Functional Reconstitution and Characterization of Recombinant Human alpha 1-Glycine Receptors*

Michael CascioDagger , Scott Shenkel§, Robert L. Grodzicki, Fred J. Sigworth§, and Robert O. Fox||**

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha -helical content and a significant fraction of polyproline structure. The small fraction of observed alpha -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 gamma -aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -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.

Upon binding of glycine, the glycine receptor (GlyR) channel becomes permeable to small anions (Cl-), 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 alpha  (48 kDa) and beta  (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).

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 beta -strands (for review see Ref. 27). Modeling of the receptor now includes mixed alpha /beta 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 alpha -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).

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 alpha 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.

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Digitonin and sodium deoxycholate were purchased from Aldrich. Soybean phospholipids, cholesterol, L-alpha -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.

Protein Expression and Purification-- A cDNA encoding the human alpha 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 alpha 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.

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 alpha 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.

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 -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.

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 alpha 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.

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 alpha 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Single Channel Recordings in Planar Bilayers-- Electrophysiological studies, in which purified alpha 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 alpha 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.

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 alpha 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 alpha 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.

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 alpha 1-GlyR by Circular Dichroism-- The observed CD spectrum of alpha 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 alpha -helix, beta -sheet, beta -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 alpha -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 beta  proteins (54).


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Fig. 4.   CD spectroscopy. CD spectra of alpha 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% alpha -helix, 37% beta -sheet, 22% beta -turn, 9% PPII, and 18% random coil. Inset, spectra of a pure alpha -helix (solid line), collagen (dashed line), and nicotinic acetylcholine receptor (dotted line). See text for spectral sources.

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 alpha -helix, beta -sheet, and beta -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 beta -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 (alpha -helix, beta -sheet and turns, PII, and unordered) showed significant improvement in the fit over our original four-parameter least squares analysis and indicated that the alpha 1-GlyR contained approximately only ~15% alpha -helix and had 9% PII conformation.

These results strongly suggest that the homomeric alpha 1-GlyR channel lacks significant alpha -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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To ensure that the reconstituted alpha 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.

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 alpha  and beta  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.

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 alpha 1-GlyR (33).

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 alpha 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 alpha 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 alpha -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 alpha 1-GlyR enriched in this latter secondary structural element, at the expense of being alpha -helical as in nAchR.

The lack of alpha -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 alpha -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 alpha -helices and beta  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 alpha /beta 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 alpha -helices and beta -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).

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 alpha 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 alpha 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 alpha 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 alpha 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 alpha -spectrin. Perhaps a similar mechanism is involved in the binding of GlyR to cytoskeletal elements. In fact, inspection of the sequences of the GlyR beta  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 alpha -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 beta -subunit in rats (6). We propose that the alpha 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).

                              
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Table I
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.

    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.

    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

    ABBREVIATIONS

The abbreviations used are: GABA, gamma -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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