Water-soluble Nicotinic Acetylcholine Receptor Formed by alpha 7 Subunit Extracellular Domains*

Gregg B. WellsDagger §, René Anandpar , Fan Wang**, and Jon Lindstrom**Dagger Dagger

From the Dagger  Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6082,  Neuroscience Center of Excellence and Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, and ** Department of Neuroscience, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6074

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the alpha 7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for alpha -bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the alpha 7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length alpha 7 AChR.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Nicotinic acetylcholine receptors (AChRs)1 are integral-membrane, pentameric ion channels in the central and peripheral nervous systems that participate in signal transmission associated with the release of acetylcholine (ACh). A considerable collection of studies of their cell biology, electrophysiology, and structure makes them the best characterized family of a superfamily of homologous neurotransmitter-gated channels that includes glycine, gamma -aminobutyric acidA, and 5-hydroxytryptamine3 receptors (1-3). Muscle-type AChRs are composed of four different subunits with the subunit composition (alpha 1)2(beta 1)delta (gamma or epsilon ) and bind the snake venom toxin alpha -bungarotoxin (alpha Bgt). Neuronal AChRs that do not bind alpha Bgt are formed from combinations of alpha 2, alpha 3, alpha 4, or alpha 6 subunits with beta 2, beta 3, beta 4, and/or alpha 5 subunits. Neuronal AChRs that do bind alpha Bgt are formed from alpha 7, alpha 8, and alpha 9 subunits, perhaps in combination with unknown subunits. When heterologously expressed, alpha 7, alpha 8, and alpha 9 form functional homomeric AChRs that appear to contain five identical subunits.

AChRs are composed of five homologous membrane-spanning subunits that are ordered around a central, cation-selective channel. The topology of AChRs that is predicted by hydrophobicity plots has received substantial experimental support (4, 5). The approximately 200 residues at the N-terminal half of each AChR subunit are extracellular, are N-glycosylated, contain sites for agonist and antagonist binding, and form the vestibule through which cations reach the transmembrane channel. Relatively little of the remainder of the primary sequence is extracellular. Three of the four transmembrane domains (M1-M3) that form the channel are grouped together following the N-terminal extracellular domain (ECD) and are separated from M4 by a large cytoplasmic loop. For the muscle-type AChR, three distinct regions of the primary sequence around amino acid residues 86-93, 149, and 190-198 of alpha 1 subunits and peptide loops around residues 34, 55-59, 113-119, and 174-180 of the gamma  or delta  subunit contribute to the ACh binding sites at the interfaces between alpha  and delta  and between alpha  and gamma  (or epsilon ) subunits, based on photoaffinity labeling and site-directed mutagenesis (6, 7). Because of primary sequence and topological similarity, homologous residues at subunit-subunit interfaces in other neuronal AChR subunits are expected to have similar roles in the agonist binding site. For example, residues of the alpha 7 subunit homologous to those of alpha 1 and to those around gamma 55 and delta 57 have been shown to contribute to the agonist binding in homomeric alpha 7 AChRs (8, 9).

Our knowledge of the molecular structure of AChRs, however, is far from complete. Electron diffraction methods using two-dimensional, tubular arrays of AChRs from Torpedo californica have successfully yielded structural details at 9 Å resolution in three dimensions (10-12) and at 7.5 Å in two-dimensional projection (13). Achieving higher resolution, however, has been elusive with membrane-bound or detergent-solubilized AChRs. No member of this superfamily of integral-membrane receptors has been crystallized, and the intact AChRs are too large (more than 200 kDa) for nuclear magnetic resonance spectroscopy.

An AChR formed from the ECD may be a suitable structural model of a full-length AChR, if the ECD can both fold and oligomerize in the absence of the remainder of the subunit. Several lines of evidence suggest that the ECD meets these requirements. First, the specific interactions between subunits that are important in assembly of the muscle-type AChR and for formation of mature acetylcholine binding sites appear to depend primarily on the ECD (14-21). The long cytoplasmic loop of alpha 1 participates in assembly subsequent to the formation of heterodimers (22). Second, membrane-tethered ECDs of mouse muscle alpha 1 and delta  form heterodimers with ligand binding sites that reflect properties of a full-length AChR (23-25). Third, sequences of alpha 7 that affect homomeric assembly also have been localized to the first half of the ECD and an area around M1 based on chimeras of alpha 7 and alpha 3 (26). According to this report, the long cytoplasmic loop of alpha 7 is not essential for oligomerization.

To determine whether AChRs formed from the ECD (residues 1-208) of alpha 7 subunits are water-soluble structural models for the ECD of the full-length alpha 7 AChR, we expressed two constructs of the ECD of the alpha 7 subunit with M1 retained and one construct of the ECD without M1 in Xenopus oocytes. The constructs with M1 were included to explore the feasibility of removing M1 by in vitro processing subsequent to in vivo synthesis. We examined ligand binding properties and velocity sedimentation profiles as indicators of global structure and local structure at the agonist binding site of the resulting AChRs, which we have designated as alpha 7 ECD AChRs. We found that each construct, including the water-soluble one without M1, assembles into an AChR with affinities for 125I-labeled alpha Bgt (125I-alpha Bgt), nicotine, and ACh that match those of the full-length alpha 7 AChR. These properties demonstrate that the alpha 7 ECD forms a water-soluble alpha 7 ECD AChR that can be a starting point for structural studies of this superfamily of ion channels.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Design of alpha 7M1, alpha 7 Enterokinase (alpha 7EK), and alpha 7 Water-soluble (alpha 7WS) Plasmids-- The full-length cDNA sequence of chicken alpha 7 (27) previously was cloned into a modified SP64T expression vector (28, 29). For alpha 7M1, which encodes the ECD of alpha 7 up to the start of M2, the alpha 7 coding sequence from the beginning of M2 to past the native stop codon was removed by digestion with BglII and BsmI. It was replaced by a double-stranded oligonucleotide cassette coding in-frame for the 19-amino acid sequence SQVTGEVIFQTPLIKNPRV and a stop signal. This sequence contained the epitope of mAb 142 from residues 2 to 17 (5), followed by a MluI restriction site in the DNA sequence. The last native residue from alpha 7 was Ile240, according to the numbering scheme of the mature chicken alpha 7 AChR subunit (27). The mAb 142 epitope was introduced for immunoblotting and for binding the subunit protein to mAb 142-coated plastic wells for solid-phase assays (5). The MluI site was included so that the epitope insert could be extended with additional residues after digestion with MluI and BsmI. It has been shown that such extension may be necessary for accessibility of the epitope by the antibody (5).

The other construct that included M1 was alpha 7EK, which was similar to alpha 7M1 except for the inclusion of a peptide spacer between the end of the ECD and the beginning of M1. To prepare alpha 7EK, a double-stranded oligonucleotide cassette coding for the 38-amino acid residue sequence TMRRRTGTVSISPESDRPDLSTFTSDDDDKILERRRTL was ligated in frame between the proximal BsmAI and distal HgaI sites that are nearly juxtaposed to the 5' side of M1 in alpha 7M1. Residues 1-6 reconstructed the end of the N-terminal extracellular domain from residues Thr203 to Thr208; the DNA sequence for residues 7-8 introduced a KpnI site; residues 9-23 were the epitope for mAb 236 (5); the DNA sequence for residues 24-25 introduced a SpeI site; residues 26-31 (DDDDKI) were the specificity sequence for EK that is modeled after its site of proteolysis on trypsinogen (30, 31); the DNA sequence for residues 32-33 introduced a XhoI site; and residues 34-38 reconstructed the native sequence of Arg205 to Leu209 at the distal end of the insert. Beginning with Tyr210 (native numbering), the remainder of the sequence was identical to alpha 7M1. The KpnI, XhoI, and SpeI restriction sites were included so that the mAb 236 epitope and the target for protease digestion easily could be modified readily. A total of 27 amino acid residues were inserted between the proximal copy of Thr208 and the distal copy of Arg205.

The third construct, alpha 7WS, was truncated at the end of the ECD of alpha 7 and did not include M1. To prepare alpha 7WS, the full-length sequence of alpha 7 was cut at the BglII site between M1 and M2. The resulting N-terminal domain sequence was cut at the BsmAI site proximal to the start of the M1 coding sequence. A double-stranded oligonucleotide cassette coding for the 22-amino acid residue sequence TMRRRTQVTGEVIFQTPLIKNP followed by a stop codon was ligated between the BsmAI site of the N-terminal sequence and the EcoRI site of a modified SP64T expression vector. Residues 1-6 of this sequence reconstructed the native sequence Arg203 to Thr208; residues 7-22 constituted the epitope for mAb 142. The oligonucleotides were synthesized using the phosphoramidite method on a MilliGen oligonucleotide DNA synthesizer.

Protein Expression in Xenopus oocytes-- DNA from each of the three plasmids was purified on a CsCl gradient. cRNA was synthesized using an SP6 mMessage mMachineTM kit (Ambion, Austin, TX) and linearized DNA. The cytoplasm of each oocyte was injected with approximately 50 ng of cRNA and incubated at 18 °C for 3-5 days in 50% Leibovitz's L-15 medium (Life Technologies, Inc.) in 10 mM HEPES, pH 7.5, containing 10 units/ml penicillin and 10 µg/ml streptomycin.

For alpha 7M1 and alpha 7EK, extraction of membrane-bound subunit protein with a buffer containing Triton X-100 was accomplished with a previously reported procedure (29). Oocytes were homogenized by hand in ice-cold buffer A (50 mM sodium phosphate, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, pH 7.5). The membrane-containing fraction was separated by centrifugation, was washed twice with buffer A, and then was extracted with buffer B (40 mM sodium phosphate, 40 mM NaCl, 4 mM EDTA, 4 mM EGTA, 4 mM benzamidine, 12 mM iodoacetamide, 2% Triton) during gentle agitation for 2 hours at 4 °C. The soluble fraction from this detergent extraction step was separated by centrifugation and was used for both Western blotting and assays of ligand binding.

For alpha 7WS, the secreted fraction was defined as the subunit protein present in the L-15 medium incubating injected oocytes. Particulates in this fraction were removed by centrifugation. The cytoplasmic fraction of alpha 7WS was defined as the subunit protein present in the soluble fraction following homogenization of the oocytes by hand in buffer A and centrifugation to sediment the insoluble, membranous component. The Triton-extracted fraction of alpha 7WS was defined as the subunit protein present in the solvent following extraction in buffer B of the membranous component from the homogenization step during gentle agitation for 2 h at 4 °C.

Immunoblotting-- Triton-extracted alpha 7M1 and alpha 7EK or secreted alpha 7WS was incubated overnight at 4 °C with mAb 142 that had been coupled to Sepharose CL-4B (Pharmacia) with CNBr (32). After this concentration step, the protein was eluted at 55 °C with 2% SDS and 20 mM beta -mercaptoethanol. Proteins were deglycosylated for 18 h at 37 °C with 1 unit of a mixture of endoglycosidase F and glycopeptidase F according to instructions of the manufacturer (Boehringer Mannheim). A sample without enzyme was run in parallel as the negative control.

Protein was denatured and reduced at 55 °C in SDS-polyacrylamide gel electrophoresis sample buffer containing 2% SDS, separated on a 13% acrylamide SDS-polyacrylamide gel electrophoresis gel, and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). After being blocked in 5% powdered milk in phosphate-buffered saline (PBS, 100 mM NaCl, 10 mM sodium phosphate, pH 7.5) containing 0.5% Triton, the membrane was incubated with 2 nM 125I-mAb 142. Specific activities of the labeled antibodies ranged from 1017 to 1018 cpm/mol. Labeling was visualized by autoradiography.

Ligand Affinities-- Immulon 4 plastic microwells (Dynatech Laboratories, Chantilly, VA) were coated with mAb 142 or mAb 236 for solid-phase assays (5). The wells were blocked with 3% bovine serum albumin in PBS. For alpha 7M1 and alpha 7EK, a volume of the Triton-solubilized protein from the equivalent of from one to three oocytes that had been injected with cRNA was added to each microwell. For the measurement of alpha Bgt affinity, 125I-alpha Bgt was added to the Triton extract and incubated overnight at 4 °C. Total volume in each well was 100 µl. The wells were washed three times with ice-cold PBS containing 0.5% Triton, and the amount of radioactivity was measured using a gamma  counter. Each data point was measured in duplicate. For the competitive inhibition assays, the wells were washed free of the Triton solution after 24 h and loaded with 125I-alpha Bgt at 4 nM in the presence of L-nicotine or ACh. The wells were incubated 8 h at 4 °C before washing and then measuring the amount of radioactivity. Each data point was measured in duplicate. Nonspecific binding was measured with Triton-solubilized protein extracts from uninjected oocytes and generally was less than 5% of the specific binding.

For alpha 7WS, the volume of L-15 medium above about six oocytes was added to mAb 142-coated microwells and incubated overnight at 4 °C for capture of the secreted alpha 7WS. The L-15 was washed away with PBS. For the measurement of alpha Bgt affinity in 0% Triton, 125I-alpha Bgt was added to buffer C (same composition as buffer B, except without Triton) and incubated overnight at 4 °C. Total volume in each well was 100 µl. The wells were washed three times with ice-cold PBS, and the amount of radioactivity was measured using a gamma  counter. Inhibition by nicotine and ACh in 0% Triton was measured after capture of the secreted alpha 7WS, washing of L-15, and loading of each well with 0.6 nM 125I-alpha Bgt and the appropriate amount of inhibitor in buffer C. Total volume in each well was 100 µl. The wells were incubated 8 h at 4 °C before washing and measuring the amount of radioactivity. Data points were measured in duplicate. Nonspecific binding was measured with L-15 medium that was used to incubate uninjected oocytes and generally was less than 10% of the specific binding. Buffer B was substituted for buffer C at the step of loading the wells with 125I-alpha Bgt or with 125I-alpha Bgt and nicotine or ACh for the ligand affinity measurements of alpha 7WS in 2% Triton.

The equilibrium dissociation constants Kd for 125I-alpha Bgt were determined by least-squares, nonlinear fitting to a Hill-type equation (Equation 1) using the graphing software KaleidaGraph (Synergy Software)
C=C<SUB>0</SUB> · <FR><NU>1</NU><DE>1+<FENCE><FR><NU>K<SUB><UP>d</UP></SUB></NU><DE>L</DE></FR></FENCE><SUP>n</SUP></DE></FR> (Eq. 1)
where C is the measured signal (counts/min), C0 is the maximal signal (which corresponds in this case to the maximum number of 125I-alpha Bgt binding sites), L is the concentration of 125I-alpha Bgt, n is the Hill coefficient. The half-maximal inhibition constants, IC50, for nicotine and ACh in the presence of 125I-alpha Bgt were determined by nonlinear fitting to Equation 2, where L is the concentration of nicotine or ACh, C0 is the maximal signal, and C1 is a constant that represents signal that is not displaced by high concentrations of agonist. We used the Cheng-Prusoff equation (Equation 3) to estimate equilibrium dissociation constants from IC50 values (33),
C=C<SUB>0</SUB> · <FR><NU>1</NU><DE>1+<FENCE><FR><NU>L</NU><DE><UP>IC</UP><SUB>50</SUB></DE></FR></FENCE><SUP>n</SUP></DE></FR>+C<SUB>1</SUB> (Eq. 2)
K<SUB>d</SUB>=<FR><NU><UP>IC</UP><SUB>50</SUB></NU><DE>1+<FR><NU>[&agr;<UP>Bgt</UP>]</NU><DE>K<SUB>d,&agr;<UP>Bgt</UP></SUB></DE></FR></DE></FR> (Eq. 3)
although other equations also have been described for that purpose (34). The Kd values shown in Table I are the average and standard error of at least three independent assays of ligand affinity unless otherwise noted. Uncertainties shown in figures are standard errors.

Sucrose Gradient Sedimentation-- Membranes from 10-20 oocytes that had been injected with the alpha 7M1, alpha 7EK, or full-length alpha 7 cRNA were extracted in buffer B. AChRs in membrane vesicles from T. californica and AChRs from the TE671 cell line (35) were solubilized in buffer B. About 200-µl aliquots of solubilized protein containing from 20 to 100 fmol of 125I-alpha Bgt binding sites were layered onto 5-ml sucrose gradients (5-20% (w/v)) in 0.5% Triton solution of 100 mM NaCl, 10 mM sodium phosphate, 5 mM EGTA, 5 mM EDTA, and 1 mM NaN3 at pH 7.5. The gradients were centrifuged 75 min at 70,000 rpm (approximately 340,000 × g) and 4 °C in a Beckman NVT90 rotor. For determining a ligand binding profile, aliquots of 11 drops each (approximately 130 µl) from the gradient were collected into Immulon 4 plastic microwells coated with mAb 142 for alpha 7M1, mAb 236 for alpha 7EK, or mAb 318 (a rat monoclonal antibody against an epitope in the cytoplasmic domain) (27) for the full-length alpha 7. The entire gradient was collected in 40 fractions. After 24 h at 4 °C, the microwells were washed and filled with from 4 to 12 nM 125I-alpha Bgt in buffer B for 6 h at 4 °C, followed by washing and quantitation of bound 125I-alpha Bgt by gamma  counting.

Processing of alpha 7WS was slightly more involved because of the low concentration of the secreted protein in the incubation medium. The protein was concentrated by binding overnight at 4 °C to alpha -toxin that had been isolated from the venom of Naja naja siamensis with ion exchange chromatography (36) and then coupled to Sepharose CL-4B with CNBr (32). The bound protein was eluted from the alpha -toxin with 200 µl of 100 mM nicotine and was layered onto 5-ml sucrose gradients (5-20%) without Triton in centrifuge tubes. The gradient was centrifuged 75 min at 70,000 rpm (approximately 340,000 × g) and 4 °C, as was done for alpha 7M1 and alpha 7EK. The entire gradient was collected in 39 fractions of 6 drops each, because the drop size was larger in the absence of detergent. After 24 h at 4 °C, the microwells were washed and filled with 1 nM 125I-alpha Bgt in buffer C for 6 h at 4 °C, followed by washing and quantitation of bound 125I-alpha Bgt by gamma  counting.

Comparison of AChR Yields-- The yield was defined as the theoretical maximal amount of bound 125I-alpha Bgt, which was the value of C0 in Equation 1 from 125I-alpha Bgt-binding assays. The AChR yield was calculated in terms of 125I-alpha Bgt binding sites per oocyte.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of alpha 7 ECD Proteins in Xenopus Oocytes-- The three variations of the ECD sequence of the alpha 7 subunit were studied (Fig. 1). The first construct, alpha 7M1, includes the N-terminal ECD, M1, and the portion of alpha 7 between M1 and M2 up to residue Ile240. It was intended to demonstrate the pharmacological properties and oligomerization behavior of the alpha 7 subunit proximal to M2 and tethered to the membrane through M1. The second construct, designated alpha 7EK, contains a peptide spacer of 27 amino acid residues that is spliced between Thr208 and Leu209 at the junction of the ECD and M1. alpha 7EK also contains M1 and terminates at Ile240, like alpha 7M1. The position of an RRR motif from 205 to 207 at the junction of the ECD with M1 suggests a structural role for these positively-charge residues; therefore, the native sequence Arg205 to Thr208 was repeated at the C-terminal end of the interposed segment before M1. The name "alpha 7EK" is derived from the DDDDKI sequence that was included in the peptide spacer for site-specific proteolysis by EK. alpha 7EK was intended to test the feasibility of removing M1 from the ECD by in vitro enzymatic proteolysis after synthesis. The third variation, alpha 7WS, was intended to be a direct route to a water-soluble alpha 7 ECD AChR. It is truncated at Thr208, just before M1 and contains no transmembrane domain. Epitopes for monoclonal antibodies mAb 142 and mAb 236 were included in the truncated constructs so that the proteins could be detected by immunoblotting and tethered through the antibodies to plastic wells for solid-phase ligand-binding assays (5).


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Fig. 1.   Designs of the three alpha 7 ECD subunit proteins alpha 7M1, alpha 7EK, and alpha 7WS. The ECD, which extends from residues 1 to 208 of the mature chicken alpha 7 AChR (27) is included in each protein. In alpha 7M1 and alpha 7EK, the first transmembrane domain, M1, extends from residues 209 to 233. The alpha 7 sequence from residues 234 to 240, which extends to the beginning of M2, is included after M1 in these two proteins. The segment labeled 142t on the C-terminal side of M1 is the epitope tag for mAb 142. Besides the mAb 142 epitope, alpha 7EK also contains an epitope tag for mAb 236, designated 236t, as well as an EK-specific protease site between the extracellular domain and M1. In contrast to the two membrane-tethered proteins, alpha 7WS does not contain M1; the mAb 142 epitope tag of the alpha 7WS protein follows directly after the ECD.

Immunoblotting of Triton X-100 extracts from oocytes injected with alpha 7M1 and alpha 7EK cRNA confirmed the expression of these proteins (Fig. 2). alpha 7M1 before deglycosylation migrated at an apparent mass of 37 kDa; alpha 7EK migrated at 40 kDa. These apparent masses were about 7 kDa higher than the values of 30 kDa for alpha 7M1 and 33.5 kDa for alpha 7EK that were calculated from amino acid compositions. The detection of predominately a single band before deglycosylation suggested that post-translational modifications were comparatively uniform on all molecules. Deglycosylation shifted each band to approximately the molecular mass of the protein calculated without modifications.


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Fig. 2.   Protein expression and glycosylation of alpha 7M1, alpha 7EK, and alpha 7WS demonstrated by immunoblotting with 125I-mAb 142. Deglycosylated protein is compared with control protein that was processed in parallel except without glycosidase. The presence (+) or absence (-) of glycosidase is noted above each lane. Each lane of alpha 7M1 and alpha 7EK contains the protein from about two oocytes; each lane of alpha 7WS contains the protein secreted from about 24 oocytes. Although alpha 7WS has the lowest molecular weight as calculated from amino acid sequences, it is the most heavily glycosylated and migrates at the largest apparent molecular weight. Triton extracts and incubation medium from uninjected oocytes were used as negative controls and showed no binding of 125I-mAb142. The positions of molecular mass markers are indicated on the left.

Without M1, alpha 7WS was secreted in soluble form into the incubation medium by oocytes that had been injected with alpha 7WS cRNA. The secreted protein displayed an apparent mass of about 41 kDa by immunoblotting, compared with a calculated mass of 26 kDa (Fig. 2). The detection of a diffuse pattern of bands before deglycosylation suggested more heterogeneous glycosylation than was present on alpha 7M1 and alpha 7EK. Deglycosylation shifted the apparent mass down to about 26 kDa, confirming that alpha 7WS had the most extensive glycosylation of the three proteins. The larger shift in apparent mass compared with alpha 7M1 and alpha 7EK also suggested that the pattern of glycosylation differed between membrane-tethered and soluble forms of the ECD. An additional observation from the immunoblot is that the yield of secreted alpha 7WS was about 10-fold less than the yields of alpha 7M1 and alpha 7EK from Triton-solubilized membrane fractions, based on a comparison of the numbers of injected oocytes needed to produce approximately equal signals on the immunoblot.

High Affinity Binding of 125I-alpha Bgt to alpha 7 ECD AChRs-- Because high affinity binding of alpha Bgt is a hallmark of the alpha 7 AChR, measurements of binding affinity with 125I-alpha Bgt were appropriate starting points for analyzing the functional and structural properties of the three alpha 7 ECD constructs. The equilibrium dissociation constant, Kd, of 125I-alpha Bgt was 1.6 nM for alpha 7M1 and 1.9 nM for alpha 7EK when measured in solid phase assays in which the Triton-solubilized proteins were bound in mAb 142-coated wells (Fig. 3 and Table I). These values compare favorably with the published Kd of 1.6 nM for 125I-alpha Bgt binding to Triton-solubilized full-length alpha 7 AChR and for alpha 7-containing AChR from chick brain (37). Binding of 125I-alpha Bgt was measured for alpha 7EK by also using the mAb 236 epitope tag to bind the protein to mAb 236-coated wells. The Kd was 2.3 nM, demonstrating that the mAb 236 epitope tag at the C-terminal end of the ECD did not interfere significantly with the function of the alpha Bgt binding site.


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Fig. 3.   Equilibrium binding of 125I-alpha Bgt to alpha 7M1 and alpha 7EK AChRs. The Triton-solubilized AChRs were bound to mAb 142-coated microwells. Assays were performed in 2% Triton X-100. The curves are the best fits of Equation 1 to the data. Each data point is the mean of duplicate determinations. Scatchard analysis of the data is shown in the insets. The Kd values are taken from Table I. Data from representative single experiments are shown.

                              
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Table I
Ligand affinities for full-length alpha 7 and alpha 7 ECD AChRs

The affinity of secreted alpha 7WS for 125I-alpha Bgt was measured first without Triton (Fig. 4 and Table I). The Kd value of 0.4 nM was about 4-fold smaller than the values for Triton-solubilized alpha 7M1, alpha 7EK, and full-length alpha 7. The presence of 2% Triton during the binding assay of alpha 7WS for 125I-alpha Bgt increased the value of Kd to 1.7 ± 0.1 nM (Fig. 4), which is in the range measured for the Triton-solubilized AChRs. Triton, however, did not significantly affect the total number of 125I-alpha Bgt binding sites. The ratio of the number of binding sites calculated from Equation 1 in the absence of Triton compared with the number of binding sites in the presence of Triton from otherwise identical pools of secreted alpha 7WS was 0.99 ± 0.13 (n = 2). Only the secreted fraction of alpha 7WS bound 125I-alpha Bgt to a measurable extent, despite the significant amount of alpha 7WS protein that was retained intracellularly. The total amount of alpha 7WS protein per oocyte that was detected in the cytoplasmic and the Triton-solubilized membrane fractions by immunoblotting was approximately equal to the amount of Triton-solubilized alpha 7M1 or alpha 7EK protein in similarly-injected oocytes. Hence all three constructs apparently were synthesized and accumulated to an approximately equal extent. The intracellular pool of alpha 7WS, however, showed no significant binding of 125I-alpha Bgt.


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Fig. 4.   Effect of Triton X-100 on the equilibrium binding of 125I-alpha Bgt to alpha 7WS. alpha 7WS was bound to mAb 142-coated microwells. The binding assays with 125I-alpha Bgt were performed without Triton X-100 (upper panel) or with 2% Triton X-100 (lower panel). The curves are the best fits of Equation 1 to the data. Each data point is the mean of duplicate determinations. Scatchard analysis of the data is shown in the insets. The Kd values are taken from Table I. In the absence of Triton, Kd was about 4-fold smaller than the values measured for alpha 7M1 and alpha 7EK. In the presence of Triton, however, the value for alpha 7WS shifted into the range for the M1-containing AChRs. Data from representative single experiments are shown.

The yield of 125I-alpha Bgt binding sites from each of the three ECD proteins was measured by the binding of 125I-alpha Bgt to AChR on mAb 142-coated wells (Fig. 5). These measurements assumed that the mAb 142 epitope in each protein was equally accessible to the antibody. The yield of 8 ± 1 fmol of 125I-alpha Bgt binding sites per oocyte with alpha 7M1 AChR was the largest among the three proteins. This value is near the value of 15 fmol per oocyte that was reported for Triton-solubilized AChR from the full-length alpha 7 subunit expressed in oocytes (29). Compared with alpha 7M1, the addition of the peptide spacer in alpha 7EK decreased the yield of AChR by about three-quarters to 1.9 ± 0.6 fmol of 125I-alpha Bgt binding sites per oocyte. The yield of secreted alpha 7WS AChR was the smallest of the three at 0.21 ± 0.02 fmol of 125I-alpha Bgt binding sites per oocyte. These comparisons indicate that insertion of residues before M1 or the absence of M1 decreases the yield of ECD AChRs.


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Fig. 5.   Comparison of yields of alpha 7 ECD AChRs from alpha 7M1, alpha 7EK, and alpha 7WS. The yield was defined as the number of 125I-alpha Bgt binding sites per oocyte. This value was the maximal amount of bound 125I-alpha Bgt that was estimated from solid phase binding assays on mAb 142-coated microwells using Equation 1 (calculated from the value of C0). At least three independent measurements were performed for each type of AChR. From 20 to 50 oocytes were pooled for the assays of alpha 7M1 and alpha 7EK. The incubation medium from 80 to 160 oocytes was the source of AChRs for the assays of alpha 7WS.

High affinity for 125I-alpha Bgt was the first step in demonstrating the structural similarity of alpha 7M1, alpha 7EK, and alpha 7WS AChRs to the full-length alpha 7 AChR. It was only an initial test of the properties that we hoped to find in a model AChR. Specifically, we were seeking a model that also was a pentamer and that had native-like affinity for agonists like nicotine and ACh. High affinity for alpha Bgt does not necessarily imply either of these properties. Muscle-type AChR illustrates this point. The human alpha 1 subunit in the absence of other muscle-type subunits binds alpha Bgt relatively tightly, with Kd of 0.6 nM compared with Kd of the pentameric muscle-type AChR of 0.1 nM (35). Instead of a pentamer, however, it exists as a monomer according to its velocity sedimentation profile relative to the profiles of fully assembled muscle-type AChR and alpha delta and alpha gamma dimers (38, 39). Moreover, it does not bind agonists with native-like affinity. In light of these characteristics for alpha 1, other experiments besides binding of 125I-alpha Bgt were required for assessing the oligomerization of the alpha 7 ECD constructs.

Velocity Sedimentation Implies alpha 7M1, alpha 7EK, and alpha 7WS Form Multimeric AChRs, Probably Pentamers-- Oligomerization of alpha 7M1, alpha 7EK, and alpha 7WS proteins was examined first with velocity sedimentation profiles in sucrose gradients. The proteins were detected by binding of 125I-alpha Bgt in solid-phase assays. These profiles were compared with full-length AChRs and monomeric alpha 1 subunits as sedimentation standards. Fully assembled, full-length alpha 7 AChR (calculated molecular mass of 272 kDa from amino acid sequence) were pentameric standards (37). Muscle-type AChR from T. californica were pentameric (i.e. a monomeric AChR with calculated molecular mass of 268 kDa) and decameric standards (i.e. a dimeric AChR with calculated molecular mass 536 kDa) (40). The monomeric alpha 1 subunit (calculated molecular mass of 52 kDa) from the human rhabdomyosarcoma cell line TE671 was the monomeric standard (35). As expected because of their much larger molecular masses, the pentameric AChR standards sedimented faster than monomeric alpha 1 subunit (Fig. 6).


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Fig. 6.   Velocity sedimentation profiles of alpha 7 ECD AChRs and full-length alpha 7 AChRs. The profiles of 125I-alpha Bgt binding to full-length alpha 7 AChR, alpha 7M1 AChR, and alpha 7EK AChR that were sedimented on a 5-20% sucrose gradient with 0.5% Triton X-100 are shown in the upper panel. Two of the arrows indicate the peaks of 125I-alpha Bgt binding to Torpedo AChR monomers (five subunits) and dimers (10 subunits) (40) that were observed in a parallel experiment. The peak of 125I-alpha Bgt binding to the human alpha 1 monomeric subunit from the TE671 cell line from a parallel experiment is indicated by a third arrow. These three proteins served as oligomerization standards for the alpha 7 ECD AChRs. The profiles for alpha 7M1 and alpha 7EK are inconsistent with monomers as the high affinity species but are consistent with pentamers. The lower panel shows the profile of the alpha 7WS AChR on a 5-20% sucrose gradient without Triton. The profile again is inconsistent with a monomer as the high affinity species but is consistent with a pentamer.

The single 125I-alpha Bgt binding peak from alpha 7M1 and from alpha 7EK also sedimented much faster than the monomeric alpha 1 subunit and slightly slower than the full-length AChRs (Fig. 6). Two conclusions can be drawn from these velocity sedimentation data in the absence of measurements of diffusion and partial specific volume. First, the position of the 125I-alpha Bgt binding peak was distant from the alpha 1 monomer standard, strongly suggesting that a multimeric species was responsible for the 125I-alpha Bgt binding. Second, this multimeric species probably was a pentamer. This conclusion about subunit stoichiometry was deduced from both the single peak and its relatively narrow distribution. The single peak implied that a multimer with a single distinct stoichiometry was the dominant species that bound 125I-alpha Bgt with nanomolar affinity. In contrast, if multiple stoichiometries such as dimers and trimers and tetramers had significant representation in the high affinity population, then one would have expected either several discrete peaks or a low, broad profile. Because of the pentameric structure of the full-length alpha 7 AChR (41), the most likely stoichiometry for alpha 7M1 and alpha 7EK AChRs also was pentameric, although minor fractions of smaller oligomers may also have been present. Moreover, it has been reported that only pentameric alpha 7 subunits bind 125I-alpha Bgt with nanomolar affinity, whereas monomeric full-length subunits and aggregated or incompletely assembled forms do not (37).

Velocity sedimentation of the secreted alpha 7WS in sucrose gradients also revealed a single 125I-alpha Bgt binding peak (Fig. 6). It sedimented slightly slower than the peaks for alpha 7M1 and alpha 7EK. As with alpha 7M1 and alpha 7EK, the position of the single, relatively narrow peak that was distant from the peak of the alpha 1 subunit did not suggest a monomeric form for alpha 7WS AChR and was consistent with the conclusion that the dominant form of alpha 7WS AChR was a pentamer.

High Affinity Binding of Nicotine and ACh to alpha 7 ECD AChRs-- High affinity binding for agonists was the second test of oligomerization and the last test of structural equivalence between the alpha 7 ECD AChRs and the full-length alpha 7 AChR. Interpretation of this test was based on an analogy with characteristics of the alpha 1 subunit of the muscle-type AChR. High affinity for agonists develops for alpha 1 only after it assembles with delta  or gamma  (39, 42), because the agonist binding site consists of structural components from both subunits at alpha delta and alpha gamma interfaces (3). Similarly, if alpha 7M1, alpha 7EK, and alpha 7WS AChRs indeed were pentamers and structural models of the full-length alpha 7 AChR, then we expected native-like affinities for nicotine and ACh.

The affinities of alpha 7M1 and alpha 7EK for the small ligands nicotine and ACh were measured by competitive inhibition of the binding of 125I-alpha Bgt (Fig. 7 and Table I). The equilibrium dissociation constants, Kd, of alpha 7M1 and alpha 7EK were 1.0 µM for nicotine and 50 µM for ACh. In the case of alpha 7EK, tethering in the solid phase assays via the mAb236 epitope tag slightly increased the values of Kd for nicotine and ACh to 1.4 µM and 60 µM, respectively. For both proteins, the Kd values are similar to those for Triton-solubilized, full-length alpha 7 and for alpha 7-containing AChRs from chick brain (Table I). Competitive binding with each ligand eliminated almost all binding of 125I-alpha Bgt, implying that the majority of the 125I-alpha Bgt binding sites also bound nicotine and ACh.


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Fig. 7.   Competitive inhibition by nicotine and ACh of 125I-alpha Bgt binding to Triton-solubilized alpha 7M1 and alpha 7EK AChRs. The AChRs first were bound to mAb 142-coated microwells. After washing, the microwells were loaded with 4 nM 125I-alpha Bgt in 2% Triton X-100 and varying concentrations of either nicotine or ACh. The curves are the best fits of Equation 2 to the data. Each data point is the mean of duplicate determinations. Kd values are taken from Table I. Similar inhibition assays were performed with alpha 7EK using mAb 236-coated microwells (Table I). Data from representative single experiments are shown.

In inhibition assays with 125I-alpha Bgt and alpha 7WS, the values of Kd for nicotine binding in the absence of Triton were 10-fold smaller than the values for nicotine binding to alpha 7M1 and alpha 7EK in the presence of Triton (Fig. 8 and Table I). The values for ACh binding to alpha 7WS without Triton were 40-fold smaller than the values for ACh binding to alpha 7M1 and alpha 7EK with Triton. The water-soluble alpha 7WS AChR without Triton, therefore, bound to small ligands more tightly than did the full-length alpha 7 AChR in Triton. The presence of 2% Triton in the inhibition assays increased the values of Kd by about 3-fold (Fig. 8). As with alpha 7M1 and alpha 7EK, the majority of the 125I-alpha Bgt binding sites of alpha 7WS AChR also bound nicotine and ACh, as shown by the elimination of almost all binding of 125I-alpha Bgt at high concentrations of agonists in either the presence or absence of Triton.


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Fig. 8.   Effect of Triton X-100 on the inhibition by nicotine and ACh of 125I-alpha Bgt binding to alpha 7WS AChR. The alpha 7WS AChR first was bound to mAb 142-coated microwells in the absence of Triton X-100. After washing, the microwells were loaded with a fixed concentration of 125I-alpha Bgt (0.6 nM with 0% Triton or 4 nM with 2% Triton) and with varying concentrations of either nicotine or ACh. The curves are the best fits of Equation 2 to the data. Each data point is the mean of duplicate determinations. Kd values are taken from Table I. Data from representative single experiments are shown.

In light of the velocity sedimentation data, these results confirm that the alpha 7M1, alpha 7EK, and alpha 7WS AChRs are oliogomers and probably are pentamers. These results demonstrate that the alpha 7 ECD with or without M1 is sufficient for the expression of a alpha 7 ECD AChR with native-like affinities for alpha Bgt and nicotinic agonists.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Structural Implications of the Ligand Binding Properties and Oligomerization of ECD AChRs-- We have shown that the N-terminal ECD of the alpha 7 AChR subunit with or without M1 forms an AChR whose ligand-binding and sedimentation properties imply that it is a structural model of the full-length AChR. In particular, the alpha 7WS AChR is water-soluble. The ECD, therefore, constitutes an autonomous unit capable of folding and assembling in the absence of the transmembrane, cytoplasmic, and other extracellular portions of the alpha 7 subunit sequence. We examined three characteristics that implied retention of the native-like structure in the three ECD AChRs: 1) high affinity binding of alpha Bgt, which is a hallmark of alpha 7 AChRs; 2) velocity sedimentation, which is most consistent with assembly of each of the three alpha 7 ECD subunits into pentamers; and 3) native-like affinities for nicotine and ACh.

First, the affinity for 125I-alpha Bgt of each of the three alpha 7 ECD AChRs was equal to or higher than that of the fully assembled, full-length alpha 7 AChR. Because the alpha Bgt binding site incorporates noncontiguous regions of primary sequence, these affinities suggest that the global structure of each alpha 7 ECD AChR models the structure of the ECD in the full-length alpha 7 AChR. The moderately higher affinity of the alpha 7WS AChR for 125I-alpha Bgt reverted back into the range of Kd values observed with alpha 7M1 and alpha 7EK AChRs when Triton was included in the assay. Triton could have exerted its effects by modifying the interaction of alpha Bgt with either the AChR or with the solvent environment, since both interactions can affect Kd. In other words, Triton may have caused structural perturbations of the AChR that affected the binding site, or 125I-alpha Bgt may have partitioned differently in Triton-containing solvent compared with detergent-free solvent.

Second, velocity sedimentation data suggested that the alpha 7 ECD subunits had assembled into multimers that probably were pentamers. Other AChRs or AChR subunits were used as oligomerization standards for estimating the extent of oligomerization of the alpha 7 ECD subunits. The single 125I-alpha Bgt binding peak for each of the ECD AChRs migrated on the sucrose gradient slightly slower than full-length, pentameric alpha 7 AChR and Torpedo AChR and much faster than the peak of monomeric alpha 1 subunit. Although differences in the amount of bound detergent (40) and in molecular shape (43, 44) will affect the sedimentation of AChRs, we do not think that these effects alone can account for the differences between the position of the monomeric subunit standard and the positions of alpha 7 ECD AChRs on the gradients. A reasonable interpretation of the position of the 125I-alpha Bgt binding peaks relative to the monomeric alpha 1 subunit peak was that these alpha 7 ECD subunits were not monomeric. Multimeric stoichiometry also is consistent with their native-like affinity for agonists. Given that full-length alpha 7 AChR are pentamers (41), the single, relatively narrow 125I-alpha Bgt binding peak from each alpha 7 ECD AChR probably arose from pentamers.

Third, affinities of the three alpha 7 ECD AChRs for nicotine and ACh also matched or were slightly higher than those of the full-length alpha 7 AChR. In the case of alpha 7WS AChR, Triton reduced the affinity of nicotine and ACh compared with that observed without Triton. The differences in affinity for agonists of alpha 7WS AChR compared with alpha 7M1 and alpha 7EK AChRs were not caused solely by Triton and may have been contributed in part by M1. Overall, these results imply that the local structure of the ACh binding site of each of the alpha 7 ECD AChRs closely matches that of the full-length alpha 7 AChR. By analogy with the behavior of the muscle-type AChR (39, 42), the binding site for small ligands is thought to form at interfaces between subunits. Therefore, native-like affinities for agonists by the alpha 7 ECD AChRs, combined with the results from velocity sedimentation, confirmed that the alpha 7 ECD subunits had assembled into multimers.

The alpha 7EK subunit demonstrated another structural property of the ECD AChRs. Splicing a peptide spacer of 27 amino acid residues between these two domains did not significantly alter ligand affinity. Therefore, strict continuity of the native primary amino acid sequence between the ECD and M1 is not required for formation of the agonist binding site or for oligomerization. In other words, an ECD that is displaced away from M1 by a peptide spacer retains its assembly and binding properties. This flexibility in design raises the possibility of first expressing a membrane-bound ECD AChR, followed by release of a water-soluble ECD AChR from M1 by enzymatic proteolysis in vitro.

Elimination of transmembrane domains either by recombinant techniques or by proteolysis of native protein has been the starting point for structural studies of other integral membrane proteins. In the family of ionotropic glutamate receptors, the soluble agonist binding domain of GluR-B and GluR-D was successfully designed by fusion of discontinuous sections of the primary sequence that apparently are separated by two transmembrane domains (45, 46). This strategy also has been successful for the x-ray crystallography of many integral membrane proteins including growth hormone receptor (47), prolactin receptor (48), tissue factor (49), interferon-gamma receptor (50), human class II histocompatibility antigen (51), insulin receptor protein-tyrosine kinase domain (52), alpha  and beta  chains of the T cell receptor (53, 54), neuraminidase (55), hemagglutinin (56), and bacterial aspartate receptor (57).

Yield of ECD AChRs-- The protein sequence between the beginning of M1 and the start of M2 (residues Leu209 to Ile240) was needed for production of AChRs from the ECD at a level comparable to that of the full-length AChR. M1 probably was the key component of this region. Compared with the yield of AChRs from alpha 7M1 AChR, the yield was smaller from alpha 7EK and even smaller from alpha 7WS. The differences were unlikely to be caused by reduced translation efficiencies of alpha 7EK and alpha 7WS, because of the extent of sequence identity among all three designs and because the amounts alpha 7EK and intracellular alpha 7WS proteins per oocyte that were detected by immunoblotting were approximately equal to the amount of alpha 7M1. Instead, the difference in the case of alpha 7EK AChR suggests some adverse effects on folding and assembly caused by the alteration of the primary sequence at the ECD/membrane interface.

The low yield in the case of alpha 7WS AChR highlighted an important role for M1. M1 has been shown to function as a tether to the ER membrane during the dimerization of truncated alpha 1 and delta subunits in the ER (23). It was successfully replaced in mouse alpha 1 by unrelated transmembrane domains with unrelated primary sequences or with a glycosylphosphatidylinositol (GPI) moiety (24). A similar role for M1 probably applies to the production of alpha 7 ECD AChRs. M1 constrains the subunits to a membrane surface, which may favor orientations between subunits that are required for assembly and may help retain them in the endoplasmic reticulum (ER) and Golgi apparatus for folding, subunit assembly, and post-translational modifications. In contrast, failure to retain the subunits on the ER and Golgi membranes removes such constraints and decreases the efficiency of these processes. In particular, M1 may enhance folding and assembly by increasing the local concentration of subunits relative to folding cofactors, such as calnexin, that are found in the ER membrane (58, 59).

Attaching the ECD to a membrane may not be the sole function of M1 for alpha 7M1 and alpha 7EK, however. Instead, the yield of AChRs may depend on particular properties of M1. Chimeras of alpha 7/alpha 3 (26) and point mutations in M1 of alpha 7 (60) demonstrate that the specific sequence used in the first transmembrane domain affects the yield of surface AChRs. Similarly, the total amount of bound 125I-alpha Bgt per oocyte varied when unrelated transmembrane domains replaced any one of the regions M1-M4 of alpha 1 in recombinantly expressed Torpedo AChRs (61).

Strategy for Structural Studies of alpha 7 ECD AChRs-- What is the optimum design of an alpha 7 ECD AChR for structural studies? The complete ECD of the alpha 7 subunit incorporating all of the amino acid sequence up to the start of M1 probably is necessary to satisfactorily reproduce the ligand-binding and assembly properties of the full-length alpha 7 AChR. Although we did not attempt to truncate the alpha 7 ECD N-terminal to Thr208, results from the mouse alpha 1 subunit suggest that there is little flexibility for moving the truncation point closer to the N terminus and into residues known to contribute to the alpha Bgt binding site (23). Truncation of mouse alpha 1 after Pro211, which is homologous with our truncation point in alpha 7WS, did not disrupt the formation of a high affinity binding site for alpha Bgt. In contrast, truncation of the mouse alpha 1 subunit after Met207 (mouse alpha 1 numbering) caused a loss of affinity for alpha Bgt.

More extreme truncations of the ECD of alpha 1 have been explored in attempts to bypass the difficulties presented by AChRs for structural studies. None, however, appears as successful in duplicating properties of full-length AChRs as the alpha 7 ECD AChRs described here. For example, peptide sequences from the region around residues 170-200 of the alpha 1 subunit have been used as potential mimics of the alpha Bgt binding site for NMR and ligand-binding studies (62, 63). The affinity for the binding of alpha Bgt, however, typically is three orders of magnitude less than with full-length AChRs (64). In addition, peptide models from the alpha 1 subunit are incapable of oligomerization and do not show significant affinity for small ligands.

Three conclusions from our investigation are relevant to a strategy for obtaining alpha 7 ECD AChRs for structural studies. First, the extracellular domain constitutes a stable, fully water-soluble AChR that quantitatively retains essential ligand-binding properties of the full-length AChR and eliminates confounding factors of detergent solubilization that hamper the crystallography of membrane proteins. Second, M1 in the alpha 7M1 and alpha 7EK constructs significantly enhances the yield of ECD AChRs compared with the yield from alpha 7WS. Although membrane tethering of the AChR subunit proteins was essential for association of the ECD of the mouse muscle-type alpha 1 subunit and the full-length delta  subunit when expressed in COS cells (24), it was advantageous but not essential for assembly of alpha 7WS AChR in oocytes. Third, the alpha 7 ECD still can form an AChR even when a peptide spacer separates it from M1 and the adjacent membrane surface.

These findings suggest that a water-soluble alpha 7 ECD AChR more likely will be produced in large amounts in two stages via a membrane-bound intermediate than directly from a water-soluble design of the ECD. The first stage is in vivo synthesis of a membrane-bound AChR that subsequently will be removed from its membrane tether by in vitro processing. The second stage is enzymatic removal of the membrane tether in vitro. alpha 7EK demonstrates that an AChR substrate can be designed for specific proteolysis within a peptide spacer between the ECD and M1. Our attempts to separate an ECD AChR from the M1 domain of alpha 7EK using EK, however, resulted in substantial nonspecific proteolysis. An alternative method that allows truncated alpha 1 subunits to dimerize with full-length delta  is substitution of M1 with a GPI moiety (24, 65). A GPI tether also leads to the expression of membrane-bound extracellular domains of chick alpha 7 on the surface of oocytes (66). A GPI tether may be preferred at the stage of enzymatic processing, because the carbohydrate portion of the anchor at the C terminus of the protein can be separated from the more distal, membrane-embedded fatty acid portion with phosphatidylinositol-specific phospholipase C (67, 68). This enzyme is expected to eliminate the risk of nonspecific enzymatic proteolysis.

In addition to alpha 7, we anticipate that ECD receptors of other members of the family of AChR subunits and other subunits of this entire superfamily of neurotransmitter-gated ion channels will mimic the structure and function of their respective full-length receptors. Demonstrations that ECD sequences are important in the assembly of both glycine receptors (69) and gamma -aminobutyric acidA receptors (70) suggest that the ECD of other members of the superfamily also will fold and assemble autonomously. The ultimately successful design of an ECD AChR for high level expression may require other design modifications. Moreover, structural aspects of the gating and ion permeation functions of transmembrane domains will have to be explored with different strategies. A water-soluble, recombinantly generated ECD AChR, however, appears to be a promising foundation for structural studies of this superfamily of ion channels.

    ACKNOWLEDGEMENTS

We thank Alexander Kuryatov for assistance with the use of alpha -toxin from N. n. siamensis and John C. Cooper and Lisa Burger for technical assistance.

    FOOTNOTES

* This work was supported in part by a grant from the Pittsburgh Supercomputing Center through National Institutes of Health National Center for Research Resources Cooperative Agreement 2 p41 RR06009.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.

§ Supported by National Institutes of Health Grant NS01903. To whom correspondence should be addressed: Dept. of Neuroscience, 235 Stemmler Hall, 36th and Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104-6074. Tel.: 215-573-2860; Fax: 215-573-2015; E-mail: wellsg{at}mail.med.upenn.edu.

par Supported by National Institutes of Health Grant NS33625.

Dagger Dagger Supported by National Institutes of Health Grant NS11323 and by grants from the Muscular Dystrophy Association, Smokeless Tobacco Research Council, and Council for Tobacco Research-USA.

1 The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; alpha Bgt, alpha -bungarotoxin; ECD, extracellular domain; EK, enterokinase; ER, endoplasmic reticulum; GPI, glycophosphatidylinositol; M1-4, transmembrane domains 1-4; WS, water-soluble; mAb, monoclonal antibody; PBS, phosphate-buffered saline.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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