extracellular M2-M3 loop (Shiang et al., 1993, 1995; Elmslie et al.,
1996; Milani et al., 1996). In two recessive traits, amino acid
exchanges have been identified within segment M1 (Rees et al.,
1994; Becker and Langosch, 1998). Moreover, homozygosity for a
null allele demonstrated that the complete loss of GLRA1 gene
function may be tolerated in the human (Brune et al., 1996).
Homologous phenotypes shown by mouse lines carrying GlyR a1
and b mutant alleles further support the causative role of GlyR
alterations in hypertonic motor disorders (Mu¨lhardt et al., 1994;
Ryan et al., 1994; Saul et al., 1994; Kling et al., 1997).
This study reports on a novel GLRA1 allele causing dominant
hyperekplexia. A missense mutation results in the substitution of
P250, which is located within the intracellular M1-M2 loop.
Recombinant a1P250T receptors displayed moderate changes in
agonist affinity yet dramatic alterations in chloride conductance,
Received June 25, 1998; revised Nov. 4, 1998; accepted Nov. 6, 1998.
This work was supported by the Deutsche Forschungsgemeinschaft, Bundesmin-
isterium fu¨r Bildung und Forschung, the European Union, and the Fonds der
Chemischen Industrie. We thank the members of family BS for participation in this
study. Generous support by P. H. Seeburg, help with Western blotting by C. Kling,
invaluable discussions with H.-G. Breitinger, and a critical reading of this manu-
script by T. Bonk are gratefully acknowledged. We thank N. Spruston for providing
Igor noise analysis macros.
Dr. Saul and Dr. Kuner contributed equally to this work.
Correspondence should be addressed to Dr. Cord-Michael Becker, Institut
f u¨r Biochemie, Universita¨t Erlangen-Nu¨rnberg, Fahrstrasse 17, D-91054 Erlangen,
Germany.
Copyright © 1999 Society for Neuroscience 0270-6474/99/190869-09$05.00/0
The Journal of Neuroscience, February 1, 1999, 19(3):869–877

defining proline (a1)250 as an important intracellular determi-
nant of GlyR channel gating.
MATERIALS AND METHODS
GLRA1 gene structure, single-strand conformation
polymorphism analysis, and sequencing of genomic DNA
The numbering scheme for GLR A1 gene structure used here follows the
designations given by Matzenbach et al. (1994) for the murine GlyR
subunit genes. This is consistent with recent revisions of the GLRA1
gene structure (Shiang et al., 1993, 1995). Genomic DNA was obtained by
phenol /chloroform extraction of peripheral blood leukocytes from par-
ticipating family members. PCR amplification of GLR A1 exons and
subsequent single-strand conformation polymorphism (SSCP) analysis at
constant temperatures (10, 15, 20, and 25°C) was performed as described
(Milani et al., 1998). After the detection of an informative polymor-
phism, amplimers of exon 7 were cloned into pBluescript II SK2 (Strat-
agene, La Jolla, CA) and subjected to DNA sequencing. Direct sequenc-
ing of genomic PCR amplimers was performed on an Applied Biosystems
Prism 377 automated DNA sequencer.
Generation and expression of GlyR a1 subunit constructs
GlyR a1 subunit cDNAs (Grenningloh et al., 1990) were cloned into a
pSP64T-derived vector (Krieg and Melton, 1984). Employing the
oligonucleotide-directed PCR mutagenesis method of Ho et al. (1993),
the point mutation C1128A coding for the mutant subunit a1 P250T was
introduced to the cDNA construct. For functional expression in Xenopus
laevis oocytes, recombinant full-length plasmids were used to generate
synthetic capped and polyadenylated cRNA using SP6 RNA polymerase
(Promega, Madison, WI). The cRNAs were purified by phenol /chloro-
form extraction, and ribonucleotides were eliminated using Chromaspin
columns (Clontech, Palo Alto, CA). RNA contents were quantified
photometrically. For expression in the human embryonic kidney cell line
(HEK 293), the a1 and a1 P250T cDNAs were cloned into the vector pCIS
in which the human cDNA was expressed under the control of the
cytomegalovirus promotor. The cells were transfected as described for 48
hr and subjected to biochemical and physiological analysis (Sontheimer
et al., 1989).
Membrane preparation and [3H]strychnine binding assay
Crude membrane fractions were prepared from transfected cells as
described (Sontheimer et al., 1989). For radioligand displacement, mem-
branes were incubated with 16.7 nM [ 3H]strychnine (DuPont NEN,
Boston, MA; specific activity 30 Ci/mmol) and increasing concentrations
of unlabeled ligands. Specific binding to membrane fractions was deter-
mined in triplicate by filtration assay using 50 mg of total protein (Kling
et al., 1997). Binding data were analyzed by a nonlinear algorithm
provided by the GraphPad program.
Electrophysiological recordings
Recording conditions and dose–response relationships. Whole-cell record-
ings (ambient temperature) from Xenopus laevis oocytes were performed
on a two-microelectrode voltage-clamp system (Kuner and Schoepfer,
1996). Oocytes were perfused with Ringer’s solution containing (in mM)
115 NaCl, 2.5 KCl, 1.8 CaCl2 , 1 MgCl2 , and 10 HEPES, adjusted to pH
7.2 with NaOH. Glycine-induced currents were recorded from outside-
out patches (Hamill et al., 1981), using an EPC-9 amplifier with Pulse
software (Heka electronics GmbH, Lambrecht, Germany). Solutions
were applied using a Piezo-driven double-barrel fast application system
(Colquhoun et al., 1992). The solutions (pH 7.2) consisted of either
Mg 21-free Ringer’s solution (external), or (in mM) 100 KCl, 2 MgCl2 ,
and 10 HEPES (internal).
Dose–response curves were constructed from peak currents induced
by seven appropriately spaced concentrations of glycine at a holding
potential of 270 mV. Data were fitted to the Hill equation to derive the
EC50 and Hill coefficient using the program Igor (WaveMetrics, Inc.,
Lake Oswego, OR). For homomeric a1 channels, EC50 values depended
on the total current expression (Saul et al., 1994), whereas such a relation
was not detectable for a1 P250T channels. With whole-cell currents ex-
ceeding 210 mA, the EC50 for glycine was 0.08 6 0.01 mM (n 5 3) in a1
channels, displaying a slightly biphasic dose–response (data not shown).
For current values more than 24 mA, the EC50 for glycine was 0.24, and
the dose–response was monophasic (see Results), consistent with obser-
vations by Taleb and Betz (1994).
Current expression levels and ion selectivity. Oocytes were injected with
cRNA solution (23 nl, 100 ng/ml) using a Nanoject Injector (Drummond
Inc., Broomall, PA). For both a1 and a1 P250T channels, the peak currents
elicited by saturating glycine concentrations (a1, 1 mM; a1 P250T, 10 mM)
were quantified in 10 different oocytes. As current expression levels may
vary among different batches of oocytes, the ratio Iwt /Imut was calculated
from the average currents determined for the same batch of oocytes.
Ratios averaged from three different batches were taken as the mean
difference in current expression between a1 P250T and a1. The reversal
potential of glycine-induced whole-cell currents was determined by
changing the voltage rampwise from 260 mV to 140 mV within 2 sec.
Ramps recorded in the absence of glycine were subtracted from ramps
recorded in the presence of glycine. Two such glycine-activated ramps
were recorded before, during, and after exposure to 50% diluted Ringer’s
solution. Reversal potentials were corrected for liquid junction poten-
tials. Assuming that cytoplasmic Cl 2 concentrations of Xenopus oocytes
are in the range of 100–110 mM, the Nernst equation predicts shifts of
12.7–15.1 mV, respectively.
Kinetic parameters and current–voltage (I–V ) curves. Whole-cell current
signals were low-pass filtered at fc 5 3.3 kHz and digitized at 10 kHz. The
current traces (decaying part: 300 msec, starting at the peak) were fitted
to single (Eq. 1: y 5 k0 1 k1 * exp(2x/t)) or double (Eq. 2: y 5 k0 1 k1
* exp(2x/t1 ) 1 k2 * exp(2x/t2 )) exponential functions to derive the
decay time constants (t). The rate of solution exchange (20–80% rise
time, typically 3 msec) was determined after each experiment by appli-
cation of 10% Ringer’s solution to the recording pipette (open-tip re-
sponse). Voltage steps were repeatedly applied with increments of 10 mV
from -100 to 1100 mV. To ascertain recovery from desensitization,
single steps were separated by 5 sec (a1) or 10 sec (a1 P250T). Glycine was
applied for 400 msec within a voltage step lasting for 600 msec.
Single-channel analysis. Single-channel currents were low-pass filtered
at 10 kHz, digitized with a modified pulse-code modulation device (Sony,
model ES 701), and recorded on videotape. For analysis, data were
replayed from tape, low-pass filtered at fc 5 2.5 kHz with the help of an
eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA),
and digitized at 10 kHz using the analog-to-digital interface of the EPC-9
(Heka). Amplitudes were determined manually using MacTAC (Skalar
Instruments, Inc., Seattle, WA). Three patches from different batches of
oocytes were analyzed for a1 constructs, and for each patch 500–1000
events were considered. Three patches expressing a1 P250T channels were
analyzed with nonstationary variance analysis as described by Spruston
et al. (1995). Briefly, the mean variance (s2) of 10–40 current responses
to pulses of 1 mM glycine was plotted as a function of the mean current
of all responses analyzed and fitted to Equation 3: s2 5 iI 2 (1/N ) * I 2
1 sb
2 (I, total current; i single-channel current; N, number of channels in
the patch; sb 2, mean background variance). popen was determined from
the relation I/N * i.
RESULTS
Occurrence of hereditary hyperekplexia in family BS was diag-
nosed clinically, and the mode of inheritance was indicative of
dominant transmission (Gabriel and Lenard, 1984). Of 17 family
members participating in genetic examination, hyperekplexia was
diagnosed in 10 subjects. In some of the cases, generalized stiff-
ness was reported in early infancy, which largely disappeared
within the first year of life. The spectrum of clinical symptoms
varied from excessive startle reactions to a predominance of
muscular hypertonia. Consistent with the guidelines of the local
committee on ethics, informed consent was obtained from all
individuals participating.
Genomic DNAs of members of family BS were subjected to
SSCP screening for GLRA1 mutant alleles. After amplification of
sequences corresponding to GLRA1 exon 7, a polymorphism
linked to the disorder was identified (Fig. 1A). Cloning of the
corresponding DNA amplimer and sequencing of nine recombi-
nants revealed a single nucleotide substitution, C1128A, encoding
a threonine residue instead of a proline in position 250 of the
mature a1 polypeptide (Fig. 1B). In addition, heterozygosity for
870 J. Neurosci., February 1, 1999, 19(3):869–877 Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating

the mutant allele was confirmed by direct sequencing of genomic
PCR amplimers (data not shown). Presence of the GLRA1P250T
allele was associated with hyperekplexia in family BS, with the
exception of one individual showing mild startle reactions in
addition to a pronounced fear syndrome, who was found to be
homozygous for the normal allele GLRA1. This patient was not
available for further physiological exploration of reflex latencies
indicative of startle disease (Brune et al., 1996). As a younger
sibling to an affected individual, however, this patient may suffer
from a behavioral disorder producing a phenocopy of
hyperekplexia.
Within the transmembrane topology predicted for GlyR sub-
unit polypeptides (Becker and Langosch, 1998), amino acid po-
sition 250 locates to the cytoplasmic loop linking transmembrane
segments M1 and M2. As noted earlier (Galzi et al., 1992),
sequence alignments show that all glycine and GABAA receptor
a polypeptides known carry a proline residue in the homologous
position (Fig. 2). Analysis of the protein secondary structure with
the Chou–Fasman algorithm (Chou and Fasman, 1974) predicted
that the substitution of P250, which is likely to confer an angular
conformation on a peptide sequence, by a threonine residue
significantly increases the propensity to form a continuous
a-helical structure (data not shown). Although the success of
predictive methods is hard to assess in individual cases, this
nevertheless suggests that the P250T mutation induces a major
change in secondary structure.
To characterize the functional properties of GlyRs comprising
the a1P250T subunit, mutant constructs were generated by site-
directed mutagenesis from wild-type a1 cDNAs. After transfec-
tion with wild-type and mutant receptor constructs, HEK 293
cells were subjected to Western blot analysis using monoclonal
antibody (mAb) 4a, which defines an epitope common to all GlyR
a subunits (Becker et al., 1988; Sontheimer et al., 1989). For both
a1 and a1P250T constructs, an immunoreactive polypeptide band
Figure 1. A, Hyperekplexia allele of the GLRA1 gene in family BS. A, Pedigree of family BS. Affected individuals are indicated by filled symbols and
unaffected individuals by open symbols. Only individuals volunteering for participation are included, and birth order was altered to avoid identification
of affected individuals. SSCP conformers of DNA samples are depicted beneath the symbols of the corresponding individuals. The asterisk denotes an
individual displaying mild startle reactions, in addition to a pronounced fear syndrome, who was found to be homozygous for the normal allele GLRA1.
B, Analysis of a normal and the hyperekplexia allele of GLRA1. The nucleotide substitution (C 3 A) corresponding to position 1128 of the cDNA
predicts the amino acid exchange P250T in the hyperekplexia a1 subunit allele (coding strand, gel lanes: G, A, T, C). The amino acid sequences (single
letter code) encoded by the two DNA ladders and reading from bottom to top are listed next to the gel patterns.
Figure 2. Alignment of amino acid se-
quences of wild-type and mutant gly-
cine, and GABAA receptor subunits. Se-
quences represent the cytoplasmic loop
between transmembrane segments M1
and M2 including the flanking regions.
The last row indicates point mutations
encoded by the GLRA1 mutant alleles.
Positions of transmembrane regions
M1 and M2 are marked. The amino
acid exchange P250T is given in bold.
Sequences were retrieved from the
EMBL nucleotide sequence database
(http://www.ebi.ac.uk/embl.html).
Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating J. Neurosci., February 1, 1999, 19(3):869–877 871

of 48 kDa was observed (Fig. 3A). No differences in staining
intensities were detectable, indicative of similar expression effi-
ciencies of these cDNA constructs. The ligand-binding properties
of recombinant a1 and a1P250T GlyRs were determined by
[ 3H]strychnine binding to membrane fractions of transfected cells
(Sontheimer et al., 1989). Equal numbers of binding sites (a1,
22.68 pmol/mg; a1P250T, 22.18 pmol/mg of membrane fraction)
became apparent for both constructs confirming the conclusion
that a1 and a1P250T subunit proteins are present in the eukaryotic
expression system at roughly equal amounts. However, the appar-
ent glycine-binding affinities derived from displacement assays
were 5.5- to 6-fold lower for a1P250T than for a1 GlyRs (Ki values,
36 6 2 mM for a1 vs 205 6 33 mM for a1P250T) (Fig. 3B). Binding
affinities for the agonists b-alanine and taurine were also reduced
with a1P250T GlyRs (data not shown).
To assess the influence of the P250T substitution on physiolog-
ical properties of recombinant GlyR channels, a1 and a1P250T
cRNAs were injected into Xenopus laevis oocytes. Whole-cell
current responses were recorded from the oocytes, and dose–
response characteristics (EC50) were established by application of
various glycine concentrations (Fig. 4A,B). For a1 channels, an
EC50 value of 0.24 6 0.02 mM glycine (mean 6 SEM, n 5 6;
currents more than 24 mA, see Materials and Methods) was
determined, whereas a1P250T channels produced half-maximal
responses at 0.54 6 0.03 mM glycine (n 5 6). The corresponding
Hill coefficients were 3.4 6 0.1 in a1 channels and 1.9 6 0.1 in
a1P250T channels (Fig. 4C). The observed differences were statis-
tically significant ( p , 0.01, unpaired Student’s t test). Further-
more, both constructs differed with respect to the maximum
current amplitude elicited by application of saturating glycine
concentrations (Fig. 4). Oocytes injected with identical amounts
(2.3 ng) of either wild-type or mutant cRNA produced currents of
11.4 6 7.3 mA (n 5 29, mean 6 SD) and 1.2 6 0.8 mA (n 5 28),
respectively. To account for the large batch-specific variability of
whole-cell current expression levels, the ratio I
a1/Ia1(P250T) was
separately assessed for each batch. The average ratio was 10 6 2
(mean 6 SEM) for three batches, indicating that mutant channels
yielded approximately 10-fold smaller currents.
In ligand-gated ion channels of the nAChR type, mutations of
an amino acid residue homologous to position GlyR a1(250)
contribute to alterations of ion selectivity (Galzi et al., 1992). By
analogy, we analyzed whether the anion selectivity of GlyR
channels is affected by the P250T substitution. Replacing the
external Ringer’s solution with a 50% diluted Ringer’s solution
revealed no significant difference in the shift of the reversal
potential between a1 channels (12.5 6 0.3 mV; n 5 4) and
a1P250T mutant channels (14.1 6 0.7 mV; n 5 4). Indeed, these
values are close to the reversal potential predicted from the
Nernst equation (13–15 mV, data not shown). As expected for a
Cl2 selective conductance, the reversal potential of a1 and
a1P250T mutant channels (224 6 2 mV; n 5 7) was close to the
Cl2 equilibrium potential of Xenopus oocytes (222 mV; Fraser
et al., 1993). Hence, mutant channels remained anion-selective,
but exhibited a moderate reduction in the apparent glycine affin-
ity and a strong reduction of the maximum current amplitude.
Comparing the traces shown in Figure 4, A and B, reveals that
a1P250T mutant channels desensitize more strongly than a1 chan-
nels. Indeed, rapid desensitization of mutant channels, eluding
detection by means of whole-cell recordings caused by large
oocytes and slow agonist application, may account for the de-
creases in whole-cell currents and increases in EC50 for glycine
that we observed. To further investigate GlyR desensitization, we
determined the macroscopic kinetic parameters of the current
response elicited by brief applications of saturating glycine con-
centrations to channels present in outside-out patches. Currents
mediated by a1 channels showed a rapidly desensitizing compo-
nent at negative potentials, whereas at positive potentials desen-
sitization was only weak (Fig. 5A). The desensitization, i.e., the
decay of the inward current in the continued presence of glycine,
could best be fitted with a double exponential function, yielding
Figure 3. Properties of the recombinant a1 P250T receptor protein. A, Western blot of membrane preparations from HEK 293. Immunostaining by
monoclonal antibody mAb 4a, which specifically recognizes GlyR a subunits, produced no detectable differences between cells transfected with the
wild-type (wt) or the mutated (P250T) a1 cDNA construct. B, Ligand-binding properties of recombinant GlyR a1 and a1 P250T receptors. Values present
displacement of [ 3H]strychnine binding by unlabeled strychnine and glycine.
872 J. Neurosci., February 1, 1999, 19(3):869–877 Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating

t1 5 12 6 3 msec and t2 5 192 6 78 msec (mean 6 SEM, n 5 5).
The current–voltage (I–V) relation of the peak current was es-
sentially linear, whereas the inward plateau current was reduced
in a voltage-dependent manner (Fig. 5B). Currents mediated by
a1P250T channels, in contrast, were strongly desensitizing at both
positive and negative potentials, without reaching a discrete pla-
teau (Fig. 5C). Consistent with the lack of the fast initial current
component, the desensitization could be fitted with a single
exponential function, giving rise to t 5 261 6 52 msec (n 5 5).
Mutant a1P250T channels exhibited a slightly outwardly rectifying
I–V relation of both peak and “plateau” current (Fig. 5D). In both
types of channels, the peak current and the plateau current
reverse direction at the same potential, indicating that the ob-
served desensitization of the current in fact reflects true desen-
sitization rather than a chloride shift (Akaike and Kaneda, 1989).
Figure 5E directly compares normalized current responses, em-
phasizing the biphasic versus monophasic desensitization of a1
and a1P250T channels, respectively. Currents mediated by a1
channels reach a plateau accounting for ;30–50% of the initial
peak current, whereas most a1P250T channels desensitized within
;1 sec, consistent with a prolonged phase of recovery from
desensitization. Taken together, the 10-fold reduction in whole-
cell current amplitudes observed does not reflect fast desensiti-
zation of the mutant channels relative to wild-type. Rather, the
extent of desensitization in mutant channels, reflecting a slower
rate of recovery from densitization, may most significantly con-
tribute to this difference.
To further elucidate whether a change in microscopic kinetic
properties or a reduction of the single-channel conductance may
account for the current reduction, we evaluated single-channel
currents of mutant and wild-type channels in outside-out patches.
Single-channel openings of a1 channels in the presence of 100 mM
glycine (Fig. 6A, top trace) exhibited a predominant conductance
state of ;80 pS as previously observed (Bormann et al., 1993). In
contrast, application of 1 mM glycine to outside-out patches con-
taining a1P250T channels elicited currents reminiscent of whole-
cell current responses, but with a very small amplitude (Fig. 6A,
bottom trace). This was consistently observed in eight patches
with currents ranging from 1 to 10 pA and might be explained by
the presence of multiple small conductances in the patch. The
current amplitudes were dependent on the glycine concentration
and returned to baseline during the continued presence of glycine
(data not shown). Although no distinct single-channel events
could be detected for a1P250T channels, the increased noise after
application of glycine is consistent with the presence of open
channels (Fig. 6A, bottom trace). Indeed, nonstationary variance
analysis (Fig. 6B,C) predicted the presence of minute, short-lived
channel conductances in outside-out patches containing a1P250T
channels with a mean conductance of 1.3 6 0.2 pS (n 5 3) and an
open probability of 0.02 6 0.01. Given the very low open proba-
bility, the error associated with these estimates of kinetic param-
eters might be large. Nevertheless, the single-channel kinetics of
a1P250T channels are strongly different from those of a1 channels
(53.5 6 12.8 pS; popen 5 0.7 6 0.4), consistent with an impairment
of channel gating. Because native GlyRs exist as a/b heteromers,
desensitization behavior and single-channel properties of hetero-
meric channels comprising either a1P250T or a1 subunits were
analyzed after coexpression with the b subunit in HEK 293 cells.
None of these properties was detectably different for a1P250T and
a1P250T/b channels (H.-G. Breitinger, unpublished observations).
DISCUSSION
Inhibitory GlyRs are ligand-gated chloride channels that repre-
sent pentameric assemblies of glycine-binding a1 polypeptides
and structural b subunits, as analyzed in spinal cord of adult
rodents (Betz, 1992). The human neurological disorder hyperek-
plexia has previously been attributed to various mutant alleles of
GLRA1, the gene encoding the GlyR a1 subunit. These hyperek-
plexia alleles of GLRA1 predict amino acid substitutions which,
according to the generally accepted model of GlyR transmem-
brane topology, reside within a region ranging from M1 to the
extracellular M2-M3 loop (Becker and Langosch, 1998). Here, we
characterize a novel GLRA1 allele resulting in dominant hy-
perekplexia in which the codon encoding proline 250 of the
normal allele is mutated into a threonine codon. After recombi-
nant expression, the exchange of this proline residue located
within the intracellular M1-M2 loop strongly diminished glycine-
induced chloride conductances rather than agonist binding.
Figure 4. Whole-cell current responses of recombinant a1 and a1 P250T
receptor channels. A, B, Whole-cell current responses at a holding poten-
tial of 270 mV elicited by different concentrations of glycine applied to
oocytes expressing homomeric a1 ( A) or a1 P250T (B) receptor channels.
Bars indicate glycine applications; concentrations are millimolar. Note
that the vertical scales for wild-type (A) and mutant (B) channels are
different. C, Dose–response curves for wild-type (circles) and mutant
(squares) receptor channels. EC50 values for glycine and Hill coefficients
are presented in Results.
Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating J. Neurosci., February 1, 1999, 19(3):869–877 873

Screening for GLRA1 mutations of a large pedigree with dom-
inant hyperekplexia resulted in the identification of the novel
allele GLRA1. Although heterozygosity for this allele was asso-
ciated with hyperekplexia, developmental as well as interindi-
vidual variations of clinical phenotypes became apparent between
affected individuals. Indeed, the spectrum of symptoms varied
from excessive startle reactions triggered by unexpected sounds to
a predominance of muscular hypertonia. Distinction has been
made between a “major” and “minor” form of this disease in
which the latter could not be assigned to GLRA1 mutant alleles
(Tijssen et al., 1995). In family BS, however, variations in pheno-
type are most likely explained by differences in genetic pen-
etrance of the GLRA1P250T allele because of as yet unidentified
background genes modulating the clinical presentation of GLRA1
mutations. The allele GLRA1P250T further adds to the genetic
heterogeneity of hyperekplexia. Although the number of hy-
perekplexia alleles as yet identified precludes any definitive con-
clusions, an interesting relationship emerges between transmem-
brane topologies and modes of inheritance of a1 subunit
mutations. The amino acid substitutions Q266H (Milani et al.,
1996), R271Q/l (Shiang et al., 1993), K276E (Elmslie et al., 1996),
and Y279C (Shiang et al., 1995) associated with dominant hy-
perekplexia cluster within or adjacent to the channel-lining M2
segment of the predicted a1 polypeptide. Heterologous expres-
sion shows that these mutations impair agonist binding and/or
channel gating of mutant receptors, suggestive of a negative
dominant effect resulting in a partial loss of function (Langosch et
al., 1994; Rajendra et al., 1994; Laube et al., 1995; Lynch et al.,
1997). In contrast, the recessive missense mutations predict ex-
changes located within M1 or the large N-terminal domain, i.e.,
S231R (Becker and Langosch, 1998), I244N (Rees et al., 1994),
and, in the spasmodic mouse, A52S (Ryan et al., 1994; Saul et al.,
1994). By both criteria, its dominant mode of inheritance and a
close proximity of the site of amino acid exchange to M2, the
allele GLRA1P250T would be assigned to the first group of mis-
sense mutations.
The proline residue affected by this mutation is conserved in
the homologous position of all GlyR and GABAA receptor a
polypeptides known (Fig. 2), suggesting a functional selection
pressure on this site for ligand-gated anion channels. On the other
hand, insertion of a proline plus an additional amino acid residue
into the corresponding region of recombinant a7 subunits of the
Figure 5. Kinetics of recombinant a1 and a1 P250T re-
ceptor channels. A, Fast application of saturating con-
centrations of glycine (thick bar) to outside-out patches
containing homomeric a1 channels. The 400 msec pulse
of 1 mM glycine elicited outward currents at 170 mV
(top trace) and inward currents at 270 mV (bottom
trace). The dotted line indicates the baseline, currents are
corrected for the leak. B, Current–voltage (I–V ) relation
of wild-type channels. Filled symbols are the I–V relation
of the peak current, and open symbols show the I–V
relation determined 400 msec after the peak (plateau).
The current reverses at ;0 mV. C, Same as in A for
homomeric a1 P250T channels, with the bar indicating the
application of 10 mM glycine. Note the different dimen-
sion of the vertical scale bar in comparison with A. D, I–V
relation of mutant channels, see B: 400 msec after expo-
sure to glycine. E, Comparison of the desensitizing
component of the current mediated by wild-type and
mutant channels. Both traces are normalized to their
respective peak currents. The horizontal bar indicates the
application of a 1.5 sec pulse of glycine. Note the differ-
ent time scale as compared with A and C. The data were
low-pass filtered at fc 5 300 Hz and digitized at 1 kHz.
874 J. Neurosci., February 1, 1999, 19(3):869–877 Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating

nicotinic acetylcholine receptor changes the channel selectivity
from cationic to anionic (Galzi et al., 1992). However, the loss of
this proline residue from recombinant GlyR a1P250T receptor
channels did not detectably alter ionic selectivity, but strongly
affected glycine-mediated current responses. Which mechanism
underlies the reduction in whole-cell current amplitudes? Based
on Western blot analysis and an equal number of ligand-binding
sites, membrane insertion of the receptor protein appears to be
undisturbed. Consistent with the assignment of determinants of
agonist binding to the large N-terminal domain (Betz, 1992),
apparent glycine binding was only weakly affected by the intra-
cellular amino acid substitution P250T. However, reductions in
apparent agonist-binding affinities may also be secondary to
changes in receptor conformations associated with gating
(Colquhoun and Farrant, 1993). Indeed, the observation that
affinities for the antagonist strychnine were not altered in a1P250T
receptors supports the notion that the ligand-binding domain
remained unaffected by the mutation. Moreover, dose–response
analysis of whole-cell currents revealed a pattern characteristic
for a gating-deficient channel (Spivak, 1995). Although the EC50
value was only slightly shifted, the Hill coefficient was decreased,
and the maximal current amplitude was strongly reduced in
a1P250T channels. Mutant channels differed from the wild-type in
their desensitization and resensitization properties, consistent
with an alteration in channel gating. Single-channel analysis sug-
gested a pronounced change in microscopic gating kinetics, com-
bined with a decrease in single-channel conductance. Taken to-
gether, the novel hyperekplexia allele GLRA1P250T defines an
intracellular determinant of GlyR channel gating. This is consis-
tent with previous observations on the recombinant a1 subunit
mutants W243A, I244N, and I244A, which exhibit increased
desensitization rates, implying that the M1-M2 loop in toto con-
tributes to GlyR desensitization properties (Lynch et al., 1997).
At present, it is not entirely clear how these changes in functional
properties relate to GlyR protein architecture. Sterical analysis
(Chou and Fasman, 1974) predicted the substitution of proline
250 by threonine to change an angular into a helical polypeptide
structure. However, the mutant a1P250A has been shown to dis-
play only slightly altered whole-cell currents (Lynch et al., 1997).
Considering the statistical nature of structural predictions and the
limited effect of the P250A mutation on channel properties
(Lynch et al., 1997), the amino acid substitution P250T may
nevertheless be speculated to disturb a hinge function of the
M1-M2 loop, which positions the adjacent segment M2, thereby
destabilizing open-channel conformations (Fig. 7). This conclu-
Figure 6. Single-channel properties of recombinant a1 and a1 P250T re-
ceptor channels. A, The top trace shows single-channel currents of homo-
meric wild-type channels recorded from an outside-out patch in the
presence of 100 mM glycine. The bottom trace shows an experiment with
a patch containing multiple homomeric mutant receptor channels, with
the arrow indicating the application of 1 mM glycine. In the continued
presence of glycine, the baseline (dotted line) was reached within 1 sec.
Note the increase in noise after the application of glycine. The holding
potential was 2100 mV. For display, data were refiltered at fc 5 1 kHz.
B, Nonstationary variance analysis of outside-out patches. The top panel
shows 10 superimposed traces with the mean current printed in gray. The
bottom panel shows the mean variance plotted versus time. C, Plot of
the mean variance obtained from a total of 30 responses in this patch as
a function of the mean current. The data were fitted with Equation 3
(see Materials and Methods), the obtained parameters are: i 5 1.6 pS;
popen 5 0.01; N 5 42.
Figure 7. Topological predictions for the cytoplasmic M1-M2 loop of
recombinant a1 and a1 P250T receptor channels. Location of transmem-
brane segments M1 and M2 are indicated.
Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating J. Neurosci., February 1, 1999, 19(3):869–877 875

sion fits into a topological model of the nicotinic acetylcholine
receptor superfamily, positioning the gate of these channels close
to the cytoplasmic end of segment M2, near the M1-M2 loop
(Wilson and Karlin, 1998). Conversely, the M2-M3 loop has been
proposed to serve as the extracellular hinge of segment M2,
thought to mediate the interaction of the ligand-binding and
channel activation site (Lynch et al., 1997).
Using recombinant analysis of the GLRA1 mutant alleles
known, it has become possible to attribute the neurological dis-
order hyperekplexia to disturbances in GlyR physiology. At
present, however, the diverse clinical phenotypes of hyperek-
plexia cannot be correlated with the distinct parameters affected
in GlyR function, such as ligand binding, intramolecular signal
transduction (activation/gating), channel conductance, and their
implications for neuronal signal processing (Jones and West-
brook, 1996). This suggests an all-or-none mechanism of GlyR
dysfunction to generate the symptoms of hyperekplexia. This
conclusion implies that the function of glycinergic inhibition in
the human is in obvious need of further investigation.
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