Identification of Residues in the Neuronal alpha 7 Acetylcholine Receptor That Confer Selectivity for Conotoxin ImI*

Polly A. Quiram and Steven M. SineDagger

From the Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To identify residues in the neuronal alpha 7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha 7-alpha 1 chimeras containing segments of the muscle alpha 1 subunit inserted into equivalent positions of the neuronal alpha 7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for alpha 7/5HT-3 over alpha 1/5HT-3 homo-oligomers. Two of these pairs, alpha 7Trp55/alpha 1Arg55 and alpha 7Ser59/alpha 1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha 7Thr77/alpha 1Lys77, is not within previously described loops of either the alpha  or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha 1 and alpha 7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha 7 and alpha 1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The two neurotransmitter binding sites of muscle nicotinic acetylcholine receptors (AChR)1 are generated by apposition of pairs of nonequivalent subunits, alpha 1/delta , alpha 1/gamma , and alpha 1/epsilon . By contrast, the binding sites of alpha 7 neuronal nicotinic receptors are generated by apposition of pairs of identical subunits, alpha 7/alpha 7 (1). Because only the alpha 7 subunit contributes to both faces of the ligand binding site, one can study the traditional alpha  and non-alpha faces by mutagenesis of a single alpha 7 cDNA.

Ligand affinities of alpha 7 neuronal and muscle AChRs differ owing to the different subunits that form their binding site interfaces. For example, the muscle-specific alpha -conotoxins MI, GI, and SI bind with high affinity to muscle receptors, whereas they bind with low affinity to alpha 7 neuronal receptors (2). On the other hand, alpha -conotoxin ImI (CTx ImI) binds with high affinity to alpha 7 receptors but binds with low affinity to muscle receptors (3). As the only known alpha 7-specific alpha -conotoxin, CTx ImI is a valuable probe of the homo-oligomeric alpha 7 binding site.

Understanding of the alpha 7 binding site has been limited by low expression of alpha 7 receptors in mammalian cell lines (4, 5). Part of the problem appears due to cell type, as neuronal cell lines promote expression of alpha 7 receptors more efficiently than non-neuronal cell lines (6). The sequence of the subunit also affects expression, as chimeras derived from alpha 7 and 5HT-3 subunits express high levels of functional homo-oligomers in non-neuronal cells (7). Joining the alpha 7 extracellular domain to the M1 junction of 5HT-3 permits expression in 293 HEK cells and preserves the pharmacology of the alpha 7 binding site (7). Thus, inserting portions of 5HT-3 sequence is a powerful tool to express receptors with an intact alpha 7 binding site.

Studies of the muscle AChR have led to a basic scaffold hypothesis to account for observations that residues in equivalent positions of the homologous gamma , epsilon , and delta  subunits contribute similarly to ligand affinity (8-10). The hypothesis postulates that owing to their high degree of homology, these subunits fold into similar peptide scaffolds such that residues equivalent in the linear sequence occupy equivalent positions in three-dimensional space. Thus, ligand affinity for a particular site is dictated by differences in primary structure rather than differences in secondary or tertiary structures.

The primary sequence of the alpha 7 subunit reveals conserved residues that contribute to both the alpha  and non-alpha faces of the ligand binding site. Within the alpha  face of the binding site, alpha 7 and alpha 1 share conserved aromatic residues that stabilize agonists, including alpha 7Tyr92, alpha 7Trp148, alpha 7Tyr187, and alpha 7Tyr195 (11-13). On the other hand, alpha 7 and non-alpha muscle subunits (gamma , epsilon , and delta ) share the conserved alpha 7Trp55, which contributes to binding of agonists and antagonists (14, 15). Thus, despite only 31-38% sequence homology with muscle subunits, alpha 7 subunits maintain conserved residues that contribute to both faces of the ligand binding site.

The experiments described herein identify residues of the alpha 7 binding site that determine selectivity for the competitive antagonist CTx ImI and examine the question of whether alpha 7 neuronal and alpha 1 muscle subunits form similar protein scaffolds. By constructing chimeras composed of alpha 7 and alpha 1 subunits, we identified three pairs of residues in equivalent positions of the subunits that confer selectivity of CTx ImI for binding sites formed from alpha 7 versus alpha 1 subunits. Moreover, exchanging these three selectivity determinants between alpha 7 and alpha 1 subunits exchanges the affinity conferred by the subunit, indicating that alpha 7 and alpha 1 subunits share similar protein scaffolds.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 125I-Labeled alpha -bungarotoxin (alpha -bgt) was purchased from NEN Life Science Products, d-tubocurarine chloride from ICN Pharmaceuticals, (+)-epibatidine and methyllycaconitine from Research Biochemicals, 293 human embryonic kidney cell line (293 HEK) from the American Type Culture Collection, and unlabeled alpha -bgt from Sigma. Human alpha 7 and rat 5HT-3 subunit cDNAs were generously provided by Drs. John Lindstrom and William Green. Sources of the human acetylcholine receptor subunit cDNAs were as described previously (16).

Synthesis and Purification of Conotoxin ImI-- Conotoxin ImI was synthesized by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems 431A peptide synthesizer. During synthesis, cysteine (S-triphenylmethyl)-protecting groups were incorporated at cysteines 3 and 12, and acetamidomethyl-protecting groups were incorporated at cysteines 2 and 8. The linear peptide was purified by reversed phase high performance liquid chromatography using a Vydac C18 preparative column with trifluoroacetic acid/acetonitrile buffers. Two intramolecular disulfide bridges were formed as follows: the cysteine S-triphenylmethyl-protecting groups of cysteines 3 and 12 were removed during trifluoroacetic acid cleavage of the linear peptide from the support resin, and the peptide was oxidized by molecular oxygen to form the 3-12 disulfide by stirring in 50 mM ammonium bicarbonate buffer, pH 8.5, at 25 °C for 24 h. The peptide was lyophilized prior to the formation of the second bridge. The acetamidomethyl-protecting groups on cysteine 2 and 8 were removed oxidatively by iodine as described (17) except that the peptide/iodine reaction was allowed to progress 16 h prior to carbon tetrachloride extraction. Residual iodine was separated from the pure product by high performance liquid chromatography, and CTx ImI was characterized by mass spectrometry.

Mutagenesis and Expression in HEK Cells-- Acetylcholine receptor subunit cDNAs were subcloned into the cytomegalovirus-based expression vector pRBG4 (18). Mutant cDNAs were constructed by bridging naturally occurring or mutagenically installed restriction sites with double-stranded oligonucleotides. The chimeras are named as follows: the first subunit is the amino-terminal sequence of the chimera, the number following gives the position of the chimeric junction, and the final subunit gives the subunit from which the carboxyl-terminal sequence of the extracellular domain is taken. The extracellular domains of all chimeras are joined at M1 to the rat 5HT-3 sequence. Chimera alpha 7/5HT-3 (alpha 7200/5HT-3) was constructed by bridging a 58-bp synthetic oligonucleotide from a TfiI site in alpha 7 to an EcoRV site in rat 5HT-3. Chimera alpha 1/5HT-3 (alpha 1205/5HT-3) was constructed by bridging a 69-bp synthetic oligonucleotide from a DraIII site in alpha 1 to a StuI site in rat 5HT-3. All constructs were confirmed by dideoxy sequencing. HEK cells were transfected with wild type or mutant cDNAs using calcium phosphate precipitation as described (18). Two days after transfection, intact cells were harvested by gentle agitation in phosphate-buffered saline with 5 mM EDTA.

Ligand Binding Measurements-- Ligand binding to intact cells was measured by competition against the initial rate of 125I-labeled alpha -bgt binding (18). The cells were briefly centrifuged, resuspended in potassium Ringer's solution, and divided into aliquots for ligand binding. Potassium Ringer's solution contains 140 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2, 25 mM HEPES, and 30 mg/liter bovine serum albumin, adjusted to a pH of 7.4 with 10 mM NaOH. Specified concentrations of ligand were added 30 min prior to addition of 3.75 nM 125I-labeled alpha -bgt, which was allowed to bind for 15 min to occupy approximately half of the surface receptors. Binding was terminated by addition of 2 ml of potassium Ringer's solution containing 600 µM of d-tubocurarine chloride. All experiments were performed at 24 ± 2 °C. Cells were harvested by filtration through Whatman GF-B filters using a Brandel cell harvester and washed three times with 3 ml of potassium Ringer's solution. Prior to use, filters were soaked in potassium Ringer's solution containing 4% skim milk. Nonspecific binding was determined in the presence of 10 nM alpha -bgt and was typically 1% of the total number of binding sites. The total number of binding sites was determined by incubation with toxin for 120 min. The initial rate of toxin binding was calculated as described previously (19) to yield the fractional occupancy of ligand. Binding measurements were analyzed according to the Hill equation: 1 - Y = 1/(1 + ([ligand]/Kapp)nH), where Y is fractional occupancy of ligand, Kapp is the apparent dissociation constant and nH is the Hill coefficient. Parameter estimates and standard errors were obtained using UltraFit (BIOSOFT). For multiple experiments, means of the individual fitted parameters and standard deviations are presented (Tables I and II).

To measure time courses of alpha -bgt dissociation, receptors were incubated with 3.75 nM 125I-labeled alpha -bgt for 120 min to achieve full occupancy, free 125I-labeled alpha -bgt was removed, and 200-µl aliquots of cells were filtered at specific times. Radioactivity at each time point was normalized to that of the maximum radioactivity at the time of toxin removal. Nonspecific binding was measured by incubation with 10 nM unlabeled alpha -bgt.

Sucrose Gradient Centrifugation-- Two days after transfection, 293 HEK cells expressing various receptors were harvested by gentle agitation in phosphate-buffered saline and resuspended in high potassium Ringer's solution. Following labeling with 3.75 nM 125I-labeled alpha -bgt for 2 h, cells were washed free of unbound radioactivity. Samples for sucrose gradient centrifugation were solubilized in 1 ml of Triton X-100 buffer (0.6% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, 35 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 µg/ml pepstatin A, pH 7.5). Extracts were layered on sucrose gradients (3-30%) and centrifuged for 22 h at 40,000 rpm, and fractions were collected and counted with a gamma  counter. Radioactivity in each fraction was normalized to that of the fraction containing the maximum radioactivity in each gradient.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of alpha 7/5HT-3 and alpha 1/5HT-3 Chimeric Receptors-- Previous studies described construction of a chimera containing the extracellular domain of chick alpha 7 joined to the M1 junction of the rat 5HT-3 subunit (alpha 7201/5HT-3) (7). The studies further showed that addition of 5HT-3 sequence maintained ligand recognition properties of the native alpha 7 binding site. We constructed a similar chimera by joining the extracellular domain of human alpha 7 to the rat 5HT-3 subunit, with the chimera junction formed at position 200 (alpha 7200/5HT-3) (Fig. 1A). To determine whether our human alpha 7/5HT-3 receptor has similar ligand recognition properties to wild type alpha 7, we expressed the constructs in 293 HEK cells and measured binding of agonists and antagonists by competition against the initial rate of 125I-labeled alpha -bgt binding. Although expression of alpha 7/5HT-3 receptors exceeds that of wild type alpha 7 receptors by 1000-fold (see Fig. 1 legend), the competitive antagonists CTx ImI and methyllycaconitine and the agonist (+)-epibatidine bind with identical affinities to the two types of receptors (Fig. 1, B-D; Table I). Thus, the alpha 7 ligand binding domain is preserved in alpha 7/5HT-3 receptors, and expression is greatly enhanced by addition of 5HT-3 sequence.


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Fig. 1.   Agonists and antagonists bind to wild type alpha 7 and alpha 7/5HT-3 receptors with similar affinities. Panel A is a schematic drawing of the wild type alpha 7 and alpha 7/5HT-3 subunits: alpha 7/5HT-3 contains alpha 7 sequence to position 200 followed by 5HT-3 sequence to the carboxyl terminus. Shaded portions represent 5HT-3 sequence. Panels B-D, 293 HEK cells were transfected with alpha 7 or alpha 7/5HT-3 subunit cDNAs, and binding of CTx ImI, methyllycaconitine (MLA), or (+)-epibatidine was determined as described under "Experimental Procedures." The curves through the data are fits to the Hill equation; means and S.E. of the fitted parameters are given in Table I. Expression of wild type alpha 7 and alpha 7/5HT-3 surface receptors was typically 7 fmol and 6 pmol per 10-cm plate, respectively.

                              
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Table I
Comparison of ligand binding parameters for wild type alpha 7 and alpha 7/5HT-3 receptors for conotoxin ImI, methyllycaconitine, and (+)-epibatidine
Data are the least squares fits to the Hill equation from the series of experiments shown in Fig. 1, B-D. Kapp is the apparent dissociation constant, nH is the Hill coefficient, and n is the number of independent experiments.

To investigate the basis of the specificity of CTx ImI for alpha 7 receptors, we needed a subunit homologous to alpha 7 and with low affinity for CTx ImI to serve as a frame of reference. We therefore constructed an analogous alpha 1/5HT-3 chimera by joining the alpha 1 subunit extracellular domain to the M1 junction of the 5HT-3 receptor. When transfected into 293 HEK cells, the alpha 1/5HT-3 cDNA leads to expression of alpha -bgt binding sites on the cell surface. Moreover, CTx ImI binds 50-fold less tightly to alpha 1/5HT-3 than to alpha 7/5HT-3 receptors (Fig. 2A). Similarly, CTx ImI binds 50-fold less tightly to adult human muscle receptors, further demonstrating neuronal specificity of CTx ImI (Fig. 2A). Thus, the alpha 1/5HT-3 receptor provides a homologous muscle-like frame of reference for investigating specificity of CTx ImI for the alpha 7/5HT-3 receptor.


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Fig. 2.   Characterization of cell surface receptors formed by alpha 7/5HT-3 and alpha 1/5HT-3 chimeras. Panel A, comparison of CTx ImI binding to surface receptors composed of wild type alpha 7, alpha 7/5HT-3, alpha 1/5HT-3 and muscle alpha 2beta epsilon delta subunits. Data are from single representative experiments; overall mean parameters and S.D. are given in Table II. Panel B, sucrose density sedimentation profiles of surface receptors formed by expressing alpha 7/5HT-3, alpha 1/5HT-3, and muscle alpha 2beta epsilon delta cDNAs in 293 HEK cells as described under "Experimental Procedures." Radioactivity in each fraction is normalized to the peak value in each gradient. Arrows and S values indicate alpha -bgt (1.3 S) and alpha 2beta epsilon delta pentamer (9 S) peaks. Panel C, time courses of 125I-labeled alpha -bgt dissociation from alpha 7/5HT-3, alpha 1/5HT-3, human muscle alpha 2beta epsilon delta , and mouse muscle alpha 2beta epsilon delta receptors. For each receptor, data are normalized to the binding at zero time. For alpha 7/5HT-3, human muscle alpha 2beta epsilon delta , and mouse muscle alpha 2beta epsilon delta receptors, the smooth curves are fits to a single exponential decay, with t1/2 given in the text; for alpha 1/5HT-3 receptors, the curve is a fit to a double exponential decay, with t1/2 values given in the text.

To determine whether surface receptors generated by alpha 7/5HT-3 and alpha 1/5HT-3 are homo-oligomers containing five subunits, we labeled them with 125I-labeled alpha -bgt and centrifuged the solubilized receptors on sucrose density gradients. We ran a parallel gradient containing the alpha 2beta epsilon delta human muscle receptor to provide a 9S pentamer standard. The alpha 7/5HT-3 receptor migrates with a sedimentation coefficient just greater than our 9S muscle receptor standard, as described previously (20), and the profile was noticeably broader (Fig. 2B). The increased molecular weight and broad profile suggest either increased glycosylation or an additional 40 kDa due to binding of alpha -bgt to the five potential binding sites. The alpha 1/5HT-3 chimera co-migrates with our 9S muscle receptor standard. Both the alpha 1/5HT-3 and human muscle receptors show significant 1.3S peaks due to free 125I-labeled alpha -bgt, indicating dissociation of some of the alpha -bgt-receptor complexes during centrifugation. These results show that both alpha 7/5HT-3 and alpha 1/5HT-3 subunits form pentameric homo-oligomers on the cell surface.

To further investigate differences in stability of the alpha -bgt-receptor complexes suggested by sedimentation analysis, we compared time courses of 125I-labeled alpha -bgt dissociation from alpha 1/5HT-3, alpha 7/5HT-3, adult mouse muscle, and adult human muscle receptors. 125I-labeled alpha -bgt dissociates from alpha 7/5HT-3 and adult mouse muscle receptors with a single slow rate constant (t1/2 = 20 h), whereas adult human muscle receptors show a more rapid single rate of dissociation (t1/2 = 2.5 h). However, 125I-labeled alpha -bgt dissociates from alpha 1/5HT-3 receptors in a biphasic manner, with a rapid component having a t1/2 of 13 min and a slow component having a t1/2 of 13.7 h. The amplitudes of the two components are approximately equal, indicating similar numbers of two classes of sites in the alpha 1/5HT-3 receptor. Thus, the kinetics of 125I-labeled alpha -bgt dissociation reveal quantitative differences in binding sites of alpha 7/5HT-3, alpha 1/5HT-3, and muscle receptors.

Determinants of CTx ImI Selectivity Identified Using alpha 7-alpha 1 Chimeras-- We constructed a series of alpha 7-alpha 1 chimeras to identify residues of the alpha 7 receptor that confer the 50-fold higher affinity of CTx ImI for alpha 7/5HT-3 over alpha 1/5HT-3 receptors. For all chimeras, we joined the extracellular domains to the M1 junction of the rat 5HT-3 subunit. Our first informative chimera contained a small insertion of alpha 1 sequence between residues 55 and 77 of the alpha 7 subunit (Fig. 3A). This short segment of alpha 1 sequence fully decreases CTx ImI affinity to that of the pure alpha 1/5HT-3 chimera (Fig. 3B), suggesting that the 55-77 segment is the entire source of high affinity in alpha 7.


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Fig. 3.   CTx ImI binding to receptors formed by alpha 7-alpha 1 chimeras. Panel A is a schematic drawing of the alpha 755alpha 177alpha 7 chimera, which contains alpha 1 sequence from residues 55 to 77. The black portion represents alpha 1 sequence; the unshaded portion represents alpha 7 sequence; and the shaded portion represents 5HT-3 sequence. Comparison of alpha 1 and alpha 7 sequence in this segment indicates candidate residues that confer CTx ImI selectivity. Panel B, CTx ImI binding to intact cells expressing alpha 7/5HT-3, alpha 1/5HT-3, and alpha 755alpha 177alpha 7 receptors.

We examined additional alpha 7-alpha 1 chimeras to determine whether the 55-77 segment is the sole source of high affinity for CTx ImI (Table II). The chimera alpha 755alpha 1103alpha 7 confers pure alpha 1-like affinity, indicating that residues between positions 78 and 103 do not contribute to selectivity. We also constructed chimeras to target two of the three regions that confer selectivity of alpha -conotoxin MI in muscle receptors (8): alpha 728alpha 131alpha 7, alpha 737alpha 154alpha 7, and alpha 7111alpha 1116alpha 7. Each of these chimeras confers pure alpha 7-like affinity, indicating no contributions of segments 28-31, 37-54, and 111-116. We also constructed chimeras targeting the carboxyl-terminal extracellular domain, including alpha 7133alpha 1153alpha 7, alpha 7164alpha 1200, and alpha 7184alpha 1200, but these did not form functional receptors. Thus, the 55-77 segment in alpha 7 appears to be the sole source of high affinity for CTx ImI.

                              
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Table II
Conotoxin ImI binding parameters for receptors containing wild type, chimeric, or point mutant subunits
Data are the least squares fits to the Hill equation from the series of experiments shown in Figs. 2-5. Kapp is the apparent dissociation constant, nH is the Hill coefficient, n is the number of independent experiments, and NE is no expression.

Dissection of the 55-77 Segment-- We constructed a series of stepwise chimeras to further localize selectivity determinants within the 55-77 segment. Starting with our reference chimera alpha 755alpha 177alpha 7, we maintained the alpha 7/alpha 1 junction at position 55 but shifted the carboxyl-terminal junction from position 77 to position 57 (Fig. 4). Surpassing only one mismatched residue, the chimera alpha 755alpha 176alpha 7 increases CTx ImI affinity toward that of alpha 7, suggesting that the pair alpha 7Thr77/alpha 1Lys77 contributes to selectivity. Shifting the junction from position 76 to 59 produces no further change in affinity, but shifting from position 59 to 57 reveals an additional increase in affinity toward that of alpha 7. The last chimera in this series, alpha 755alpha 157alpha 7, falls 3-fold short of pure alpha 7-like affinity, indicating that residues 55-57 contribute the remaining increment of selectivity. Thus, the stepwise chimeras reveal at least three subsets of selectivity determinants within the 55-77 segment; one is between positions 55 and 57, the second is between positions 57 and 59, and the third is the pair alpha 7Thr77/alpha 1Lys77.


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Fig. 4.   Dissection of selectivity determinants for CTx ImI starting with the base chimera alpha 755alpha 177alpha 7. CTx ImI affinities for alpha 1/5HT-3 and alpha 7/5HT-3 receptors are shown by the vertical dashed lines, and the error bars indicate S.D. Affinities of the chimeric receptors are expressed as the log of the ratio of the dissociation constant of the chimera divided by that of the alpha 7/5HT-3 receptor. At right is a schematic representation of the chimeras in this series, with alpha 7 sequence unshaded, alpha 1 sequence black, and 5HT-3 sequence shaded. The text below indicates amino acid sequences in the 55-77 segment with alpha 1 sequence in boldface and underlined, and alpha 7 sequence in plain text. Data are means ± S.D. of at least three experiments.

Point Mutants of Selectivity Determinants-- We further localized selectivity determinants by constructing point mutations in the alpha 7/5HT-3 cDNA. Beginning with the 55-57 segment, the point mutation alpha 7W55R decreased CTx ImI affinity by the same amount observed in the stepwise chimeras. Similarly, mutating within the 57-59 segment, alpha 7S59Q decreases affinity by the same amount observed in the stepwise chimeras. Finally, the mutation alpha 7T77K decreases affinity as observed in the chimeras. Thus, three residues, alpha 7Trp55, alpha 7Ser59, and alpha 7Thr77, contribute to CTx ImI selectivity for alpha 7 (Fig. 5).


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Fig. 5.   CTx ImI binding to alpha 7/5HT-3 and alpha 1/5HT-3 receptors containing point mutations of selectivity determinants. For each mutant receptor, CTx ImI affinity is expressed as in Fig. 4. NE, no expression. To the right is a schematic of the alpha 755alpha 177alpha 7 chimera. The text below indicates the amino acid sequences of the 55-77 segment, with mutant residues underlined, alpha 1 sequence in boldface, and alpha 7 sequence in plain text.

We next combined mutations of two or three determinants into one receptor to look for interactions between the determinants and to ask whether the set of three determinants fully accounts for selectivity of CTx ImI. The three possible double mutations, alpha 7(W55R/S59Q), alpha 7(W55R/T77K), and alpha 7(S59Q/T77K), decreases affinity in an additive manner (Table II), indicating that these pairs of residues contribute independently. Moreover, the triple mutation alpha 7(W55R/S59Q/T77K) fully decreases affinity to that observed for both the alpha 755alpha 177alpha 7 and alpha 1/5HT-3 chimeras (Fig. 5). Thus, alpha 7Trp55, alpha 7Ser59, and alpha 7Thr77 confer CTx ImI selectivity for alpha 7 receptors. The overall results support the basic scaffold hypothesis because exchange of the selectivity determinants from alpha 1 to alpha 7 exchanges the affinity for CTx ImI.

Exchange of Selectivity Determinants between the alpha 7 and alpha 1 Subunits-- To further confirm that alpha 7W55R, alpha 7S59Q, and alpha 7T77K confer CTx ImI selectivity, we sought to convert alpha 1 to alpha 7 affinity by constructing the converse mutations in the alpha 1/5HT-3 subunit (Fig. 5). Two of the three point mutants, alpha 1Q59S and alpha 1K77T, maintain good levels of expression and increase affinity for CTx ImI, as expected from the chimeras. Combining these two mutations into a single receptor with alpha 1(Q59S/K77T) increases CTx ImI affinity in an additive manner, again showing that these determinants contribute independently. However, the third point mutation, alpha 1R55W, does not express alone nor when present in the triple mutant alpha 1(R55W/Q59S/K77T). The residual affinity between the double point mutation alpha 1(Q59S/K77T) and alpha 7/5HT-3 equals that conferred by the point mutant alpha 7W55R, further suggesting that basic scaffold of the alpha 1 subunit is similar to that of the alpha 7 subunit.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We probed the binding site of the alpha 7 receptor using the neuronal-specific toxin CTx ImI together with chimeras containing portions of alpha 1 sequence substituted into the extracellular domain of alpha 7. The results reveal three pairs of residues in equivalent positions of the subunits that confer selectivity of CTx ImI for alpha 7 over muscle-like AChRs. Because exchange of these residues between alpha 7/5HT-3 and alpha 1/5HT-3 exchanges affinity for CTx ImI, the extracellular domains of alpha 7 and alpha 1 subunits appear to fold into similar basic scaffolds. This basic scaffold hypothesis was originally developed to explain ligand selectivity conferred by subunits with high homology, such as gamma  and delta  subunits (49% identity), epsilon  and delta  subunits (47%), and gamma  and epsilon  subunits (53%) (8-10, 18, 21). We find that the hypothesis extends to the less homologous alpha 7 and alpha 1 subunits, which are only 38% identical.

To investigate the basis for neuronal specificity of CTx ImI, we needed to achieve high levels of expression of receptors with alpha 7 binding sites and a homologous, muscle-like frame of reference. As described by others (7), we find that substituting 5HT-3 sequence from the M1 domain to the carboxyl terminus markedly increases expression, yet preserves the ligand recognition properties of the alpha 7 binding site. The increased expression was initially surprising because the extracellular domain was widely known for its importance in receptor assembly (22-24). However, studies of alpha 7-alpha 3 chimeras show that formation of homo-oligomers requires matching of particular residues in the M1 and M2 transmembrane domains (25). Thus, our results further confirm that the region carboxyl-terminal to M1 contributes to assembly of homo-oligomers.

Another surprise is that our alpha 1/5HT-3 construct forms homo-oligomers on the cell surface. This observation contrasts with expression of the alpha 1 subunit alone, which remains monomeric and retained within the cell. Thus, 5HT-3 sequence between M1 and the carboxyl terminus promotes homo-oligomer formation. We found differences, however, between alpha 1/5HT-3 and alpha 7/5HT-3 homo-oligomers in their kinetics of alpha -bgt dissociation. alpha -bgt dissociates from alpha 7/5HT-3 homo-oligomers with a single slow rate constant, whereas the toxin dissociates from alpha 1/5HT-3 homo-oligomers with one rapid and one slow rate constant. Thus, despite the presence of only one type of subunit, binding sites in alpha 1/5HT-3 homo-oligomers appear to be nonequivalent. The origin of nonequivalent sites is not known, but they may arise during the course of subunit folding and oligomerization that produces a fully assembled pentamer with high affinity for alpha -bgt (26). Because acquisition of the toxin binding site is associated with a protein folding event, and folding requires interaction with specific subunits, successive addition of alpha 1/5HT-3 subunits may produce unusual interactions, leading to nonequivalence of the binding sites.

In addition, we find that alpha 1/5HT-3 and alpha 2beta epsilon delta muscle receptors bind CTx ImI with similar low affinities. Because the muscle receptor contains the stabilizing residues epsilon Trp55/delta Trp57, and the alpha 1/5HT-3 homo-oligomer contains the destabilizing residue alpha 1Arg55, one might expect higher affinity of the muscle receptor compared with that of alpha 1/5HT-3. Resolution of the apparent paradox likely lies in the different contributions of the (-) face in the two types of receptors, because they contain identical (+) faces. Potentially, residues flanking the three determinants identified here, but unique to the epsilon  and delta  subunits, may decrease affinity, despite the presence of epsilon Trp55/delta Trp57 in the muscle receptor. The unique residue differences may cause small changes in the protein scaffold and prevent interaction between CTx ImI and epsilon Trp55/delta Trp57, as well as other determinants of affinity. In addition, the low affinity may result from reorientation of CTx ImI at the binding site, and the low affinity may be due to stabilization by residues common to the alpha 1, epsilon , and delta  subunits.

We chose CTx ImI to probe the alpha 7 binding site because it is a constrained two-loop structure owing to its two disulfide bridges, similar to the muscle-specific alpha -conotoxins (Fig. 6). CTx ImI contains four amino acids in its first loop and three in its second, whereas muscle-specific alpha -conotoxins contain three and five amino acids in its first and second loops, respectively. In addition, CTx ImI contains basic residues in both loops (Arg7 and Arg11) and an acidic residue in the first loop (Asp5), unlike muscle-specific alpha -conotoxins; these unique residues may further contribute to neuronal specificity of CTx ImI.


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Fig. 6.   Comparison of alpha -conotoxin ImI with muscle-specific alpha -conotoxins MI, GI, and SI.

Photoaffinity labeling and mutagenesis studies establish that the ligand binding sites of the muscle AChR contain contributions of both alpha  and non-alpha subunits. Residues of the alpha  or (+) face of the binding site cluster into three linearly separate regions, leading to a three-loop model of the alpha  subunit contribution to the binding site. Similarly, residues of the non-alpha or (-) face of the binding site cluster into four separate regions of the linear sequence, leading to a four-loop model of the non-alpha contribution to the site (reviewed in Refs. 27 and 28). Studies of the highly homologous delta , epsilon , and gamma  subunits show that residues in equivalent positions of the subunit make equivalent contributions to ligand affinity (8-10, 18, 21). Thus, the (-) face contributed by these subunits harbors virtually superimposable peptide scaffolds. Here, we extend the basic scaffold hypothesis to the less homologous alpha 7 and alpha 1 subunits by showing that exchanging selectivity determinants between alpha 7 and alpha 1 subunits exchanges affinity for CTx ImI.

Two of the three pairs of alpha 7 selectivity determinants, alpha 7Trp55/alpha 1Arg55 and alpha 7Ser59/alpha 1Gln59, are within one of the four loops that contribute to the (-) face of the binding site. The contribution of alpha 7Trp55 was first demonstrated by photoaffinity labeling with d-[3H]tubocurarine, which labeled the equivalent residues gamma Trp55/delta Trp57 in Torpedo receptors (29). Subsequent mutagenesis of alpha 7201/5HT-3 homo-oligomers revealed contributions of the equivalent residue in chick alpha 7Trp54 to agonist and antagonist affinity (15). The second pair in this segment, alpha 7Ser59/alpha 1Gln59, is equivalent to the residues in non-alpha subunits in muscle receptors, epsilon Asp59/delta Ala61, which contribute to dimethyl-d-tubocurarine selectivity of the adult receptor (9). Thus, approximately half of the neuronal specificity of CTx ImI is due to stabilization by one of the four loops at the (-) face of the subunit.

The third pair of selectivity determinants, alpha 7Thr77/alpha 1Lys77, is not contained within previously described loops of either the alpha  or non-alpha faces of the binding site. One member of this pair, alpha 1Lys77, is immediately carboxyl-terminal to the main immunogenic region, which extends from the tip of the extracellular lobe of the AChR (30). alpha 1Lys77 may be far enough from the main immunogenic region that it can fold back to the binding site. We cannot say whether alpha 7Thr77/alpha 1Lys77 contributes directly or allosterically to the binding site. However, the positively charged alpha 1Lys77 may repel one of the arginine side chains in CTx ImI, whereas alpha 7Thr77 may be neutral or stabilize CTx ImI through hydrogen bonding. We observed equal and opposite changes in free energy of binding with alpha 7T77K and alpha 1K77T, suggesting direct contributions to affinity. A long range interaction would not be expected to show such equal and opposite free energy changes because it would have to propagate through intervening residues.

The overall results reveal three pairs of equivalent residues in the alpha 7 and alpha 1 subunits that confer selectivity of CTx ImI and show that the extracellular domains of alpha 7 and alpha 1 subunits fold into similar basic scaffolds. The precise contacts between CTx ImI and alpha 7 await experiments that mutate residues in both the toxin and the receptor.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Receptor Biology Laboratory, Dept. of Physiology and Biophysics, Mayo Foundation, 200 First St. S.W., Rochester, Minnesota 55905. Tel.: 507-284-5612; Fax: 507-284-9420; E-mail: sine.steven{at}mayo.edu.

1 The abbreviations used are: AChR, acetylcholine receptor; CTx ImI, alpha -conotoxin ImI; HEK, human embryonic kidney; alpha -bgt, alpha -bungarotoxin; 5HT, 5-hydroxytryptamine.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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