From the Department of Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261, the § Department of
Cellular and Molecular Physiology, Yale University School of Medicine,
New Haven, Connecticut 06510, the ¶ Howard Hughes Medical
Institute and Department of Molecular Biophysics and Biochemistry, Yale
University, New Haven, Connecticut 06511, and the
Department
of Human Biological Chemistry and Genetics, University of Texas Medical
Branch, Galveston, Texas 77555-1055
Received for publication, December 5, 2000
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ABSTRACT |
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By utilizing a baculoviral expression system
described previously (Cascio, M., Schoppa, N. E., Grodzicki,
R. L., Sigworth, F. J., and Fox, R. O. (1993)
J. Biol. Chem. 268, 22135-22142), functional
recombinant homomeric human Ligand-gated channels act in mediating signal transduction rapidly
at the synapse. Members of this family of channels include receptors
for both inhibitory neurotransmitters such as glycine and
Upon binding of glycine, the glycine receptor (GlyR) channel becomes
permeable to small anions (Cl Only limited structural information has been available for the GlyR, as
well as other members of the ligand-gated ion channel superfamily since
the native protein subunits are typically produced in very low
abundance. The member that is best characterized to date is the
nicotinic acetylcholine receptor (nAchR), which may be purified from a
naturally abundant source, the electric organ of Torpedo
electric fish. The structure of nAchR has been imaged in the closed
(10, 11) and opened state (12). From extensive affinity labeling and
mutagenesis studies, a fairly detailed model of the agonist-binding
site has evolved, suggesting that six loops from adjacent subunits form
the agonist-binding pocket in the nAchR (see Refs. 13 and 14 and
references therein). Similar approaches have mapped homologous binding
sites on the receptors for GABA (15, 16), glycine (17-20), and
serotonin (21). Additionally, residues localized to the transmembrane
domains of these receptors have been identified by cysteine scanning
methods (for review see Ref. 22) as well as using lipophilic
cross-linking agents (23-25). However, despite these and other studies
on nAchR and other members of this neuroreceptor superfamily, the
details of the molecular architecture of these ion channels remain
refractory and subject to debate (for review see Ref. 26). Initial
models for the ligand-gated channel superfamily were derived
immediately upon the cloning of the constituent nicotinic acetylcholine
receptor subunits based on hydropathy plots of the primary structure
and evolved somewhat in subsequent years. The current generally
accepted model for the family of ligand-gated channels contains four
transmembrane helices, despite a paucity of structural evidence
supporting this view. Although labeled "putative," the
presumed existence of these transmembrane helices is widely accepted.
However, some structural studies have questioned this paradigm,
indicating that the membrane spanning domains may also include
Baculovirus expression systems have been found to be of general use in
overexpressing membrane proteins that are typically expressed in
nonfunctional form in simpler bacterial systems (31, 32). We (33) and
others (34) have applied a baculoviral expression system to produce
sufficient quantities of Given that we can overexpress an active glycine receptor on the surface
of insect cells and purify relatively large amounts of protein (on the
order of mg/liter of cell culture) which binds agonist and antagonist
competitively (33), it remains to be shown that this solubilized and
purified protein-detergent complex retains functionality. In this
report it is shown that the purified protein can indeed be
reconstituted into lipid vesicles as an active ligand-gated channel.
Reconstitution into lipid vesicles was effected by gel filtration, and
the activity was characterized at the level of single channels by
electrophysiological measurements in black lipid membranes and
macroscopically by measurement of the quenching of an internalized
fluorescent probe by ion flux via channel opening. These results are
significant in that they indicate later biophysical and biochemical
characterization of reconstituted homomeric GlyR may be correlated to
native structure. In this study, we also examine the secondary
structure of the glycine receptor in lipid vesicles by CD. CD
spectroscopy is a useful tool for examining the structure of membrane
proteins; it is extremely sensitive to small changes in the folding of
the peptide backbone and provides quantitative information on the net
secondary structure of membrane proteins in a lipid environment, that
of small unilamellar vesicles. These studies provide the first
quantitation of glycine receptor secondary structure. Similar to the
conclusions obtained in coupled proteolytic and mass spectrometric studies of reconstituted GlyR (29), these studies also indicate that
the four transmembrane helix model for ligand-gated channels may be
erroneous. The structural architecture of GlyR may be archetypic for
the family of ligand-gated channels given the significant sequence and
proposed topological similarities (1, 35). Therefore, subsequent
characterizations of GlyR topology and structure may additionally
provide insight into the general conserved mechanisms used for channel design.
Materials--
Digitonin and sodium deoxycholate were purchased
from Aldrich. Soybean phospholipids, cholesterol,
L- Protein Expression and Purification--
A cDNA encoding the
human GlyR Reconstitution--
Reconstitution protocols were adapted
from Garcia-Calvo et al. (39). All reconstitution procedures
were carried out at 4 °C. Aliquots of purified detergent-solubilized
Microscopic Activity Assays--
Aliquots of purified
reconstituted preparations prepared for CD measurements were further
diluted into a hypertonic solution, such that the final buffer was 120 mM KCl, 5 mM EDTA, 5 mM EGTA, 25 mM KPi, pH 7.4, with an additional 600 mM sucrose constituent, freeze-thawed once, and then
bath-sonicated at room temperature for 30 s. Small aliquots of the
final vesicle suspension were stored frozen at
Membrane currents were recorded with a Warner BC-525 amplifier and
stored on videotape using an Instrutech VR-10 PCM encoder. Recordings
were transferred digitally to a Macintosh computer and filtered with a
Gaussian filter to a bandwidth of 50-200 Hz for analysis.
Macroscopic Activity Assays--
Vesicles containing
Emission spectra were initially collected on a Spex Fluorimax DM3000
fluorimeter using SPQ, with 344 nm excitation and emission monitored at
443 nm, using a 1-cm path length quartz cell (Hellma). Emission spectra
were collected for 30 s at 100-ms intervals. After ~5 s of
base-line measurement, 100 µl of vesicle samples were rapidly diluted
20-fold into the cuvette containing iso-osmotic quench buffer (80 mM potassium gluconate, 40 mM NaI, 5 mM EDTA, 5 mM EGTA, 25 mM
KPi, pH 7.4, ± 100 µM glycine). The
base-line curves observed before the addition of SPQ-containing
aliquots and t0, the time when sample was
introduced into the cuvette, were matched, and the data were averaged
over the entire scan. Mixing was done by magnetic stirring using
a Glas-Col microsubmersible stirring. While the DM3000 fluorimeter
provided sensitive quantitation of activity, the slow mixing precluded
accurate quantitation of the time constant for the rapid quenching upon
channel opening. To determine more accurately the channel kinetics,
additional studies were conducted using a stopped-flow fluorimeter and
are described below. In these later studies, SPQ was replaced with MQAA
as an indicator since it is more efficiently quenched and more polar
(less apt to leak out of the vesicles). For MQAA, excitation wavelength
was 350 nm, and emission was monitored at 460 nm. The observed channel
activity was reproducible in all studies.
The mass flux studies presented herein were conducted using an Applied
Photophysics Stopped-flow Spectrakinetic Monochrometer, using
conditions identical to those above except that vesicle samples were
diluted with an equal volume of quench buffer of equivalent composition
except ±200 µM glycine. Collection of the data was
partitioned such that half of the time points were collected in the
first 200 ms, and the other half was collected over 2 s. To
estimate the number of oligomers incorporated per vesicle, phosphate
and protein assays were performed on each sample. Phospholipid concentration was determined by the method of Fiske and Subbarow (42).
GlyR concentration was determined by modified Lowry assay (43) in which
the protein concentrations relative to standard curves of bovine serum
albumin were correlated to the concentration as determined by
quantitative amino acid analyses on representative samples. Vesicle
size was measured by light scattering on a Nicomp model 279 Submicron
Particle Sizer (Pacific Scientific).
CD Spectroscopy--
Vesicles containing purified human
Measurements were made using an Aviv 62DS spectropolarimeter. At least
10 reproducible spectra were collected for each preparation, averaged,
and smoothed using a Savitzky and Golay filter (45). All reported
spectra were base-line corrected (by subtraction of similarly
collected, averaged, and smoothed base lines of vesicles identically
prepared, except without purified protein) and are the average of two
independent preparations. All measurements were taken over the
wavelength range from 300 to as low as 185 nm, with a 0.5-nm step size,
at room temperature using a 0.1-cm path length quartz cell (Hellma).
For one sample, ellipticity measurements below 195 nm were precluded
due to solvent absorption. The CD spectra of the protein in the near UV
region were analyzed by two independent methods. In the first method a
linear, unconstrained least squares curve-fitting procedure (46) was
employed using a reference set derived from 15 water-soluble proteins
(47). The second method of analysis utilized singular value
decomposition (SVD) (48) in which poly(L-proline)-type
(PII) conformations were incorporated in the analyses using
the self-consistent methodology of Sreerama and Woody (49) using the
program SELCON3.
Single Channel Recordings in Planar
Bilayers--
Electrophysiological studies, in which purified
Occasionally another channel type was observed, having a similar
conductance at negative potentials, but a higher conductance (200 pS)
at positive potentials and a reversal potential near zero. The
application of 1 µM strychnine in one experiment did not
block this channel activity.
Bulk
We can use the fluorescence data to determine the total activity of the
reconstituted glycine receptors in the small unilamellar vesicles.
Reconstituted GlyR was diluted into liposomes such that many of the
vesicles did not contain any protein; this ensured that the vast
majority of vesicles did not contain more than one GlyR since, upon
activation and opening of a single channel, quenching of the internal
fluophore would prevent the determination of the activity of the other
GlyRs in that particle. Since we can measure the protein and lipid
content of the samples and the size of the vesicles, we can determine
the amount of activity from comparing the total fluorescence after
glycine-gated quenching to the total fluorescence after complete
quenching after detergent addition. After dilution, sample phospholipid
concentration was determined by phosphate assay, and protein
concentration was determined by corrected Lowry assay (quantitative
amino acid assay indicated that the protein concentrations as
determined by modified Lowry assay are ~5% lower than actual
concentration). For example, for one preparation light scattering
measurements of the vesicles indicated Gaussian distributions with an
average radius of 74 nm, and lipid:protein levels indicated ~43% of
the vesicles contained a pentameric receptor. From these measurements,
the fraction of the preparation exhibiting activity was extrapolated to
be ~100%. This was not unexpected since every solubilized receptor
applied to the gel filtration column was capable of competitively
binding antagonist and agonist as a requisite step in their
purification. However, given that the measurements and assumptions used
in these determinations all have associated errors, it remains possible that some small fraction of the reconstituted protein is inactive.
Secondary Structure of Reconstituted
Upon examinations of globular protein structure, it has become apparent
that left-handed type II polyproline helices (PII) are a
common structural motif (for review see Ref. 55). A significant fraction of residues not classified as
These results strongly suggest that the homomeric To ensure that the reconstituted It has been reported that GlyR purified from native tissue and
reconstituted into giant liposomes show two well defined ion channel
activities, one of which is rectifying (57). It was speculated that
variations in activity arose from subunit heterogeneity (i.e. variable stoichiometry of subtypes of To ensure that the bulk population of the reconstituted protein
exhibits the functionality demonstrated at the single channel level in
the black lipid membrane studies, mass flux assays similar to those of
García-Calvo et al. (39) were conducted. These studies were conducted in lieu of strychnine binding studies since these latter studies only assay for the retention of ligand binding capability. As a consequence of the method of optimizing solubilization conditions (i.e. the mixed micelles containing protein,
lipid, and detergent are specifically bound by the
aminostrychnine-agarose matrix and competitively eluted with glycine),
by definition, the solubilized receptors retain ligand binding
activity. Since the ligand-binding site of the ligand-gated ion channel
superfamily is fairly complex and has been shown to involve multiple
loops (for review see Ref. 14), the amino-terminal domain is assumed to
be in a native conformation. However, some subset of the
membrane-spanning domains and intracellular loops may become denatured
upon solubilization or reconstitution of GlyR. Therefore, reconstituted
protein was assayed directly for activity by measuring flux of a
fluorescence quencher upon ligand binding. These studies indicated that
most, if not all, of the reconstituted receptors effectively retained activity.
One concern, however, was that in these mass flux assays the channels
were only partially blocked by physiologic levels of strychnine.
Aliquots of vesicles containing GlyR were incubated for 15-30 min with
micromolar levels of strychnine prior to dilution into quench buffer.
While this preincubation with strychnine inhibited channel activation,
this inhibition was not complete (data not shown). However, the single
channel studies in black lipid membranes indicate that strychnine
effectively blocked any activity in the reconstituted channels (Fig.
2B). A possible explanation for this incomplete inhibition
in the mass flux assays is lowered effective strychnine concentration
due to its association with hydrophobic surfaces such as vessel walls
and with vesicles lacking protein. In our hands, similar difficulties
in strychnine partitioning were also observed in single cell patch
clamping experiments of insect cells expressing If we assume that the spectra of the respective ligand-gated receptors
reflect differences in secondary structure of the native channels, from
where might these differences arise? Although the sequences of the
human The lack of The presence of PII helices, as indicated by analyses of
the CD spectrum of the protein reconstituted into lipid vesicles, prompted us to examine more closely the sequence of the
1-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and
reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single
channel recordings of planar bilayers and in flux assays of small
unilamellar vesicles, providing evidence that the recombinant homomeric
receptor may be functionally reconstituted. This reconstitution is
significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of
structure-function. Circular dichroism spectroscopy of reconstituted
GlyR indicated a low
-helical content and a significant fraction of
polyproline structure. The small fraction of observed
-helix is
insufficient to accommodate the four helical transmembrane domains
proposed in models for this receptor. By inference, other members of
the homologous ligand-gated channel superfamily, which include the ionotropic
-aminobutyric acid, acetylcholine, and serotonin
receptors, may also be erroneously modeled, and alternate models should
be considered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid (GABA)1
and excitatory neurotransmitters such as acetylcholine and serotonin (1). These membrane protein channels are amphiphilic molecules which,
in response to neurotransmitter binding, transiently form pores through
the lipid membrane where they are embedded, allowing the passive
movement of small ions down their concentration gradient. This flux
changes the electrical potential across the membrane, thus affecting
the probability of the opening of voltage-gated channels.
), whose passive flux causes
hyperpolarization of the cell. Strychnine, a convulsive alkaloid whose
neurotoxic effect is attributed to blocking of the glycinergic
transmission in the central nervous system, is the most potent known
specific antagonist to the GlyR. By exploiting the strong antagonistic
binding of strychnine (Kd ~ 5 nM),
this ligand-gated channel was the first channel isolated and purified
from mammalian nervous tissue via affinity chromatography on an
aminostrychnine matrix (2). Cross-linking studies indicate that the
native channel is a pentameric assembly of
(48 kDa) and
(58 kDa) subunits (3). These receptors copurify with gephyrin, an
associated 93-kDa peripheral polypeptide that is essential for GlyR
clustering (4-6). Receptor heterogeneity arises from variable subunit
subtypes (7, 8) as well as from alternative RNA splicing (9).
-strands (for review see Ref. 27). Modeling of the receptor now
includes mixed
/
topology in the transmembrane segments (28).
Additionally, recent proteolytic studies conducted on reconstituted
glycine receptors yielded an accessibility profile that precludes M1
and M3 from being transmembrane
-helices (29). Studies on
reconstituted nAchR also indicated that the classical model of M1 as a
transmembrane helix is incompatible with labeling patterns obtained by
derivatization with thiol-reactive compounds (30).
1-GlyR protein for subsequent
biochemical and biophysical characterization. In whole cell and
inside-out patch clamp experiments the insect-expressed homomeric
receptors do indeed form a functional ligand-gated strychnine-inhibited chloride channel. Utilization of the baculovirus system that
successfully overexpresses functional receptors is highly significant
since it frees one from the limiting constraints of working with
naturally abundant proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphatidylcholine, phenylmethylsulfonyl fluoride,
benzamidine, benzethonium chloride, aprotinin, strychnine, lysozyme,
bovine serum albumin, 5-bromo-4-chloroindolyl phosphate, and nitro blue
tetrazolium were purchased from Sigma. Alkaline phosphatase-conjugated
goat anti-rabbit antibody was purchased from Promega. Sf9 insect
cells, the pBlueBac transfer vector, and wild-type Autographa
californica nuclear polyhedrin baculovirus were purchased from
Invitrogen. All media and antibiotics were purchased from JRH
Biosciences. 1-Palmitoyl-2-oleoylphosphatidylethanol-amine (PE) was
purchased from Avanti Polar Lipids. Sephadex G-100 and Sephacryl G-100
were purchased from Amersham Pharmacia Biotech.
1-GlyR (kindly provided by Dr. D. B. Pritchett, University of Pennsylvania) was inserted into a pBlueBac
transfer vector (Invitrogen) and recombinantly incorporated into
baculovirus and purified as described previously (33). All procedures
involving insect cell culture were performed as described by Summers
and Smith (36). Sf9 insect cells were grown in suspension under
natural atmosphere at 27 °C in spinner flasks (gently stirred at 50 rpm) in Hink's TNM-FH insect media supplemented with 10% (v/v) fetal
bovine serum and 0.1% Pluronic F-68, 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml fungizone. Independent
preparations consisted of 750 ml of cells at an initial density of
1 × 106 cells/ml were infected with GlyR baculovirus
at a high multiplicity of infection (>5), harvested 3 days
post-infection by gentle centrifugation, and stored at
70 °C until
needed. Solubilization of the membrane components in a solubilization
buffer containing 1% digitonin and 0.1% sodium deoxycholate and
subsequent purification of the GlyR by affinity chromatography on
aminostrychnine agarose were as described previously (33). Protein
purification was monitored by SDS-polyacrylamide gel electrophoresis
(37) and Western immunoblotting, using an affinity-purified antibody
against a decapeptide corresponding to the amino-terminal 10 residues
of
1-GlyR, samples of which were organically extracted
(38) before electrophoresis. The final buffer conditions of the
solubilized purified protein was 1% digitonin, 0.1% sodium
deoxycholate, 1 M KCl, 10 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, 200 mM glycine,
1.5 mg/ml phosphatidylcholine, and 25 mM KPi,
pH 7.4.
1-GlyR (1-2 ml) were applied to a Sephadex G-100 column
(~45 ml of swollen resin, 1.2 × 25 cm) pre-equilibrated with
the reconstitution buffer, 25 mM KPi, pH 7.4. Sample was eluted with reconstitution buffer by gravity flow at a flow
rate of ~4.0 ml/h. Eluted protein was collected with the void volume,
as observed by A280 and verified by Western
immunoblotting of eluant fractions applied to nitrocellulose via a dot
blot apparatus (Scott Laboratories). The absence of detergent from
these fractions was verified by thin layer chromatography. Silica
plates were spotted with eluant fractions, run in either CHCl3/methanol/H2O (65:25:4) or ethyl
acetate/methanol (4:1) and developed by acid hydrolysis. Each pool of
reconstituted protein in lipid vesicles isolated from a given
preparation was partitioned into 3 aliquots for subsequent
characterization via microscopic and macroscopic activity assay and CD
spectroscopy, thereby ensuring the equivalence of the experimental samples.
70 °C. To form
membranes, a glass applicator coated with the PE/decane solution and
dipped into the vesicle suspension was used to form a membrane across
the ~200-µm aperture of a Teflon partition as described previously
(40). The bathing solutions were chosen to match those used in previous
whole cell recordings (33). The "front" chamber, which was held at
zero potential, contained 150 mM NaCl, 5 mM
KCl, 4 mM MgCl2, 2 mM
CaCl2, 10 mM HEPES and the back chamber
contained 140 mM CsCl, 1 mM EGTA, and 10 mM HEPES. Glycine or strychnine were added to the front chamber as noted.
1-GlyR protein were prepared as described above, except
conditions were scaled up as follows: 10-15 ml of purified sample were
applied to a 2.6 × 40-cm Sephacryl S-100 column, and a flow rate
of ~ 0.5 ml/min was maintained by peristaltic pumping. The
reconstituted GlyR vesicles were fused with sonicated liposomes (22.5 mg/ml crude soybean phospholipid, 4.5 mg/ml cholesterol) by freeze-thaw
sonication in the presence of either
6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ, Molecular
Probes) or N-(carboxymethyl)-6-methoxyquinolinium bromide
(MQAA, Molecular Probes), I
-quenched fluorescent probes,
under conditions yielding a final concentration of 120 mM
potassium gluconate, 5 mM EDTA, 5 mM EGTA, 25 mM KPi, pH 7.4. To form small tight unilamellar
vesicles of approximately uniform diameter, samples were
probe-sonicated immediately before fluorescence measurement. Samples
were jacketed in an ice bath and sonicated using a Branson Sonifier 450 microtip at setting 6, with 50% pulses (1/2 s on and 1/2 s off) applied in three 20-s bursts with the sample allowed to
thermally equilibrate between bursts. Aggregates were removed by
centrifugation at 15,000 × g for 10 min. Flux assays
were adapted from García-Calvo et al. (39), with the
major modification being the elimination of the additional gel
filtration step to remove external probe. Blank control vesicles
without protein were prepared as above except solubilization buffer
without protein was applied to the gel filtration column. External
quenching of the fluorescent indicator by iodide was complete within
the instrumental dead time, similar to that observed for chloride (less
than 2 ms) (41). Given the rapid quenching, external fluorophores were
not removed after vesicle loading due to the large reduction in
fluorescent signal observed after the additional gel filtration
required to remove external MQAA or SPQ (data not shown). This
reduction was probably due to slow leakage of the internal fluorescent
indicator driven by equilibration during the time-consuming chromatography.
1-glycine receptor were reconstituted as described
above, providing a vesicle suspension in 25 mM
KPi buffer, pH 7.4. Samples were additionally
probe-sonicated to yield small unilamellar vesicles using a Branson
Sonifier 450 microtip at setting 6, with 50% pulses (1/2 s on
and 1/2 s off) applied in three 20-s bursts with the sample
jacketed in an ice bath and allowed to thermally equilibrate between
bursts. Aggregates were removed by centrifugation at 15,000 × g for 10 min, and the isolated supernatant was optically
clear. Incorporation of the reconstituted protein into small vesicles
(much smaller than the wavelength of the incident light) with high
lipid-to-protein ratios minimizes differential light scattering and
absorption flattening effects, respectively, two potential artifacts
arising due to the particulate nature of the lipid-protein complexes
(44). Typically, samples of reconstituted receptor was not diluted CD
measurements and had a final concentration of 0.08-0.1 mg/ml. Blank
vesicles without protein were made by injecting equivalent volumes of
solubilization buffer alone to the gel filtration column (as described
above), and (protein-free) vesicles eluting in the void volume were
treated equivalently for use as blanks.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-glycine receptors reconstituted into lipid vesicles
were incorporated into planar bilayers, showed functional glycine-gated
and strychnine-inhibited ion channels. The voltage dependence of single
channel current in symmetrical solutions was nearly linear, with a main
conductance level of 79 pS (Fig. 1),
corresponding to the ~70-pS conductance observed in patch clamp
recordings of Sf9 cells under comparable conditions (33). As has
been reported for native glycine receptor channels (50) and
homo-oligomeric
1-channels transfected into human
embryonic kidney cells (20, 51), subconductance levels were observed.
The most prominent had a conductance of 47 pS but represented only
about 8% of the total channel open time (Fig. 1C).
Increasing glycine concentration increased the frequency and duration
of bursts of channel openings (Fig.
2A), whereas addition of 1 µM strychnine to the same side of the membrane produced a
complete block of channel activity (Fig. 2B).
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Fig. 1.
Voltage dependence of single channel
currents. A, currents recorded at various potentials in
the presence of 100 µM glycine. Representative recordings
at membrane potentials from 100 to + 60 mV, with the closed channel
current indicated by a dotted line for each trace are shown.
Filter bandwidth was 200 Hz. B, current-voltage relationship
for the main conductance level (solid circles) and the most
prominent subconductance level (open circles), from the
recording shown in A. Least squares fits yielded
conductances of 79 and 47 pS (lines). C,
amplitude histograms of channel activity. All-points histograms were
accumulated from record segments 19 and 10 s in duration at
80
and +80 mV, respectively; filter bandwidth was 200 Hz.
Arrows indicate the levels of the most prominent
subconductance state. A fit of three Gaussian components (smooth
curve) assigns an area to the subconductance peak that is 7.8 and
8.3%, respectively, of the peak corresponding to the main conductance
level.
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Fig. 2.
Ligand dependence of currents.
A, representative recordings from a bilayer as glycine was
added successively to the front chamber to give the indicated
concentrations. Membrane potential was 60 mV. Note the increase in
burst duration and the appearance of overlapping events at the higher
concentrations. Filter bandwidth was 200 Hz. B, block of
channel currents by 1 µM strychnine added to the front
chamber. Channels were activated by 100 µM glycine;
membrane potential was
40 mV, and filter bandwidth was 50 Hz.
Bars above the trace indicate the times when the
recording was blanked as strychnine was added and stirring occurred.
After stirring, no channel events were observed for the remainder of
the recording.
1-GlyR Activity in Reconstituted Vesicles by
Fluorescence Quenching--
Although the single channel recordings as
described above provide information with respect to the elementary
characteristics of the reconstituted
1-GlyR channels,
they do not provide an assessment of the activity of the bulk
population. Flux assays, as measured by the quenching of SPQ
fluorescence via I
entry into vesicles through the open
anionic receptor channel, were used to assess activity macroscopically.
Initial fluorescence decay curves were conducted by rapidly mixing the
vesicles into an excess of quench buffer. In the absence of glycine, a
subset of the vesicles were leaky, and fluorescence quenching could be fit by a single exponential decay curve with a time constant on the
order of 0.2 s
1. However, the dead time of
mixing precluded accurate measurement of the time constant for the
rapid quenching of the internal SPQ as a consequence of glycine gating
of the channels. To quantitate this component, a stopped-flow
fluorimeter was used. MQAA was substituted for SPQ due to its more
advantageous properties (e.g. quantum efficiency, charge
properties). In the absence of glycine, the time constant was
reproducibly determined to be ~0.2 s
1 as
observed previously. Initial fluorescence, Fo, was determined by extrapolation of the averaged decay curve to time 0. Fluorescence is reported as relative fluorescence,
Ft/Fo. Given the determined
values for Fo and the time constant for nonspecific
channel opening or leakage, parallel studies in which agonist was
included in the quenching buffer could not be described well by a
single exponential decay function. Rather, in the presence of 100 µM glycine, external I
quenched
fluorescence in a biphasic manner (see Fig.
3 for representative spectra). However,
the quenching was so rapid that most of it occurred in the
instrumentation dead time (~20 ms), and there was a large associated
error in the curve-fitting to this fast component. The time constant
for the rapid quenching caused by ligand-gated channel activity was
observed to be >50 s
1.
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Fig. 3.
Bulk flux assay. Reconstituted GlyR
vesicles are fused via cycles of freeze-thaw sonication with sonicated
liposomes (5:1 phospholipid/cholesterol, w/w) in the presence MQAA, the
fluophore that is quenched by I , as described in the
text. Excitation was at 344 nm, and emission was monitored using a 440 nm filter. An equal volume of vesicles was diluted into quench buffer
(identical buffer except 80 mM potassium gluconate, 40 mM NaI), in the presence (solid line) or absence
(dotted line) of 200 µM glycine. The
representative experiment shown in the figure is the average of five
repeats with the lines extrapolated from least square fitting of the
data. Quenching in the absence of ligand was fit as a sum of a constant
and a single exponential, and the relative fluorescence was determined
by dividing the observed fluorescence by the value at time 0, as
extrapolated by the fit. Quenching in the presence of glycine was fit
as a sum of a constant and two exponentials, given an initial
fluorescence and the slower time constant as determined in the absence
of ligand. In the presence of glycine, quenching was essentially
complete in <100 ms.
1-GlyR by
Circular Dichroism--
The observed CD spectrum of
1-GlyR reconstituted into small unilamellar
phosphatidylcholine vesicles is shown in Fig.
4. The spectrum exhibits a very strong
positive ellipticity at the lower limits of detection for these
samples, and a broad weak negative band was centered at ~210-225 nm.
This spectrum is qualitatively similar to that observed in studies
conducted on reconstituted nAchR ((52) see inset to Fig. 4),
a fellow member of the ligand-gated ion channel superfamily of
receptors; however, upon analysis, some significant differences are
observed between the two receptors with respect to the calculated
secondary structure. In fact, no linear combination of the CD reference
spectra of Yang and co-workers (47) could adequately fit the
experimental GlyR spectrum; this was reflected by a large normalized
root mean square deviation (
0.1) for the fitted to the
experimental spectrum. The reference data base consisted of spectra
representative of the
-helix,
-sheet,
-turn, and the unordered
component, referred to as random coil, deconvoluted from the CD
spectra of 15 water-soluble proteins with well resolved structure. This
type of analysis has been shown to determine accurately the
fraction of
-helix in a protein (53) but, like all CD methods, is
less reliable in determining the secondary structure on nonhelical
components. Similarly, SVD analysis of the GlyR spectrum also failed to
fit the experimental curve. This failure may be due to a relative
absence of the structures found in the examined protein in the
reference data base (i.e. the protein fold exhibited by this
membrane protein is not well represented in the reference data set). A
similar failure of current CD methods of analyses to determine
accurately secondary structure is observed in examining all
proteins (54).
View larger version (16K):
[in a new window]
Fig. 4.
CD spectroscopy. CD spectra of
1-GlyR reconstituted into phosphatidylcholine vesicles.
The spectra are base-line corrected and are the average of 10 reproducible scans. Collected large vesicles from gel filtration void
volume were probe-sonicated to make the vesicles small and unilamellar.
Buffering was at 25 mM KPi, pH 7.4, and scans
were taken at 0.5 nm intervals from 300 to 194 nm. All measurements
were taken at room temperature in a 0.1-cm path length cell. The
calculated secondary structure was 15%
-helix, 37%
-sheet, 22%
-turn, 9% PPII, and 18% random coil. Inset,
spectra of a pure
-helix (solid line), collagen
(dashed line), and nicotinic acetylcholine receptor
(dotted line). See text for spectral sources.
-helix,
-sheet, and
-turns are in the PII conformation. Recently, this
structural class was incorporated in the analysis of the CD spectra of
proteins and, as a consequence of its characteristic spectrum, was
found to quantitate successfully this class of structural fold (49). The experimental spectrum most resembles the extended
-structure found in the cell adhesion promoting peptide from collagen (56) that
contains extended polyproline-like structure. SVD analysis of the
experimental spectrum using the five-component basis set (
-helix,
-sheet and turns, PII, and unordered) showed significant improvement in the fit over our original four-parameter least squares
analysis and indicated that the
1-GlyR contained
approximately only ~15%
-helix and had 9% PII conformation.
1-GlyR
channel lacks significant
-helix content. These measurements provide the first indication of GlyR net secondary structure and raise serious
questions on the validity of models of glycine receptor topology that
are based primarily on sequence analysis in which the transmembrane
domains were modeled as
-helices. More generally, given the sequence
and structural homology between members of the ligand-gated
superfamily, the current model for ligand-gated channels, which is
predicated primarily on the presence of the empirically predicted four
transmembrane helices per subunit, may require re-evaluation and is
further discussed below.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-GlyR in small
unilamellar vesicles that are characterized structurally are
functional, ligand-gated channels, equivalent aliquots of the
reconstituted channel were used for single channel studies, mass flux
assays, and CD studies. In both single channel studies and mass flux
assays, the reconstituted receptor was found to be gated with glycine and inhibited by strychnine. Receptor activity at both a microscopic (i.e. single channel activity) and macroscopic
(i.e. mass flux activity) level provides assurance that the
substrate used in subsequent CD studies is indeed in a native conformation.
and
subunits) or differences in post-translational modification. In black
lipid membrane studies we predominantly observed the rectifying form, and channel properties are very similar to those observed in patch clamp studies of receptors in cell membranes. Our planar bilayer reconstitution results suggest that these variations in activity arise
in the absence of sample heterogeneity and may reflect variations in
protein phosphorylation or glycosylation.
1-GlyR
(33).
1-subunits of the GlyR and the nAchR are fairly
similar (19% identity, 46% similarity), the regions of high homology
are fairly discrete. If the regions of high homology corresponding to
the loops comprising the agonist/antagonist-binding site and the four
spans of high hydrophobicity (69% similarity) are removed from
consideration, then the identity and similarity is reduced to 13 and
34%, respectively. Perhaps the variation in secondary structures
arises from differences in the structure of regions of reduced
homology. In the ligand-gated ion channel superfamily, the
putative large cytoplasmic loop between the M3 and M4 regions of the
receptors is the area with the greatest sequence divergence, with the
loop size varying among members from ~80 to 265 amino acids. This
region is composed of ~20% of
1-GlyR structure. In
fact, recent modeling of the secondary structure of the proposed
extramembranous domains of the nAchR using various empirical prediction
schemes predicted the presence of multiple consensus helical stretches
not found in GlyR or GABA receptors (58). The reference spectra of
-helix and that of a poly(L-proline)II-type helix (or
the spectrally similar collagen-like fold) are dramatically different
(see inset, Fig. 4), especially in their very large ellipticities in the low UV region where their peaks are of opposite sign. Some of the differences between the spectra of the two
ligand-gated channels might be attributable to regions of
1-GlyR enriched in this latter secondary structural
element, at the expense of being
-helical as in nAchR.
-helicity as determined by CD spectroscopy is somewhat
unexpected; members of the family of ligand-gated channels are
typically modeled as a pentameric assembly of subunits, with each
subunit having four transmembrane
-helices. The rationale for this
model was based primarily on hydrophobicity plots of the sequences of
the ligand-gated channels, as well as a wealth of accumulated
biochemical data, principally from studies of the nAchR (for review see
Ref. 59). However, while these biochemical studies test and support a
four transmembrane helix model, they do not prove it; critical
evaluation in the absence of structural data is more problematic. Our
CD data for GlyR, along with Fourier transform infrared spectroscopic
studies showing that the nAchR transmembrane domains contain both
-helices and
structure (60), are among the first spectroscopic
data that strongly suggest that this model may be erroneous. Whereas
the calculated secondary structure as determined by CD in this study
cannot be assigned to specific regions of the molecule, and thus cannot
provide direct evidence supporting an
/
arrangement of the
transmembrane domain, these studies clearly indicate that there is
insufficient helical content (10-15%) to accommodate four
transmembrane helices per subunit (which would require a minimum of
~20%). Studies using proteolytic enzymes as probes of topology also
indicate that the four helix transmembrane model is erroneous and
suggest that the transmembrane domains contain a mixture of
-helices
and
-sheets (29). The potential pitfall of mapping transmembrane
topology via sequence analysis has been similarly illustrated in
investigations of the glutamate receptor in which it was shown that,
contrary to previous models, the putative second transmembrane domain
does not span the bilayer but rather forms a re-entrant loop (61, 62).
Hydropathy analysis has also been shown to be potentially misleading
with regard to the transmembrane topology of other membrane proteins,
e.g. the voltage-dependent K+
channel (63) and the P-glycoprotein transporter (64).
1-subunit. This class of secondary structure has
recently been shown to occur commonly in globular proteins (55).
Although prolines need not be an obligatory component of this type of
fold, the sequence of the
1-subunit is indeed enriched
in proline content (5.2%), as are the sequences of other GlyR subunits
and other members of the ligand-gated channel superfamily.
Interestingly, the
1-GlyR subunit contains stretches of
amino acids that compose a consensus SH3 binding region (Table
I). This consensus sequence has an XPpXP motif, where X represents an
aliphatic residue and p represents a preference for proline (65) and,
depending on classification (type I or II), typically contains a basic
amino acid three residues before the initial proline (class I) or two
residues after the final proline (class II) of this motif (66). In the
case of the
1-GlyR, the consensus sequence (residues
365-371, PPPAPSK) most closely resembles a class II ligand and
would be expected to bind in the "minus" conformation (67). It was
first noted by Rotin and co-workers (68) that cytoskeletal association
via SH3 binding domains could represent a novel mechanism for correctly targeting membrane proteins to their appropriate site in polarized or
specialized cells. They found that an SH3 binding region of the rat
epithelial Na+ channel interacts with an SH3 domain of
-spectrin. Perhaps a similar mechanism is involved in the binding of
GlyR to cytoskeletal elements. In fact, inspection of the sequences of
the GlyR
subunit, as well as other members of the ligand-gated ion
channel superfamily, reveal many of these subunits to contain similar
consensus SH3 binding regions (Table I). Inspection of the sequence of
the N-methyl-D-aspartic acid receptor
(69) also reveals a multiplicity of putative SH3 binding regions. The
association of these subunits with cytoskeletal elements possessing SH3
domains (as shown for
-spectrin, nonmuscle myosin 1b, and
Saccharomyces cerevisiae protein ABP-1, for reviews see
Refs. 65 and 66) may be a general mechanism in neuroreceptor targeting
and clustering. It has been shown that gephyrin, a GlyR-associated
protein that links the receptor to subsynaptic tubulin, interacts with
a cytoplasmic loop (lacking an SH3 consensus sequence) of the
-subunit in rats (6). We propose that the
1-subunit
might also interact, possibly via SH3 interactions, with other linking
elements to affect clustering and/or localization. A similar
multiplicity of linking components has been identified that interacts
with the heterologous N-methyl-D-aspartic acid receptor (which is composed of NR1 and NR2 subunits);
various components interact specifically with different subunits and/or splice variants of this receptor (70-72).
SH3-binding ligand motifs
The ongoing studies indicate that the homomeric GlyR has been
purified and reconstituted in an active form. This functional homogenous channel is produced in sufficient quantities for biophysical and crystallographic studies. Further studies are currently being conducted and foreshadow a molecular understanding of glycine receptor
function, as well as that of the other members of the physiologically
important superfamily of ligand-gated receptors.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Narasimha Sreerama and Dr. Robert Woody for providing SELCON3. We also thank Dr. Mark Zeidel for providing access to the Applied Photophysics Stopped-flow Spectrakinetic Monochrometer and Dr. Larry Coury and Dr. John Mathai for their considerable assistance, patience, and expertise in using this equipment as well as in light scattering studies. Quantitative amino acid analyses were conducted by Myron Crawford at the W. M. Keck Foundation, Biotechnology Resource Laboratory, Yale University. We also thank Dr. B. A. Wallace for providing the experimental spectrum of the nicotinic acetylcholine receptor. We also acknowledge Shared Instrumentation Grant 1S10RR11998 from the National Institutes of Health which provided support for the circular dichrometer.
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FOOTNOTES |
---|
* This work was supported by the Howard Hughes Medical Institute (to R. O. F.) and National Institutes of Health Grants NS21501 (to F. J. S.), GM51911 (to M. C.), and GM55851 (to R. O. F).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.
** To whom correspondence should be addressed: Dept. of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.658 Basic Science Bldg., Galveston, TX 77555-0647. Tel.: 409-772-2163; Fax: 409-747-4745; E-mail: fox@bloch.utmb.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010968200
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ABBREVIATIONS |
---|
The abbreviations used are:
GABA, -aminobutyric acid;
GlyR, glycine receptor;
SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium;
MQAA, N-(carboxymethyl)-6-methoxyquinolinium bromide;
SVD, singular value decomposition;
PII, left-handed type II
polyproline;
SH3, src homology 3 domain;
nAchR, nicotinic acetylcholine
receptor.
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