Amino Acid Determinants of a 7 Nicotinic Acetylcholine Receptor Surface Expression*

Transient transfection has not been a successful method to express the a 7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. transfection in detectable surface receptor expression. Cell lines stably expressing the a 7 nicotinic acetylcholine receptor detectable, albeit variable, levels of surface receptor expression. a 7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of a 7–5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the a 7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.

The ␣7 nicotinic acetylcholine receptor (nAChR) 1 is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors that includes the nAChRs, the cation-permeable 5-hydroxytryptamine type 3 (5HT 3 ) receptor, and the inhibitory glycine and GABA A receptors. All members possess the conserved subunit topology of a large extracellular aminoterminal domain followed by four transmembrane domains, the third and fourth of which are separated by the cytoplasmic loop, a highly variable stretch of amino acids both in composition and length (for reviews, see Refs. [1][2][3]. ␣7 subunits are capable of forming homooligomeric receptors for which ␣-bungarotoxin is a specific and high affinity label (4,5). Upon activation, ␣7 nAChRs conduct a significant amount of Ca 2ϩ (P Ca 2ϩ /PNa ϭ 20) (5). Thus, depending on their anatomical location, ␣7 nAChRs may influence neurotransmitter release and synaptic integration. In the mammalian brain, small populations (compared with predictions from ␣-bungarotoxin binding density) of this receptor have been functionally located to presynaptic and postsynaptic sites by recording from individual neurons under conditions of rapid drug application and removal (6 -13).
Heterologous expression of the ␣7 nAChR would facilitate investigations of the structure-function relationships of this receptor; however, transient transfection of neuronal and nonneuronal cell lines with ␣7 cDNA results in a surface receptor expression level that is below the detection limit of an 125 I-␣bungarotoxin binding assay. The failure to detect receptors with this assay indicates that less than 1,000 ␣7 nAChR toxin binding sites are on the cell surface. It is possible to select for cell clones that express abundant ␣7 nAChRs. There are numerous examples of surface ␣-bungarotoxin binding sites on cell lines that stably express the ␣7 nAChR cDNA as well as on primary neuronal cultures (14 -22). The establishment of stably transfected cell lines, however, does not amend itself to mutational analysis of proteins in that rapid evaluation of mutated cDNA phenotypes is not feasible with this method. Recent work by Rakhilin et al. (23) indicates that cells that do express surface ␣7 nAChRs detectable with 125 I-␣-bungarotoxin binding express ␣7 subunits in two disulfide-bonded conformations, whereas cells that do not express surface ␣-bungarotoxin binding sites predominantly express ␣7 subunits in a single disulfide-bonded conformation. Apparently, the formation of ␣-bungarotoxin binding sites from ␣7 subunits requires cellular factors that are insufficient in many clones of a cell line and types of cells.
An ␣7-5HT 3 chimeric receptor is an example of a receptor that expresses substantial surface receptors in a transient transfection experiment (48). The ␣7-5HT 3 chimeric receptor forms from subunits composed of the extracellular ligand binding domain of the ␣7 nAChR and the four transmembrane domains and the cytoplasmic loop of the 5HT 3 receptor (5HT 3 R). The junction between the two subunit types occurs at a shared valine residue at position 201 (48), and the receptor is called the V201 chimeric receptor.
The ␣7 nAChR and the V201 chimeric receptor provide us with tools to study the molecular basis for the differences between the surface expression level of these two receptors. We tested the hypothesis that amino acid residues within the subunits of ligand-gated ion channels function as surface expression determinants by regulating the distribution of receptors between surface and intracellular pools. We constructed a series of ␣7-5HT 3 chimeric subunit cDNAs, expressed them in transfected cells, and measured the level of surface receptor expression with an [ 125 I]␣-bungarotoxin binding assay. This approach has identified amino acids that determine the number of ␣7-5HT 3 chimeric surface toxin-binding receptors without a concomitant change in total number of toxin-binding receptors produced by the cell. Analogous surface expression determinants were identified in the ␣7 subunit that increase ␣7 nAChR surface expression, as measured with ␣-bungarotoxin binding. We have identified an additional structure-function relationship based on the amino acid sequence of ligandgated ion channel subunits; the amino acid sequence of ligandgated ion channel subunits regulates the number of toxinbinding receptors on the cell surface.

EXPERIMENTAL PROCEDURES
Unless otherwise specified, all chemicals were purchased from Sigma. Cell culture reagents were purchased from Sigma and Life Technologies, Inc. Restriction enzymes were purchased from New England BioLabs (Beverly, MA) and Roche Molecular Biochemicals. Rat ␣7 nAChR cDNA was cloned as described (5). Mouse 5HT 3 A serotonergic receptor subunit cDNA was a gift from D. Julius (24). Purified preparations of Torpedo californica nAChR were a kind gift of S. Pedersen (Baylor College of Medicine, Houston, TX). Cell lines were obtained from ATCC (Manassas, VA). Xenopus laevis were purchased from Nasco (Fort Atkinson, WI).

Chimera Construction and Site-directed Mutagenesis
␣7-5HT 3 chimeric subunit cDNAs were constructed by a three-step polymerase chain reaction (PCR) protocol using Pfu polymerase (Stratagene, La Jolla, CA) as follows. Oligonucleotide primers were designed to construct the junction between ␣7 nAChR and 5HT 3 R subunit cDNA sequences. These primers were synthesized as forward (5Ј-3Ј) and reverse (3Ј-5Ј) oligonucleotides comprised of half ␣7 nAchR subunit sequence and half 5HT 3 R subunit sequence flanking the junction between the two subunit sequences (as designed for each chimeric subunit; Table I and Fig. 2). The ␣7 nAChR subunit sequence was amplified with the reverse primer of this type in conjunction with an ␣7 nAChRspecific forward primer (Table I). 5HT 3 R subunit sequence was amplified with the forward version of the chimeric primer in partnership with a 5HT 3 R subunit-specific reverse primer (Table I). The amplicons from these two PCRs were purified and placed into a third PCR in order to amplify the chimeric sequence between the ␣7 nAChR-specific and 5HT 3 R-specific oligonucleotides; the amplicons purified from the previous PCR served as template, and the forward ␣7 nAChR-specific and the reverse 5HT 3 R-specific oligonucleotides amplify the chimeric sequence. This is possible because the PCR products from the previous reaction contain overlapping sequence (generated during the first PCR) and therefore can anneal and prime for cDNA synthesis. The latest amplicon was digested with SacI and Bsu36I restriction enzymes and ligated with a similarly digested and dephosphorylated (U. S. Biochemical Corp.) ␣7-5HT 3 chimeric subunit cDNA in an eukaryotic expression vector, pcDNAI/Amp (Invitrogen, Carlsbad, CA).
A chimeric subunit cDNA was constructed in which the first transmembrane domain (transmembrane domain I, amino acid residues 201-235) of the ␣7 nAChR was replaced with homologous sequence from the 5HT 3 R. This was achieved using a three-step PCR similar to that described above.
Site-directed mutagenesis was carried out using PCR techniques. A three-step PCR protocol was utilized. Mirror image forward and reverse oligonucleotides were designed to contain a mutation of choice (Table  II). The forward mutagenizing primer was coupled with the abovementioned 5HT 3 R-specific reverse primer in a PCR using the appropriate ␣7-5HT 3 chimeric subunit cDNA as template. Similarly, the reverse mutagenizing primer was coupled with the ␣7 nAChR-specific oligonucleotide in a PCR with the same ␣7-5HT 3 chimeric template. Each of the mutant amplicons was purified and mixed in a PCR with the forward and reverse primers specific for the ␣7 nAChR and 5HT 3 R subunit cDNAs, respectively. The purified product from this amplification was digested with SacI and Bsu36I restriction enzymes and then ligated with similarly digested and dephosphorylated ␣7-5HT 3 chimeric subunit cDNA.
FLAG epitope-tagged 5HT 3 R (5HT 3 -FLAG) subunit cDNA in the pcDNAI/Neo (Invitrogen) was a gift from P. Seguela (McGill University, Montreal, Canada). This construct was described previously (5). The subunit cDNA was subcloned into pcDNAI/Amp at HindIII and XbaI restriction sites. A FLAG epitope-tagged V201 chimeric cDNA was generated by subcloning.
The sequence of all mutated cDNA constructs was verified by dideoxy sequencing (26) as performed by SeqWright (Houston, TX).

Cell Transfection
COS Cells-COS cells were transfected with receptor subunit cDNA by the DEAE-dextran method as described previously (27,28). Briefly, 1 ϫ 10 6 cells/150-mm culture dish were transfected with 5 g of the appropriate cDNA in 10 ml of Dulbecco's modified Eagle's medium containing 1% antibiotics, 1% fetal bovine serum, 100 M chloroquine disulfate, 0.04% DEAE-dextran. Following 4 h of incubation, cells were treated for 2 min with 10% Me 2 SO in PBS followed by transfer to complete medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% antibiotics). Twenty-four h following transfection, cells were trypsinized, counted, and plated to 24-well dishes coated with poly-L-lysine (Sigma, M r Ͼ 300,000) at 25,000 cells per well. Receptor expression was assayed 24 h later.
Neuroblastoma Cells-Adenoviral transfection was performed according to a modified version of Cristiano et al. (29). Briefly, complexed adenovirus, plasmid cDNA, and poly(L-lysine) in PBS were incubated for a total of 40 min at room temperature. The mixture was added to wells of a 48-well plate containing 25,000 SK-N-SH neuroblastoma cells per well in a total volume of 400 l of complete medium. After a 2-h incubation at 37°C, 5% CO 2, the transfection mixture was aspirated and replaced with complete medium. Cells were assayed 48 h later.

125
I-␣-Bungarotoxin Binding Assay-Surface receptors were assayed by binding 125 I-␣-bungarotoxin. Transfected cells were rinsed twice with PBS and then preincubated in PBS, 1% bovine serum albumin with (nonspecific binding samples, in triplicate) or without (total binding samples, in triplicate) 10 M cobratoxin for 30 min at room temperature. This incubation was followed by the addition of 125 I-␣-bungaro- toxin (NEN Life Science Products) to 5 nM and incubation for 1 h at room temperature. Cells were rinsed four times with 1 ml of PBS and dissolved in 0.5 ml of 0.1 N NaOH, and isotope was quantified by liquid scintillation counting. Toxin binding dose-response data were generated by performing 125 I-␣-bungarotoxin binding using concentrations of toxin ranging from 0.1 to 10 nM as described above. Total receptors were measured under the same conditions as above, except binding was performed in solubilization buffer (10 mM sodium phosphate, pH 7.4, 50 mM NaCl, 2 mM EDTA/EGTA, 1% Triton X-100), and toxin-bound cell lysates were rinsed over filters. GFC filters (Whatman) pretreated in 3% polyethylenimine, 10 mM sodium phosphate, pH 7.4, followed by 2% bovine serum albumin block in 10 mM sodium phosphate, pH 7.4, were used. Filters were rinsed four times with 4 ml of 10 mM sodium phosphate, pH 7.4, 50 mM NaCl, 1% bovine serum albumin. After drying, filters were treated with 1 ml of Soluene (Packard) for 30 min at 60°C and then liquid scintillation-counted in the presence of 7 ml of Ultima Gold (Packard).
FLAG Antibody Binding Assay-COS cells transfected with FLAG epitope-tagged V201 chimeric or 5HT 3 R subunit cDNA were rinsed twice with PBS and fixed in 4% paraformaldehyde, 4% sucrose in PBS for 10 min at room temperature. Cells were rinsed twice in PBS and blocked in 6% casein in PBS for 30 min at 37°C. Mouse anti-FLAG antibody (Research Diagnostics Inc., Flanders, NJ) was diluted to 10 g/ml in blocking solution and incubated with cells for 2 h at 37°C. Cells were rinsed three times with PBS. Biotinylated anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA) was diluted to 5 g/ml in blocking solution and incubated with cells at 37°C for 30 min. Cells were rinsed three times in PBS. Cells were then incubated with 0.63 pM 125 I-Streptavidin (Amersham Pharmacia Biotech) for 1 h at room temperature. After rinsing three times with PBS, cells were solubilized in 0.1 N NaOH and liquid scintillation-counted. Nonspecific binding was determined as above, except the primary antibody was omitted.
Receptor Purification and Quantitative Immunoblot-␣-Bungarotoxin binding proteins were purified using ␣-cobratoxin affinity resin according to the method of Kemp et al. (30). Briefly, transfected COS cells were rinsed with PBS and then harvested by scraping in PBS containing Complete protease inhibitor (Roche Molecular Biochemicals). Cells were pelleted by centrifugation and then resuspended in solubilization buffer containing Complete protease inhibitor. Cell lysate was rocked at 4°C for 1 h and then centrifuged for 1 h at 100,000 ϫ g. The supernatant was collected and mixed overnight at 4°C with agarose-conjugated cobratoxin (Sigma) equilibrated with solubilization buffer. The cobratoxin resin was rinsed four times with solubilization buffer, and toxin receptors were eluted with SDS-polyacrylamide gel electrophoresis sample buffer (300 mM Tris-Cl, pH 6.8, 30% glycerol, 8% SDS, 1 M urea, 1 M dithiothreitol, 1% ␤-mercaptoethanol).
Samples were resolved with SDS-polyacrylamide gel electrophoresis (4% stacking, 8% separating gels) transferred to Immobilon membrane (Millipore, Bedford, MA), and immunodetected with antibodies specific for the extracellular domain of the ␣7 nAChR subunit (31,32). All antibodies were diluted in PBS, 0.1% Tween 20, and 5% nonfat dry milk. Blots were rinsed in PBS and 0.1% Tween 20. One to 200 ng of bacterial recombinant protein encoding the ␣7 nAChR extracellular domain was applied to each gel in order to generate a standard curve and quantify toxin-purified protein. Rainbow molecular weight markers (Amersham Pharmacia Biotech) were loaded to each gel to estimate the molecular weight of protein samples. Immunoreactivity was visualized with peroxidase-conjugated secondary antibody (Cappel Organon Teknika, West Chester, PA) followed by ECL reagent (Amersham Phar-macia Biotech) and film exposure (Eastman Kodak Co.). Purified receptor bands on exposed film were quantified with densitometry analysis (NIH Image; National Institutes of Health) in comparison with an ␣7 nAChR recombinant protein standard curve.

Sucrose Gradient Fractionation
Lysates of approximately 1 ϫ 10 7 cells were prepared as above, preincubated with 20 nM 125 I-␣-bungarotoxin, and applied to a 5-20% linear sucrose gradient prepared in 10 mM sodium phosphate, pH 7.4, 50 mM NaCl, 1% Triton X-100. Gradient and loaded sample were sedimented at 60,000 rpm (SW 60; Beckman Corp., Palo Alto, CA), 4.5 h at 4°C. An aliquot of purified T. californica nAChR preincubated with 20 nM 125 I-␣-bungarotoxin was run in parallel as a standard for toxinbound pentameric and dipentameric receptor. Fractions were collected from the bottom of the centrifuge tube. Odd numbered fractions were assayed for radioactivity by liquid scintillation counting, and even numbered fractions were resolved with SDS-polyacrylamide gel electrophoresis and immunodetected as described.

Oocyte Expression and Electrophysiological Recordings
Oocytes were prepared, injected, and recorded according to Chen and Patrick (28). Currents induced by 3 s of agonist application (300 M nicotine) were recorded. The V201 chimera served as a control, and the oocytes injected with the V201 chimera subunit cDNA had currents of 300 nA or greater when agonist was applied. Recorded currents were filtered at 300 Hz using an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Data were acquired and analyzed using Axodata, Axograph (Axon Instruments), and Igor (Wave Mechanics) software on an Apple Macintosh IIci computer.

RESULTS
For much of the data to be described, 125 I-␣-bungarotoxin binding was utilized to quantify receptor expression level. Because the ligand binding domain of the chimeric receptor is contributed by the ␣7 subunit and ␣7 nAChRs and V201 chimeric receptors exhibit equivalent affinity for ␣-bungarotoxin (33), iodinated ␣-bungarotoxin is a quantitative label for both ␣7 nAChRs and ␣7-5HT 3 chimeric receptors. Thus, the term "receptor" is defined as an ␣-bungarotoxin binding site and "expression level" refers to the number of toxin binding sites produced by a cell.
Surface Receptor Expression Level Is Low for ␣7 nAChR and Is High for 5HT 3 R and V201 Chimeric Receptors-We evaluated the surface expression levels of the V201 chimeric receptor, the 5HT 3 R, and the ␣7 nAChR. COS cells were transfected with ␣7 nAChR or V201 chimeric subunit cDNAs, and surface receptor expression was quantified 2 days post-transfection. ␣7 nAChR surface expression is below the detection limit of a 125 I-␣-bungarotoxin binding assay (Fig. 1A). In contrast, cells transfected with the V201 chimeric subunit cDNA express tens of thousands of surface toxin binding sites (Fig. 1A). These results demonstrate again that the V201 ␣7-5HT 3 chimeric receptor expresses many more toxin binding sites on the cell surface than the ␣7 nAChR.
We then determined whether the high surface expression level of the V201 chimera reflects a property of wild type 5HT 3 R's or is unique to the chimera. We compared the surface expression level of wild type 5HT 3 R with that of the V201 chimeric receptor. The 3Ј-end of each subunit cDNA was tagged with a sequence encoding the FLAG peptide, and surface receptor expression was measured on transfected cells with a FLAG antibody-125 I-streptavidin binding assay. Surface receptor expression levels were equivalent for the 5HT 3 R and the V201 chimeric receptor, indicating that the surface expression phenotype of the V201 chimera is conferred by 5HT 3 sequence (Fig. 1B). Based on the subunit sequence of the V201 chimera, 5HT 3 R surface expression determinants are contained within amino acids 201-449 of the subunit.
The Difference between V201 Chimeric Receptor and ␣7 nAChR Surface Expression Levels Involves a Postreceptor Assembly Mechanism-We measured total ␣7 nAChR expression and total V201 chimeric receptor expression to determine whether the number of surface receptors is simply a result of higher levels of expression of the V201 chimeric receptor. Cells expressing the V201 receptor express approximately 3.5 times more toxin binding sites than cells expressing the ␣7 nAChR. 65,000 Ϯ 15,000 toxin binding sites are detected in extracts of cells transfected with ␣7 cDNA (n ϭ 30), while extracts of cells expressing the V201 chimeric receptor contain 224,000 Ϯ 23,000 toxin binding sites per cell (n ϭ 41; Fig. 1C). Less than 1% of ␣7 nAChRs expressed are on the cell surface, whereas 30% of V201 chimeric receptors are surface receptors (Fig. 1D). This 30-fold difference demonstrates that the number of surface toxin binding sites produced by the ␣7 nAChR and the V201 chimeric receptor does not simply result from greater V201 chimeric receptor production. These results indicate that the mechanism underlying the different surface expression levels of these two receptors lies downstream of receptor assembly and processing events that produce a toxin binding site. We propose that the differential surface expression of the two receptor types is determined by the amino acid sequence of their subunits. To state it another way, sequence differences carboxyl-terminal to residue 201 between ␣7 and V201 chimeric subunits underlie the surface expression differences observed for these receptors.
Mutational Analysis of ␣7-5HT 3 Chimeric Receptors Reveals Five Surface Expression Determinants-We tested the idea that subunit sequence determines the level of surface receptor expression by identifying amino acids in ␣7-5HT 3 chimeric receptor subunits that act as surface expression determinants. Initially we mapped the 5HT 3 portion of the V201 chimera necessary for surface receptor expression. Chimeric subunit cDNAs were engineered in which the junction between ␣7 and 5HT 3 sequence moved progressively toward the 3Ј-end of the cDNA. Based on secondary structure analysis (34), the junction between ␣7 and 5HT 3 amino acid sequence for each of these chimeric subunits was positioned at amino acids 235, 241, 267, and 271 ( Fig. 2A). Each cDNA construct was expressed in COS cells, and surface receptor expression was tested by a 125 I-␣bungarotoxin binding assay. Each of the engineered chimeric cDNAs failed to express surface toxin binding sites on transfected COS cells. Receptor protein was produced, since immunoblot of these cell lysates with anti-␣7 antibodies detected subunit protein, 2 indicating that ␣7-5HT 3 chimeric receptor There is no significant difference p Ͼ 0.05 between the two receptors' surface expression level according to two-tailed Student's t test. C, number of toxin binding sites (mean Ϯ S.E.) expressed in lysates of COS cells transfected with ␣7 nAChR or V201 chimeric receptor subunit cDNAs. D, percentage of the total number of toxin binding sites Ϯ S.E. on the cell surface. surface expression requires part or all of 5HT 3 amino acids 201-235. We searched for surface expression determinants in the region 201-235 by constructing cDNA subunits in which the junction between ␣7 and 5HT 3 sequence occurs at the amino acids shown in Fig. 2B. Expression of cDNAs encoding these new chimeras in COS cells and subsequent surface 125 I-␣-bungarotoxin binding revealed surface expression determinant domains that we termed domains I and II.
Surface expression determinant domain I lies within the amino acid sequence 201-208. Surface receptor expression is maintained but reduced in the L208 chimera as compared with the V201 chimera (Fig. 2B). No additional surface expression determinants are located within the amino acid sequence 208 -216 because surface receptor expression levels are maintained from the L208 to I216 chimeric receptors. Surface expression determinant domain II is located within the amino acid sequence 216 -219. Surface expression is lost when the V219 chimera is expressed by COS cells.
The V219 chimeric receptor surface expression phenotype is not the result of a failure in receptor assembly. Sucrose gradi-ent fractionation of lysates from cells transfected with V219 chimeric subunit cDNA demonstrates that pentameric receptors form (Fig. 7, and see below). Therefore, amino acid regions 201-208 and 216 -219 of these subunits contain one or more amino acid residues that determine the number of chimeric receptors on the cell surface.
An alignment of ␣7 and 5HT 3 amino acids 201-235 identifies 5 amino acids that are nonconserved within domains I and II (Fig. 3). We tested these amino acids as candidates for surface expression determinants by point-mutating each within V201 and V219 chimeric receptor subunits. The V201 chimera was chosen because surface expression determinant domains I and II are encoded by 5HT 3 sequence, and in transfected cells, this subunit cDNA expresses high numbers of surface receptors. Conversely, the V219 chimera was used because surface expression determinant domains I and II are encoded by ␣7 sequence and, when expressed in transfected cells, do not express detectable surface toxin binding sites. The predictions for these experiments are as follows: 1) point mutation of a surface expression determinant from a 5HT 3 amino acid to an ␣7 amino FIG. 2. Schematic of chimeric receptor subunits used to map surface expression determinant domains I and II. ␣7-5HT 3 chimeric receptor subunit cDNAs were engineered to encode chimeric subunit proteins as illustrated. Open bars, ␣7 sequence; stippled bars, 5HT 3 sequence. Each chimeric receptor is named according to the amino acid (single letter code) and the position in ␣7 sequence at which the chimera junction occurs (arrows). Surface receptor expression was evaluated as described under "Experimental Procedures." n Ն 6 for each construct tested. TMD, transmembrane domain. A, ␣7-5HT 3 chimeric subunits identify amino acid region 201-235 from 5HT 3 R as necessary for chimeric receptor surface expression. B, measurement of the number of surface toxin binding sites for ␣7-5HT 3 chimeric receptor subunits within amino acid region 201-235 reveal two surface receptor expression determinant domains. *, significance (p Ͻ 0.001) comparing the L208, L212, and I216 chimeras with the V201, V219, A223, and L231 chimeras (Tukey multiple comparisons test). acid will decrease surface expression of the V201 chimera, and 2) mutation of an ␣7 residue to a 5HT 3 residue at a surface expression determinant position will increase V219 chimera surface expression.
Surface expression determinants were identified at two positions in the V201 chimera and at five positions in the V219 chimera. The P207T and S218C mutant V201 chimeric receptors expressed 32 and 68% fewer surface receptors, respectively (Fig. 4A). I202T, M203I, and V219I mutated V201 chimeric receptor surface expression was equivalent to the parent V201 chimera (Fig. 4A). V219 chimeric receptor surface expression was increased by any one of the five point mutations introduced. Surface toxin binding sites were detected for the T202I, M203I, T207P, C218S, and V219I mutated V219 chimeric receptor point mutants (Fig. 4B).
We find that mutation at amino acids amino-terminal to and within the first transmembrane domain of the subunit have a profound effect on ␣7-5HT 3 chimeric receptor expression. Five surface expression determinants have been identified for the V219 chimeric receptor, two of which have a significant influence on V201 chimeric receptor expression as well. Mutation of a surface expression determinant from an ␣7 amino acid to a 5HT 3 amino acid increases chimeric receptor expression, whereas mutation of an expression determinant from a 5HT 3 residue to an ␣7 residue decreases chimeric receptor expression.
Mutation of Surface Expression Determinants Affects Chimeric Receptor Distribution-We have shown that the surface expression phenotypes of the ␣7 nAChR and the V201 chimeric receptor are not the result of a mass action mechanism and are likely to have their effects downstream of receptor processing events that produce a toxin binding site. We hypothesized that the observed surface expression levels are determined by the amino acid sequence of each receptor's subunits. We have identified five surface expression determinants that significantly alter the surface expression level of ␣7-5HT 3 chimeric receptors. Next, we tested if surface expression determinants defined for ␣7-5HT 3 chimeric receptors function through a mass action mechanism by quantifying the total number of receptors produced by each chimeric subunit cDNA. We performed 125 I-␣-bungarotoxin binding to lysates of transfected cells. Our results show that the total number of toxin binding sites per cell is equivalent for each set of chimeric receptors (Table III). Therefore, changes in surface expression level are due to receptor redistribution between intracellular and surface pools, and the different surface expression levels measured for the chimeras are most likely achieved through a receptor transport mechanism.
The V201 chimeric receptor and the point-mutated forms of this receptor produce approximately 200,000 toxin binding sites per cell. Although the V219 chimeric receptor produces far fewer toxin binding sites per cell, the original and point-mutated forms of this receptor are produced at equivalent levels (Table III). Since the total number of receptors per cell is equivalent for each class of chimeric receptor, one can express the number of surface receptors as a percentage of the total number of toxin binding sites measured (Table III). This quotient describes the extent to which surface receptor expression is affected by mutation of an expression determinant; insertion of a 5HT 3 amino acid increases surface expression and insertion of an ␣7 amino acid decreases surface expression.
Expression Determinants Control ␣7 nAChR Surface Expression-Are the determinants identified for ␣7-5HT 3 chimeric receptors relevant to native receptors? Wild type ␣7 nAChR subunit cDNA was mutated to a 5HT 3 amino acid at each of the five nonconserved positions within surface expression determinant domains I and II, and surface receptor expression was measured as before. In four out of five cases, surface expression of mutated ␣7 nAChR subunit cDNA increased to levels detectable in a toxin binding assay. On transfected COS cells, ␣7 T202I, M203I, C218S, and V219I expressed 1,000 -3,000 toxin binding sites per cell (Fig. 5A), an expression level that is just above the detection limit of the assay. These residues determine surface expression of wild type ␣7 nAChRs.
Modest but reproducible and measurable increases in ␣7 nAChR surface expression can be achieved with point mutations at four surface expression determinant positions in and near the first transmembrane domain. This level of receptor expression is much lower than that of the V201 chimera. One possibility is that more than one ␣7 nAChR surface expression determinant must be mutated to a 5HT 3 amino acid in order to achieve a V201 chimeric receptor-like (or 5HT 3 R-like) surface expression phenotype. A second possibility is that additional surface expression determinants lie elsewhere in the 5HT 3 subunit. These two possibilities were tested by evaluating the surface expression level of a chimeric receptor formed from a subunit in which residues 201-235 (containing surface expression determinant domains I and II) of ␣7 were replaced with homologous sequence from the 5HT 3 R. This receptor expresses 2,381 Ϯ 684 surface toxin binding sites per cell, an expression level comparable with the point-mutated ␣7 nAChRs (Fig. 5B). Therefore, the surface expression level of the V201 chimeric receptor (and wild type 5HT 3 R) is probably due to a cooperative interaction between the expression determinants identified and additional determinants that lie elsewhere in the subunit.
Surface Expression Level Is an Intrinsic Property of the Receptor-We tested whether the surface expression determinants identified for ␣7-5HT3 chimeric receptors are dependent on the type of cell used for receptor expression. A comparison was made of the surface expression level of V201 receptor, V219 receptor, and point mutants generated from them between COS cells and SK-N-SH neuroblastoma cells. Surface ␣-bungarotoxin binding sites have been reported for these cells (35). We, however, did not detect any surface receptors on the mock-transfected cells used for these experiments. In all cases, surface receptor expression was lower on SK-N-SH cells than on COS cells (Fig. 6A). When the data are plotted as a percentage of V201 surface expression, the relative level of expression of each receptor is equivalent between COS and SK-N-SH cells (Fig. 6B). The surface expression level of wild-type and ␣7 point mutants expressed on SK-N-SH cells were also tested. As was the case for the chimeric receptors, receptor surface expression level was reduced compared with COS cells. 2 These results demonstrate that the surface expression phenotype exhibited by these receptors is a structure/function property of the receptor.
Chimeric Receptor Toxin Binding Unchanged by Point Mutations-Interpretation of the surface binding data would be confounded if the mutations introduced into V201 and V219 chimeric receptors alter the apparent binding affinity of chimeric receptor for toxin. Toxin binding to COS cells transfected with V201 chimeric subunit cDNA and the point mutants generated from this construct was performed in order to estimate the apparent affinity of each mutant for ␣-bungarotoxin. Surface toxin binding was performed with concentrations of radiolabeled ␣-bungarotoxin that ranged from 0.1 to 10 nM. Normalized specific toxin binding was plotted against the log dose of toxin, and the apparent K D and Hill coefficient values were determined with Scatchard analysis (Table IV). No significant differences were found for these parameters compared with the V201 chimeric receptor. These results show that mutations at surface expression determinant positions within the receptor subunit do not affect the apparent affinity of these receptors for toxin, only the number of surface receptors capable of binding toxin.
Oligomerized Chimeric Receptor Subunits Bind Toxin-The fact that V219 chimeric receptor expression is not detected with a surface toxin binding assay although receptors are detected in cell lysates raises the issue of whether the V219 chimera is binding toxin as a monomer or an oligomerized receptor. Either V219 chimeric subunits do not associate properly to form receptor, thus preventing transport to the cell surface, or the V219 chimeric receptor, properly folded and assembled, is not transported to the surface. We distinguished between these two possibilities by performing sucrose gradient fractionation of lysates from COS cells transfected with V201 or V219 chimeric receptor subunit cDNA. Thirty fractions (15 drops/fraction) were collected from each gradient, and the radioactivity in each fraction was counted. Purified T. californica nAChR is used as a size marker and runs as two peaks corresponding to radiolabeled ␣-bungarotoxin-bound to monopentamer (ϳ9 S, fraction 13) and dipentamer (13 S, fraction 6) forms of the receptor. Both V201 and V219 chimeric receptors resolved as single peaks in the gradient three fractions up the gradient from the ϳ9 S peak of the T. californica nAChR (Fig. 7). In replicates of the experiment, the V201 peak was observed in 1-3 fractions up the gradient, indicating that the chimeric receptors have a lower S value than the T. californica nAChR. The small hump  in the V201 peak was not observed in replicates of the experiment and is attributed to experimental variability. Although heterooligomeric non-neuronal forms of the nAChR exhibit assembly intermediates of monomer, dimer, trimer, and tetramer complexes (35,36,37), such assembly intermediates of ␣7 and ␣7-5HT 3 chimeric receptors have not been shown to bind ␣-bungarotoxin (23,47). In these studies, subunit protein of the M r (Fig. 7, arrowheads) that represents the toxin-binding species was not detected in gradient fractions on either side of the peak fractions, further supporting the notion that only oligomerized chimeric subunits bind ␣-bungarotoxin and that the toxin binding assay measures assembled receptors. Expression Level of Toxin Binding Sites Correlates with Expression Level of Receptor Protein-We tested whether the toxin binding assay directly measures the number of receptors produced. There are two possible mechanisms to account for the observation that the number of toxin binding sites in lysates of transfected cells is conserved among V201 mutant receptors: 1) cells transfected with V201 chimeric and mutated V201 chimeric cDNAs express equivalent numbers of receptor that bind a fixed number of toxin molecules, or 2) receptor expression level is nonequivalent between cells transfected with V201 chimeric and mutated V201 chimeric cDNAs, and the number of toxin molecules bound by each population of mutant V201 chimeric receptor is different.
Quantitative purification of receptors with a toxin-agarose resin allows us to determine if an equivalent amount of receptor protein is present in each receptor population. If this is the case, it will confirm that surface expression determinants affect surface receptor number through redistribution of receptors between surface and intracellular pools. The V201 chimera and the point-mutated forms of this receptor were toxin-purified and analyzed by quantitative immunoblot (as described under "Experimental Procedures"). Densitometric measurement of the band in the eluate sample indicates that an equivalent amount of V201, V201 I202T, and V201 P207T receptors capable of binding toxin are produced in a transfection experiment (Fig. 8). The three ␣7-5HT 3 chimeric receptors tested exhibit different surface expression phenotypes, yet the total number of receptors capable of binding toxin is equivalent, as is the protein comprising the toxin binding sites.
Toxin-purified receptor protein is approximately 5% of the total. It should be noted that equivalent amounts of receptor protein were immunodetected in each sample's starting material. It should also be noted that the toxin binding species for these receptors is of a higher M r than the majority of immunoreactive protein in the starting material. The smaller M r species in the starting material represents truncated or degraded subunits that do not bind toxin, since these bands are not present in the toxin-purified material.
Purification of V219 and V219 C218S chimeric receptors with this method yielded visually equal amounts of receptor protein (n ϭ 3). 2 Densitometry quantification was not performed on these samples, because the band intensities on the immunoblot films were below the linear detection range of emulsified film. These observations are in agreement with the finding that the total number of ␣-bungarotoxin binding sites measured in transfected cell lysates is equivalent for each receptor class and supports the hypothesis that surface expression determinants affect receptor transport.
When V201 S218C chimeric receptors were purified with toxin-coupled agarose, much less receptor protein was obtained. The toxin purification results obtained for the V201 S218C chimeric receptor indicate that much less receptor is produced when this mutant chimera is expressed (Fig. 8). These data are inconsistent with the toxin binding data for this receptor (which is equivalent to the V201 chimeric receptor and other mutants; see Table III) and the purification results obtained with the V201, V201 I202T, and V201 P207T chimeric receptors. An alternative explanation is that under extended incubation times (1 h at 24°C versus 5 or 16 h at 4°C), interaction between the V201 S218C chimeric receptor and toxin is less stable than its counterparts.
Functional Expression of Wild-type ␣7 nACh and ␣7-5HT 3 Chimeric Receptors-Xenopus oocyte expression of V201 chimeric and ␣7 nAChRs served as controls for ion-conducting functional screening of chimeric and point mutant subunit cDNAs used in this study (Table V). Of the subunit cDNAs tested, V201 I202T, ␣7 T202I, P207T, C218S, and V219 C218S mutants formed functional receptors in this expression system. Of the mutant cDNAs that did express functional receptors in Xenopus oocytes, the peak current amplitudes for the ␣7 T202I, FIG. 5. Surface expression determinants function in wild-type ␣7 nAChR. A, point mutation of wild-type ␣7 to 5HT 3 amino acids results in detectable surface toxin binding. The mean number of surface binding sites per cell Ϯ S.E. for wild-type ␣7 and the T202I, M203I, T207P, C218S, and V219I ␣7 point mutants were as follows: Ϫ377 Ϯ 445, 4,630 Ϯ 862, 3,506 Ϯ 785, 103 Ϯ 669, 2,731 Ϯ 1,399, and 5,299 Ϯ 3,457, respectively. *, significance (p Ͼ 0.05) as compared with wild-type ␣7 (two-tailed Student's t test). n Ն 6 for all cDNAs tested. B, replacement of surface expression determinant domains I and II in wild-type ␣7 subunit with 5HT 3 R subunit sequence results in surface receptor expression. Replacement of the wild-type ␣7 subunit sequence 201-235 with homologous sequence from the 5HT 3 R subunit results in increased surface receptor expression but not greater than observed for single point mutations. *, significance (p Ͻ 0.001) as compared with wild-type ␣7 (two-tailed Student's t test). n ϭ 12.
T207P, and C218S mutants were significantly different from control (Table V). Functional receptors were detected for the V219 C218S mutant, whereas the V219 chimera did not exhibit function (Table V). At the nicotine concentration used, receptor function is not significantly altered by the V201 I202T mutation.

DISCUSSION
Cells express many more surface chimeric ␣7-5HT 3 receptors than ␣7 nAChRs without a concomitant increase in total receptor number. Therefore, changes in surface receptor number are not due to a mass action effect of increased total recep-tor expression. These observations indicate that the amino acid sequence of receptor subunits determines surface receptor expression level. To test this idea, we constructed a series of ␣7-5HT 3 chimeric receptor subunit cDNAs, expressed them in cells, and quantified receptor expression with an 125 I-␣-bungarotoxin binding assay. Toxin binding is a quantitative measurement of receptor expression level, since toxin binds to ␣7 nAChRs and ␣7-5HT 3 chimeric receptors with equivalent affinity (33). For clarity in this discussion, the term "receptor" is defined as an entity that binds ␣-bungarotoxin, and "expression level" refers to the number of toxin binding sites produced by a cell. Our studies identified amino acid positions within receptor subunits that affect surface receptor expression through a mechanism that redistributes receptors between surface and intracellular pools. We conclude this because, following point mutation of receptor subunits, we observe changes in surface receptor number without observing changes in total receptor number. Receptor number was demonstrated to be equivalent for each class of receptor studied through toxin binding assay and quantitative receptor purification. Furthermore, only assembled receptors bind toxin, and the mutations introduced into these receptors did not affect apparent receptor affinity for toxin binding. These surface receptor expression  Fig. 1. B, when the data are normalized to the surface expression level of the V201 chimera in each cell type, the relative expression is not significantly different between the two cell types (p Ͼ 0.05); two-tailed Student's t test. determinants are an intrinsic property of the protein because they function independently of the cell type and transfection method used for receptor expression. Mutation of surface expression determinants in the ␣7 nAChR increases ␣7 surface expression; therefore, we propose that this also occurs through a redistribution of ␣7 nAChRs between intracellular and surface pools. We have identified amino acid substitutions that ameliorate the very low surface expression level of the ␣7 nAChR.
The surface expression phenotype of the ␣7-5HT 3 V201 chimeric receptor is conferred by 5HT 3 R subunit sequence because the V201 chimera and the 5HT 3 R exhibit equivalent surface expression levels. Therefore, the expression determinants defined for V201 also apply to the 5HT 3 R. We propose that one mechanism regulating the surface expression level of ligandgated ion channels in general, and ␣7 nACh and 5HT 3 receptors specifically, is dependent on the amino acid sequence of their subunits.
Cell-dependent and Cell-independent Factors Govern Surface Receptor Expression-In vitro expression studies with the ␣7 nAChR indicate that surface expression of the ␣7 nAChR depends, in part, on cell-dependent factors. Transient transfection of cells with ␣7 nAChR subunit cDNA does not produce detectable surface ␣-bungarotoxin binding sites despite intracellular receptor being present. There are numerous examples of surface ␣-bungarotoxin binding sites on primary neuronal cultures as well as on cell lines that stably express the ␣7 nAChR cDNA (14 -22) in addition to Xenopus oocyte expression of ␣7 nAChRs, which results in sufficient numbers of receptors for detection with electrophysiological methods as well as toxin binding (33). However, these studies reported large variability in the level of ␣7 nAChR surface expression achieved with primary cultures and cell lines (range from referenced work (14)(15)(16)(17)(18)(19)(20)(21)(22): 2,000 -40,000 surface toxin binding sites/cell). Clearly, cellular factors influence the level of ␣7 nAChR surface expression. There is also evidence that cells make ␣7 subunit protein but that it fails to bind ␣-bungarotoxin as an oligomerized receptor. Rakhilin et al. (23) report that ␣7 nAChRs expressed in cells produce ␣7 subunits in different disulfide-bonded conformations that either form ␣-bungarotoxin binding sites or do not. Certainly, the differences in total toxin binding sites between the V201 chimera, V219 chimera, and ␣7 nAChR may result from subunit sequence effects on the formation of a toxin binding site. However, such a mechanism cannot account for our observation that point mutation of a receptor subunit changes the number of surface toxin binding sites without a concomitant change in total number of toxin binding sites produced by the cell. Our work measures the influence of subunit amino acid sequence on surface receptor expression downstream of subunit folding and assembly into ␣-bungarotoxin binding receptors.
The surface expression phenotypes exhibited by the receptors tested in this study are independent of the transfection method and the cell type used for receptor expression. Chimeric receptor subunits expressed in either COS fibroblast or SK-N-SH neuroblastoma cells exhibit the same rank order of surface receptor numbers, although, overall, SK-N-SH surface expression was lower. Surface expression of ␣7 nAChRs on SK-N-SH cells was below the detection limit (ϳ1,000 toxin binding sites/cell) of our binding assay, probably due to the lower expression level in these cells. The mechanism(s) by which mutations change surface receptor expression involve cellular factors, but there is an intrinsic component dependent on the amino acid sequence of the receptor subunit. We have demonstrated a structure-function relationship between the sequence of ligand-gated ion channel subunits and the level of surface receptor expression.
Possible Surface Receptor Determinant Mechanisms-Surface expression determinants act to redistribute receptors between surface and intracellular pools, since the mutations that affect surface receptor expression level do not change the total number of toxin-binding receptors produced by the cell. Surface expression determinants may act through a receptor transport mechanism: receptor transport to the plasma membrane following subunit assembly and receptor maturation in the endoplasmic reticulum and Golgi apparatus or receptor transport (endocytosis) as part of a surface receptor recycling and/or degradation mechanism. One can imagine an intracellular receptor pool positioned either immediately downstream of the Golgi apparatus or endocytosis from the plasma membrane. Functioning as sink and source, an intracellular pool could effect changes in surface receptor numbers without changing the total number of receptors.
Effects on receptor surface expression resulting from point mutation of ligand-gated ion channel subunits indicate that a conformational change has occurred in these receptors. The mutations that increase surface receptor expression (mutations to a 5HT 3 R amino acid) may act to stabilize a conformation of the receptor that either promotes or retards a molecular interaction with transport and retention machinery, respectively. Those mutations that increase receptor retention (mutations to an ␣7 amino acid) could function to either decrease a molecular interaction with protein transport components or increase an interaction with protein retention components. Protein transport and retention components can be part of the Golgi and endoplasmic reticulum or function nearer the plasma membrane, i.e. components of the post-Golgi and endocytic pathways. With the possible exception of the threonine-to-proline mutation at position 207, these mutations are conservative in terms of charge and side chain bulk, suggesting that subtle changes in the amino acid sequence of receptor subunits can significantly change surface receptor expression levels. Elucidation of the molecules and the mechanism underlying the effect that these mutations have on receptor surface expression FIG. 7. 125 I-␣-bungarotoxin binding labels V201 and V219 receptors resolved with sucrose gradient fractionation. Radioactivity in fractions collected from the bottom of a 5-20% linear sucrose gradient. The T. californica electric organ monomeric (ϳ9 S) and dimeric (ϳ13 S) nAChRs served as size standards (arrows). Fractions were subject to immunoblot in order to detect subunit protein. Shown are fractions corresponding to the peak radioactivity fraction (fraction 16) and a fraction near the top of the gradient (fraction 28), where monomeric subunits migrate. Protein of the M r that represents the toxin-binding species is present in fraction 16 and is absent from later fractions (arrowheads).
will provide insight into fundamental aspects of receptor expression.
Electrophysiology-Although many chimeric and point-mutated subunit cDNAs produce surface toxin binding sites when expressed in COS cells, many of these constructs did not express functional receptors in Xenopus oocytes. The functional phenotype observed for these receptors may reflect the exist-ence of receptor subunits that fold, assemble, and are transported properly (hence cell surface toxin binding), yet no longer gate in the presence of agonist or bind nicotine. Further experimentation is needed to demonstrate such a scenario.
Average peak current recordings of mutant constructs revealed significant changes compared with controls. These differences may reflect the number of receptors expressed, channel conductance, channel permeability, or receptor sensitivity to agonist. For example, the larger whole cell currents exhibited by the ␣7 mutants could result from more surface receptors, as is the case for COS cell expression, or changes in channel conductance. Effects on Ca 2ϩ permeability must also be considered, since ␣7 nAChR currents generated in oocytes are boosted by the Ca 2ϩ -induced Cl Ϫ conductance (38). Presumably, this is not a concern with recordings of the chimeric receptors, since transmembrane domain II is encoded by 5HT 3 sequence, and 5HT 3 Rs do not conduct Ca 2ϩ (24).
Functional Consequence of Surface Expression Determinants in Nicotinic Cholinergic Transmission-In brain, the number of ␣7 nAchRs measured with ␣-bungarotoxin binding is in discordance with the number of ␣7 nAChRs that are measured with electrophysiology techniques (7,9,10,39,40). In certain brain areas, the density of ␣-bungarotoxin binding sites suggests that ␣7 nAChR activation would produce a signal easily measured with contemporary electrophysiology techniques. Since ␣-bungarotoxin binding to sections and lysates of brain tissue does not distinguish between intracellular and surface FIG. 8. The amount of toxin-binding receptor is equivalent for the V201 chimera and generated point mutants. A, sample immunoblot for the quantification of toxin-purified receptor protein from COS cell lysates expressing V201 and V201 point mutants. SM, starting material; FT, flow-through; MW, molecular weight. B, ␣7 standard curve used to quantify ␣7 immunoreactive protein in the blot depicted in A. Density measurements are depicted as the mean of triplicate measurements, and the S.E. bars plotted are smaller than or equal to the size of the symbols. C, summary table of yield of receptor protein toxin-purified from lysates of COS cells transfected with the V201 class of chimeric subunit cDNAs. No significance (p Ͼ 0.05) was found when V201 was compared with P207T (two-tailed Student's t test). pools of receptor, the ␣7 nAChRs in the brain that are not functionally measured could represent mostly an intracellular pool. Large intracellular pools of receptor may be a common theme for neuronal nAChRs. Immunohistochemical techniques used to localize a subclass of non-␣7 nAChR in brain sections shows the majority of labeled protein is associated with a cytoplasmic compartment, in addition to immunoreactive protein at synaptic and perisynaptic regions (41)(42)(43)(44)(45)(46). In transiently transfected cells, the majority of ␣7 nAChRs are part of an intracellular pool (our results, and Ref. 43). The discrepancy between the density of ␣-bungarotoxin binding sites in brain and the magnitude of ␣7 nAChR-generated currents can be resolved if many of them are intracellular. The idea that ␣7 nAChR surface expression is a regulated process in neurons is consistent with the previously discussed channel properties. ␣7 nAChRs in brain conduct Ca 2ϩ upon activation, a powerful and potentially cytotoxic signaling molecule. The greater the number of ␣7 nAChRs at the synapse, the larger the Ca 2ϩ signal upon receptor activation. The determinants we have defined within the amino acid sequence of ␣7 nAChR subunits results in an intrinsically low level of ␣7 nAChR surface expression and is accompanied by a large intracellular pool of receptor. Mobilization of ␣7 nAChRs, either recently assembled or internalized, to the synapse from this intracellular pool will boost the Ca 2ϩ signal and increase synaptic efficacy to subsequent receptor activation.