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Research ArticleCELLULAR AND MOLECULAR

Identification of Residues That Confer α-Conotoxin-PnIA Sensitivity on the α3 Subunit of Neuronal Nicotinic Acetylcholine Receptors

Drew Everhart, Edward Reiller, Armen Mirzoian, J. Michael McIntosh, Arun Malhotra and Charles W. Luetje
Journal of Pharmacology and Experimental Therapeutics August 2003, 306 (2) 664-670; DOI: https://doi.org/10.1124/jpet.103.051656
Drew Everhart
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Edward Reiller
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Armen Mirzoian
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J. Michael McIntosh
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Arun Malhotra
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Charles W. Luetje
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Abstract

Neuronal nicotinic receptors composed of the α3 and β2 subunits are at least 1000-fold more sensitive to blockade by α-conotoxin-PnIA than are α2β2 receptors. A series of chimeric subunits, formed from portions of α2 and α3, were coexpressed with β2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of α3. Analysis of receptors formed by a series of mutant α3 subunits, in which residues that differ between α3 and α2 were changed from what occurs in α3 to what occurs in α2, allowed identification of three determinants of α-conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of α-conotoxin-MII and κ-bungarotoxin sensitivity on the α3 subunit revealed overlapping, but distinct, arrays of determinants for each of these three toxins. When tested against an EC50 concentration of acetylcholine, the IC50 for α-conotoxin-PnIA blockade was 25 ± 4 nM for α3β2, 84 ± 7 nM for α3P182Tβ2, 700 ± 92 nM for α3I188Kβ2, and 870 ± 61 nM for α3Q198Pβ2. To examine the location of these residues within the receptor structure, we generated a homology model of the α3β2 receptor extracellular domain using the structure of the acetylcholine binding protein as a template. All three residues are located on the C-loop of the α3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.

Nicotinic acetylcholine receptors (nAChRs) are located at the neuromuscular junction and throughout the central and peripheral nervous systems. The nAChRs, together with GABA-, glycine-, and serotonin-gated ion channels, constitute a superfamily of receptors known as cys-loop receptors. These receptors are pentameric assemblies of subunits, each possessing an extracellular “cys-loop” (two cysteines, separated by 13 residues, forming a disulfide bond) (Corringer et al., 2000). The cys-loop seems to be involved in the coupling of agonist binding and channel gating in these receptors (Kash et al., 2003). The agonist binding sites of cys-loop receptors have long been thought to be located at interfaces between the extracellular domains of various subunits. This idea was confirmed by the recently reported crystal structure of the molluscan acetylcholine binding protein (AChBP), a soluble pentameric protein with homology to the extracellular domain of nAChRs (Brejc et al., 2001; Smit et al., 2001).

Neuronal nAChRs are assembled from a family of at least 12 distinct subunits, α2–10 and β2-4 (Corringer et al., 2000). When studied in exogenous expression systems such as Xenopus oocytes, a variety of functional subunit combinations can be expressed, each displaying unique pharmacological properties (Role, 1992). These pharmacological differences can be exploited to study the structural determinants of receptor subtype specificity. Neuronal nAChRs can form as pentameric homomers of a single subunit (such as α7 receptors), as simple heteropentamers of one type of α subunit and one type of β subunit (such as α4β2 receptors) and as complex heteropentamers of three or more subunits (such as α3α5β4 receptors) (Whiting et al., 1991; Flores et al., 1992; Conroy and Berg, 1995; Chen and Patrick, 1997; Drisdel and Green, 2000).

Venoms from the Conus genus of predatory marine snails contain a multitude of peptide neurotoxins, with peptides of the α-conotoxin family displaying various selectivities for muscle and neuronal nAChRs (McIntosh et al., 1999). The α-conotoxins MII from C. magus, PnIA from C. pennaceus, and GIC from C. geographus all selectively antagonize α3β2 neuronal nAChRs (Cartier et al., 1996; Hogg et al., 1999; Luo et al., 1999; McIntosh et al., 2002). We previously identified residues on both α3 (K185 and I188) and β2 (T59) that confer sensitivity to MII (Harvey et al., 1997). Recently, it became clear that MII also antagonizes neuronal nAChRs containing α6, a subunit highly homologous to α3 (Gerzanich et al., 1997). Whether PnIA and GIC antagonize α6-containing receptors is not currently known. The similar ability of PnIA and MII to antagonize α3β2 receptors is interesting, because although PnIA and MII have the same disulfide-bonding pattern, they differ at 9 of the 12 noncysteine residues. These two toxins could act at the same site on the α3β2 receptor or, as has been demonstrated for antagonism of α7 receptors by the α-conotoxins ImI and ImII (Ellison et al., 2003), could act at different sites. We used a series of chimeric and mutant α subunits to identify residues that confer sensitivity to PnIA and to determine the relationship, if any, to the residues that determine MII sensitivity.

Materials and Methods

Materials.Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). The care and use of X. laevis frogs in this study were approved by the University of Miami Animal Research Committee and meet the guidelines of the National Institutes of Health. RNA transcription kits were from Ambion (Austin, TX). Collagenase B was from Roche Diagnostics (Indianapolis, IN). All other reagents were from Sigma-Aldrich (St. Louis, MO). PnIA was synthesized, and proper disulfide bond formation was achieved as described previously (Luo et al., 1999).

Expression of Neuronal nAChRs in X. laevis Oocytes. cDNA clones encoding rat α2, α3, α4, β2, and β4 subunits, as well as α subunit chimeras and mutants, were used as templates for cRNA synthesis. Chimeric and mutant subunits were constructed as described previously (Luetje et al., 1993, 1998; Harvey et al., 1996). Our notation for chimeric subunits is to list the source of the amino-terminal portion, followed by the residue number in the amino acid sequence where the chimeric joint is made (numbering taken from the mature α3 subunit sequence), and then followed by the source of the carboxyl-terminal portion. For example, the chimeric subunit α2-195-α3 is composed of α2 sequence from the amino terminus until residue 195, after which it is composed of α3 sequence. Our notation for mutant subunits is to list the naturally occurring residue followed by the position of that residue, followed by the change that was made. For example, α3I188K is an α3 subunit in which isoleucine 188 has been changed to a lysine.

m7G(5′)ppp(5′)G capped cRNA was synthesized in vitro from linearized template DNA. Mature X. laevis frogs were anesthetized by submersion in 0.1% 3-aminobenzoic acid ethyl ester and oocytes were surgically removed. Follicle cells were removed by treatment with collagenase B for 2 h at room temperature. Stage V oocytes were individually injected with 10 ng of cRNA in 50 nl of water and incubated at 18°C in Barth's saline (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM CaNO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 100 μg/ml gentamicin, and 15 mM HEPES, pH 7.6) for 2 to 7 days. RNA transcripts for each subunit were injected at a molar ratio of 1:1.

Electrophysiological Methods. Current responses were measured under two-electrode voltage clamp, at a holding potential of –70 mV, using a TEV-200 voltage-clamp unit (Dagan, Minneapolis, MN) and an OC-725C voltage-clamp unit (Warner Instrument, Hamden. CT). Micropipettes were filled with 3 M KCl and had resistances of 0.3 to 2.0 MΩ. Current responses were captured, stored, and analyzed on a Macintosh IIci computer using a data acquisition program written with LABVIEW (National Instruments, Austin, TX) and LIBI (University of Arizona, Tucson, AZ) software (Luetje et al., 1993) and on a Macintosh G3 computer using AxoGraph 4.6 software (Axon Instruments, Inc., Foster City, CA). Oocytes were perfused at room temperature (20 –25°C), in a chamber constructed from 1/8-inch inner diameter Tygon tubing, with perfusion solution (115 mM NaCl, 1.8 mM CaCl2, 2.5 mM KCl, 0.1 μM atropine, and 10 mM HEPES, pH 7.2). Perfusion was continuous (except during toxin incubations) at a rate of ∼20 ml/min. ACh was applied diluted in perfusion solution. Toxin was applied diluted in perfusion solution supplemented with 100 μg/ml BSA. In preliminary time-course experiments with 100 nM and 1 μM PnIA, we found that a 5-min incubation was sufficient for blockade of α3β2 receptors to reach equilibrium (data not shown). In Figs. 1 to 3, PnIA blockade was determined by comparing the ACh-induced peak current response after a 5-min incubation with toxin, to the average of three ACh-induced peak current responses preceding the toxin incubation. In these experiments, ACh was applied immediately after the toxin incubation with no toxin included in the ACh application. This allowed toxin to be conserved during initial screening of mutant and chimeric receptors. Although PnIA is thought to be a competitive antagonist of neuronal nAChRs, under these conditions (application of ACh alone after toxin incubation), ACh and toxin are not in direct competition. Thus, the ACh concentrations used to screen the various wild-type, chimeric, and mutant receptors need not be equipotent. ACh concentrations used ranged from the EC10 to the EC75.

    Fig. 1.
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Fig. 1.

α-Conotoxin-PnIA is selective for α3β2 neuronal nicotinic receptors. A, current responses of Xenopus oocytes expressing α3β2 (left) and α2β2 (right) receptors to application of 10 μM ACh (bars) before and after 5-min incubation with 1 μM PnIA. Scale bars, 400 nA (α3β2), 60 nA (α2β2), 10 s. B, PnIA inhibition of α3β2 (filled squares), α2β2 (open squares), α3β4 (open circles), and α4β2 (open triangles) receptors. The response to ACh (EC10 < ACh < EC75) for each receptor after a 5-min incubation with various concentrations of PnIA is presented as a percentage of the preincubation ACh response. Each point is the mean ± S.E.M. of three to nine oocytes. The α3β2 data were fit as described under Materials and Methods (IC50 = 68 ± 12 nM, nH = 0.8 ± 0.1).

    Fig. 3.
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Fig. 3.

Proline 182, isoleucine 188, and glutamine 198 are important for α-conotoxin-PnIA sensitivity. PnIA sensitivity of α3 mutants coexpressed with β2. Current responses to ACh (EC10 < ACh < EC75) after 5-min incubation with 1 μM PnIA is presented as a percentage of the preincubation ACh response (mean ± S.E.M.; n = 3–9). Significant differences from α3β2 were *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

For accurate determination of IC50 values in Fig. 4, coapplication of ACh and toxin after toxin incubation was necessary. In these directly competitive conditions, the ACh concentration used to activate the various receptors should be equipotent. ACh dose-response curves were constructed as described previously (Harvey and Luetje, 1996). We used a concentration of ACh at the EC50 value for each receptor (70 μM for α3β2, 25 μM for α3P182T β2, 35 μM for α3I188K β2, 80 μM for α3N191D/E194A β2, and 35 μM for α3Q198P β2). PnIA blockade was determined by comparing the peak current response to ACh and toxin coapplication after a 5-min incubation with toxin, to the average of three ACh-induced peak current responses preceding the toxin incubation. BSA was not included in the posttoxin incubation coapplication of ACh and toxin because preliminary experiments showed modest (23 ± 9%; n = 3), rapidly reversible (<1 s) inhibition of α3β2 receptors by BSA (100 μg/ml).

    Fig. 4.
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Fig. 4.

α-Conotoxin-PnIA inhibition of wild-type and mutant receptors. The response to an EC50 ACh concentration after a 5-min incubation with various concentration of PnIA and 100 μg/ml BSA is presented as a percentage of the preincubation response to ACh. Each point is the mean ± S.E.M. of three to five oocytes. The data are fit as described under Materials and Methods, and fit values are presented in Table 1. Some error bars are obscured by symbols. ACh concentrations used for each receptor were 70 μM for α3β2, 25 μM for α3P182Tβ2, 35 μM for α3I188Kβ2, 80 μM for α3N191D/E194Aβ2, and 35 μM for α3Q198Pβ2.

Data Analysis. Data were fit using PRISM 3cx software (Graph-Pad Software Inc., San Diego, CA). ACh dose-response data were fit using the equation I = Imax/[1 + (EC50/X)n], where I is the current response in the presence of agonist concentration X, Imax is the maximum current, EC50 is the agonist concentration producing the half-maximal current response, and n is the Hill coefficient. PnIA dose-inhibition data were fit using the equation: I = Imax/[1 + (X/IC50)n], where I is the current response in the presence of antagonist concentration X, Imax is the maximum current, IC50 is the antagonist concentration producing the half-maximal inhibition, and n is the Hill coefficient. IC50 values derived from Fig. 4 were used to determine differences in toxin sensitivity between α3β2 receptors and receptors formed by mutant subunits. In Fig. 4, an EC50 value for ACh was used in testing the toxin sensitivity of wild-type α3β2 and the various mutant receptors. Thus, the IC50 values are directly comparable.

Statistical significance was determined using a one-way analysis of variance followed by the Newman-Keuls post-test.

Generation of Homology Models. Sequence alignment of the amino-terminal extracellular region of the rat nicotinic subunits (α2, α3, and β2) with the AChBP was performed using the ALIGNX module of VECTOR NTI 5 (InforMax, Inc., Bethesda, MD). The alignments between the AChBP and α2, α3, and β2 exhibited identity values of 21.7, 20.8, and 18.4%, respectively. Although the sequence identity between the AChBP monomer and the neuronal nAChR extracellular domains is relatively low, the presence of highly conserved ACh binding residues in the AChBP (Brejc et al., 2001) and the nicotinic pharmacology of the AChBP (Smit et al., 2001) suggest that homology modeling of neuronal nAChR extracellular domains using the AChBP structure is appropriate (Le Novere et al., 2002).

Three-dimensional models were constructed using the program MODELLER 6 (Sali and Blundell, 1993) on a Silicon Graphics Indi-go2 Extreme workstation. The script “model” was used with neuronal nAChR subunit/AChBP alignments. Disulfide bonds in the AChBP template structure were explicitly included during homology model refinement. The amino-terminal extracellular domain sequences of the α3 (or α2) and β2 subunits were modeled, using the AChBP pentamer structure (PDB ID: 1I9B) to get initial coordinates for an α3β2 (or α2β2) pentamer (subunit ordering of αβαββ). Five to 10 models for each type of receptor were produced with energy refinement handled within the program. Various levels of refinement were assayed to find the protocol that produced the lowest energy structures. Conditions were optimized such that resulting structures exhibited energies in line with current published nicotinic receptor homology models (Le Novere et al., 2002).

Models were inspected visually and with PROCHECK (Laskowski et al., 1993) for inappropriate stereochemistry (e.g., clashing side chains and disallowed torsion angles). In only a few cases did residues require manual adjustment using the O software package (Jones et al., 1991). Further minimization was then carried out using the CNS software package (Brunger et al., 1998). Twenty cycles of conjugate gradient were performed until minimization was complete. CNS minimized structures were then reanalyzed by PROCHECK to check for stereochemical soundness. The image in Fig. 5 was produced using RIBBONS (Carson, 1997). Coordinates for the α3β2 and α2β2 models may be obtained at http://chroma.med.miami.edu/pharm/faculty_Luetje.html.

    Fig. 5.
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Fig. 5.

Location of proline 182, isoleucine 188, and glutamine 198 within a homology model of α3β2. A ribbons representation of the α3 subunit is displayed in dark gray, whereas the β2 subunit is shown in light gray. Determinants of PnIA sensitivity that we have identified are highlighted in black, with the side chains shown as balls and sticks. All three residues are part of the C loop of the α3 subunit (β strands 9 and 10 are indicated). Conserved aromatic residues at the ACh binding site (α3: tyr93, trp149, tyr190, and tyr197; β2: trp57) are shown in gray. Scale bar, 10 Å.

The orientations of the C-loops in different models were compared in LSQMAN (Kleywegt, 1999). Models were aligned using α-helix 1 and β strands 1 to 8 (numbered as in Brejc et al., 2001) of each monomer as a core region. Differences in the positions of α carbons within the C-loop were then measured. Each of the five to 10 different models produced for each receptor were also examined in O for differences in side chain orientation.

Results

α-CTx-PnIA Is Selective for α3β2 Receptors. We examined the sensitivity of several heteromeric neuronal nAChRs to blockade by PnIA (Fig. 1). The current response of an α3β2-expressing oocyte to 10 μM ACh is inhibited after a 5-min incubation with 1 μM PnIA (Fig. 1A, left). In contrast, the current response of an α2β2-expressing oocyte to 10 μM ACh is unaffected by 1 μM PnIA (Fig. 1A, right). In Fig. 1B, we show that although the α3β2 receptor was inhibited, with an IC50 value of 68 ± 12 nM, receptors containing a different α subunit (α2β2or α4β2) or a different β subunit (α3β4) were insensitive to 1 μM PnIA. Even 10 μM PnIA failed to significantly inhibit α2β2 receptors. These results are similar to what has been reported previously (Luo et al., 1999). We used this large difference in sensitivity between the α3β2 and α2β2 receptors as a probe to identify residues on the α3 subunit that confer high PnIA sensitivity. We selected a concentration of 1 μM PnIA to screen a series of receptors formed by chimeric and mutant subunits, because this toxin concentration substantially blocked α3β2 receptors but had no effect on α2β2 receptors.

Determinants of α-CTx-PnIA Are Located within Several Regions of the α3 Extracellular Domain. To determine which regions of the α3 sequence are responsible for PnIA sensitivity, a series of chimeric α subunits were expressed in combination with β2. When the amino-terminal extracellular domain of α3 is replaced by α2 sequence (α2-215-α3), the resulting receptor is insensitive to 1 μM PnIA (Fig. 2A). Conversely, when the entire amino-terminal extracellular domain of α2 is replaced by α3 sequence (α3-215-α2), the resulting receptor is as sensitive to 1 μM PnIA as wild-type α3β2 receptors (Fig. 2B). Thus, determinants of PnIA sensitivity are localized to the amino-terminal extracellular domain of the α3 subunit.

    Fig. 2.
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Fig. 2.

α-Conotoxin-PnIA sensitivity of receptors formed by chimeric α subunits. A, chimeras, constructed of various lengths of α3 sequence followed by α2 sequence, coexpressed with β2. B, chimeras, constructed of various lengths of α2 sequence followed by α3 sequence, coexpressed with β2. C, a chimera, constructed of α4 sequence followed by α3 sequence, coexpressed with β2. Current response to ACh (EC10 < ACh < EC75) after 5-min incubation with 1 μM PnIA is presented as a percentage of the preincubation ACh response (mean ± S.E.M.; n = 3–9). Significant differences from α3β2: **, p < 0.01; ***, p < 0.001. Significant differences from α2β2: †††, p < 0.001. Additional significant differences include the following: α2-121-α3β2 versus α2-84-α3β2(p < 0.01), α2-181-α3β2 versus α2-121-α3β2 (p < 0.05), α2-195-α3β2 versus α2-181-α3β2 (p < 0.001), α2-215-α3β2 versus α2-195-α3β2 (p < 0.001), α3-195-α2β2 versus α3-215-α2β2 (p < 0.001), and α4-183-α3β2 versus α4β2 (p < 0.001).

To localize critical residues to smaller regions of the α3 extracellular domain, we determined the effect of replacing smaller portions of the subunit sequence. In Fig. 2A portions of the α3 subunit were replaced with α2 sequence and receptors formed by the resulting chimeras were tested for loss of toxin sensitivity. Although replacement of the first 84 residues of α3 with α2 sequence (α2-84-α3) had no effect on PnIA sensitivity, replacement of the first 121 residues of α3 with α2 sequence (α2-121-α3) resulted in a significant loss of toxin sensitivity (Fig. 2A). This result suggests that at least one determinant of PnIA sensitivity lies within region 84 to 121 of the α3 subunit. Additional significant losses of PnIA sensitivity occur upon replacement of the first 181, 195, and 215 residues of α3 with α2 sequence (α2-181-α3, α2-195-α3, and α2-215-α3, respectively), suggesting the presence of additional determinants of PnIA sensitivity within the 121 to 181, 181 to 195, and 195 to 215 regions of the α3 subunit (Fig. 2A).

In Fig. 2B, portions of the α2 subunit were replaced with α3 sequence, and receptors formed by the resulting chimeras were tested for gain of toxin sensitivity. Replacement of the first 195 residues of the α2 subunit with α3 sequence resulted in a chimera (α3-195-α2) that formed receptors significantly more sensitive to PnIA than wild-type α2β2 receptors, but significantly less sensitive than receptors formed by the α3-215-α2 chimera (Fig. 2B). This result suggests the presence of determinants of toxin sensitivity within regions 1 to 195 and 195 to 215. Chimeras in which the first 84, 121, or 181 residues of α2 are replaced with α3 sequence could not be tested because these chimeric constructs fail to form functional receptors (Luetje et al., 1993).

Three Residues on the C-Loop of α3 Are Determinants of α-CTx-PnIA Sensitivity. Within the identified portions of the α3 extracellular domain, the residues of interest are those that differ between α3 and α2. To test the role of each of these residues, we determined the PnIA sensitivity of receptors formed by each of a series of mutant α3 subunits, in which the residue of interest was changed to what occurs in the α2 subunit. Within regions 181 to 195 and 195 to 215, the number of mutations to be tested is eight and five, respectively. Regions 84 to 121 and 121 to 181 are more problematic, with 13 and 15 mutations to be examined, respectively. In previous work with α-CTx-MII (Harvey et al., 1997), we found that although the α4 subunit is more homologous to α2 than to α3, receptors formed by an α4-183-α3 chimera are highly sensitive to toxin block but α2-181-α3-containing receptors display a loss of toxin sensitivity. This result suggested that α4 contains determinants of α-CTx-MII sensitivity that α2 lacks. In Fig. 2C, we find that the same is true for PnIA sensitivity. Although the α2-181-α3-containing receptors are less sensitive to PnIA block than wild-type α3β2, the α4–183-α3 receptors are as sensitive to toxin as α3β2. This result suggests a strategy for identifying determinants of PnIA sensitivity. Within region 84 to 181 of α3, residues that differ in α2, but are identical in α4, will be examined. This decreases the number of mutations to be examined within this region from 28 to eight.

Each mutant α3 subunit was coexpressed with β2 and tested for sensitivity to 1 μM PnIA. Although most of the mutations failed to affect toxin sensitivity, three mutations (P182T, I188K, and Q198P) caused significant losses in sensitivity and a double mutation (N191D/E194A) caused a significant increase in toxin sensitivity. All of these residues lie within regions 181 to 195 and 195 to 215. These two regions constitute the “C-loop” of the α3 subunit, a region known to be a critical component of the ACh binding site (Corringer et al., 2000; Brejc et al., 2001). We failed to identify any determinants within region 84 to 181. This may be due to multiple residues making only small contributions, or to a failure of our screening strategy in this region.

The four mutant α3 subunits that formed receptors with significant changes in toxin sensitivity (α3P182T, α3I188K, α3Q198P, and α3N191E/E194A) were examined in more detail in Fig. 4. To obtain accurate and directly comparable IC50 values for inhibition of wild-type and mutant receptors, we used a different protocol (see Materials and Methods). EC50 values for ACh at each receptor were derived from dose-response curves. These values are shown in Table 1 and are not significantly different. Responses to ACh (EC50) after toxin incubation were measured in the continued presence of toxin. This prevented any changes in toxin off-rate from affecting our measurements. The P182T, I188K, and Q198P mutations each caused a significant loss of PnIA sensitivity. Receptors formed by the α3P182T subunit were 3-fold less sensitive to PnIA blockade (IC50 = 84 ± 7 nM) than α3β2 (IC50 = 25 ± 4 nM). The α3I188K subunit formed receptors that were 28-fold less sensitive to toxin (IC50 = 700 ± 92 nM) than α3β2. The α3Q198P subunit formed receptors that were 35-fold less sensitive to PnIA (IC50 = 870 ± 61 nM) than α3β2. The toxin sensitivity of receptors formed by the α3N191E/E194A subunit (IC50 = 17 ± 4 nM) was not significantly different from the sensitivity of wild-type α3β2, contrasting with the significantly higher sensitivity of this double mutant to blockade at a high concentration of toxin (1 μM) in Fig. 3. This difference is due to the higher Hill coefficient of the inhibition curve for the N191E/E194A mutant (1.0 ± 0.1) compared with α3β2 (0.6 ± 0.1).

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TABLE 1

Acetylcholine and α-CTx-PnIA sensitivity of receptors formed by wildtype and mutant α3 subunits

EC50 and nH values for ACh, and IC50 and nH values for PnIA, were derived as described under Materials and Methods.

Despite the high sequence homology between the α3 and α6 subunits, only one of the three residues we have identified as determinants of sensitivity (I188) is present in α6. This suggested that receptors formed by α6 might not be sensitive to blockade by PnIA. Although functional receptors containing the rat α6 subunit can be difficult to express (Gerzanich et al., 1997), functional receptors can be formed by using a chimera constructed from the α6 extracellular domain and the remainder of α3 (Kuryatov et al., 2000). We found that such a chimera of rat α6 and rat α3 expressed more efficiently with β2 and β3, than with β2 alone. We tested the PnIA sensitivity of this α6-207-α3β2β3 receptor using the same protocol as in Fig. 1. Current responses to 1 μM ACh after 5-min incubation with 1 μM PnIA were 0.4 ± 0.05% (mean ± S.D.; n = 3) of the preincubation ACh response, indicating that α6-containing receptors are highly sensitive to PnIA. We also found that κ-bungarotoxin (κ-Bgt), another “α3β2-selective” toxin, effectively blocked the α6-207-α3β2β3 receptor with an IC50 of 3.2 ± 0.7 nM (n = 3).

Location of Determinants of α-CTx-PnIA Sensitivity within a Homology Structure of α3β2. Homology models of receptor extracellular domains were generated as described under Materials and Methods, using the structure of AChBP (Brejc et al., 2001) as template. An ACh binding interface of our α3β2 receptor model is shown in Fig. 5. Conserved aromatic residues at the ACh binding site (α3: tyr93, trp149, tyr190, and tyr197; β2: trp57) are shown in gray. Proline 182, isoleucine 188, and glutamine 198 of α3 are shown in black. The side chains of I188 in α3, K188 in α2 and K188 in α3I188K all extend toward the ACh binding site, but are relatively unconstrained in our models. When multiple models are compared, the positions of the distal atoms in these side chains varied by as much as 5 Å. The side chains of Q198 in α3, P198 in α2, and P198 in α3Q198P are near to, but extend away from, the ACh binding pocket. The glutamine at position 198 is moderately constrained, with the positions of the distal atoms varying by as much as 2.5 Å between models. As expected, the proline side chain atoms at position 198 are tightly constrained, varying by less than 1 Å between models. The side chains of P182 in α3, T182 in α2, and T182 in α3P182T are fairly distal from the binding pocket. The proline side chain atoms at this position were tightly constrained (<1-Å variation), whereas the threonine side chain was less constrained (up to 2.5-Å variation).

Discussion

Neuronal nicotinic receptors containing α3 or α2 subunits have dramatically different sensitivities to antagonists. For example, α3β2 receptors are at least 1000-fold more sensitive to blockade by PnIa, MII, and κ-Bgt than are α2β2 receptors (Fig. 1; Luetje et al., 1993; Cartier et al., 1996). Through analysis of receptors formed by chimeras of the α3 and α2 subunits, we found that determinants of PnIA sensitivity are widely distributed throughout the extracellular domain of the α3 subunit. By examining receptors formed by a series of mutant α3 subunits, we have identified three of these determinants as proline 182, isoleucine 188, and glutamine 198. Changing each of these residues in turn to what occurs in the α2 subunit results in 3-, 28-, and 35-fold losses of PnIA sensitivity, respectively. To assess the location of these residues within the receptor structure, we generated a homology model of the extracellular domain of the α3β2 receptor, using the atomic coordinates of the AChBP (Brejc et al., 2001) as a template. All three residues are located on the C-loop (β strands 9 and 10) of α3, with isoleucine 188 positioned nearest the ACh binding pocket.

The PnIA and MII toxins are both members of the α4/7 subfamily of α-conotoxins (four and seven residues between the cysteines) and have nearly identical peptide backbone conformations (Hu et al., 1996; Shon et al., 1997; Hill et al., 1998). Although both toxins antagonize α3β2 neuronal nAChRs, they differ at nine of 12 noncysteine residues, suggesting that the toxins interact with the receptor in different ways. Just such a difference became apparent in our work with chimeric subunits. Although both toxins interact with residues spread across a large portion of the α3 extracellular domain, PnIA seemed to interact with residues within region 195 to 215 (Fig. 2), whereas MII did not (Harvey et al., 1997). Within region 195 to 215 (β strand 10), we identified Q198 as a determinant of PnIA sensitivity (Figs. 3 and 4). Interestingly, although Q198 is not a determinant of MII sensitivity, it is a determinant of κ-Bgt sensitivity (Luetje et al., 1993). Within region 181 to 195 (β strand 9), I188 is a determinant of sensitivity to PnIA (Figs. 3 and 4), MII, and κ-Bgt (Harvey et al., 1997; Luetje et al., 1998). In contrast, P182 is a determinant of PnIA sensitivity (Figs. 3 and 4) but not MII or κ-Bgt sensitivity, whereas K185 is a determinant of MII and κ-Bgt but not PnIA sensitivity (Harvey et al., 1997; Luetje et al., 1998). It seems then that although PnIA and MII interact somewhat differently with the α3 subunit, they interact with the same general region (the C-loop). This contrasts with the situation for α-conotoxins ImI and ImII, which seem to interact with completely distinct regions of the α7 subunit (Ellison et al., 2003).

Although in initial work MII seemed to be selective for the α3β2 receptor (Cartier et al., 1996), it later became clear that MII also antagonizes neuronal nAChRs containing the α6 subunit (Gerzanich et al., 1997; Kuryatov et al., 2000). This is not surprising given the high homology between the α3 and α6 subunits and in particular the conservation of both identified determinants of MII sensitivity (K185 and I188) in the α6 subunit (Lamar et al., 1990). In contrast, only one of three identified determinants of PnIA sensitivity is conserved in α6 (I188), suggesting that α6-containing receptors might not be sensitive to this toxin. However, we found the α6-207-α3β2β3 receptor to be highly sensitive to PnIA blockade. This result, together with the proximity of I188 to the ACh binding site in our α3β2 ECD model, suggests position 188 as a critical determinant of pharmacological specificity in neuronal nAChRs.

To examine the physical location and potential role of each identified residue within the receptor, we constructed homology models of the α3β2 and α2β2 receptor extracellular domains. We also constructed models for each of the mutant α3β2 receptor extracellular domains. Our models did not differ substantially from the structure of the template (AChBP). Comparison of our α2, α3 and β2 structures with the AChBP monomer structure yielded Cα root mean squared deviations of 0.62, 0.65, and 0.62 Å, respectively. This is similar to what has been reported (0.71 Å) for a similarly modeled structure of the α7 neuronal nAChR extracellular domain (Le Novere et al., 2002). Of course, our models are static structures, whereas the receptors themselves (and most likely the AChBP) are dynamic. It is unclear what state of the receptor these models represent, and thus our models should be seen as approximations of the interaction site for the toxin.

Our identification of I188 as a major determinant of PnIA, MII, and κ-Bgt sensitivity, and the proximity of this residue to the putative ACh binding pocket, suggest it as a major determinant of pharmacological specificity on the α3 subunit. It is likely that this residue interacts directly with the toxin, or that substitution with lysine is directly repulsive to the toxin. The roles of P182 and Q198 are less clear. It is possible that these positions are interacting directly with the toxin. The approximate distance between I188 and P182 (19 Å) could be spanned by PnIA at its longest dimension (approximately 18 Å), but it is also possible that the loss of a proline at position 182, or the gain of a proline at position 198, might alter the conformation of the C-loop. To examine this possibility, we aligned a core region of the α3 and α2 subunits from our best models (see Materials and Methods). A comparison of the C-loops of α3 and α2 yielded a Cα root mean squared deviations of 0.28Å, suggesting little difference in C-loop orientation between the two subunits. We also examined the orientation of two conserved residues in the C-loop that form part of the ACh binding pocket, Y190 and Y197. The positions of the hydroxyl oxygens of each of these tyrosines differed by less than 1 Å between the α3 and α2 subunits and the aromatic rings had identical orientations. Because these results are based on modeled structures, we cannot say for certain that the C-loops of α2 and α3 have similar conformations. However, we can say that the differing residues at positions 182 and 198 of α3 and α2 do not require a major reorientation of the C-loops of these subunits. Another possibility is that the loss (or gain) of prolines at positions 182 and 198 causes a change in the flexibility (or rigidity) of the C-loop, and may alter the dynamics of this region upon ACh binding.

Through analysis of receptors formed by chimeric and mutant subunits, we have identified three determinants of α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nAChRs. Our development of a homology model of the α3β2 receptor extracellular domain shows these determinants to be located within the C-loop of the α3 subunit. Continued use of these techniques to refine our understanding of the structure of the ligand binding sites on neuronal nAChRs should aid in the development of subtype-selective ligands.

Acknowledgments

We thank Floyd Maddox and Ana Mederos for excellent technical assistance.

Footnotes

  • This work was supported by National Institutes of Health Grants DA08102 (to C.W.L.) and MH53631 (to J.M.M.). A.M. was supported in part by awards from the Florida Biomedical Research Foundation (BM030) and the American Heart Association, Florida/Puerto Rico Affiliate (SDG-0130456B). D.E. was supported in part by T32-HL07188.

  • DOI: 10.1124/jpet.103.051656.

  • ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine binding protein; ACh, acetylcholine; BSA, bovine serum albumin; MII, α-conotoxin-MII; PnIA, α-conotoxin-PnIA; κ-Bgt, κ-bungarotoxin.

  • ↵1 Current address: Cornell Veterinary School, Ithaca, NY 14853.

    • Received March 28, 2003.
    • Accepted May 5, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics

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Journal of Pharmacology and Experimental Therapeutics: 306 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 306, Issue 2
1 Aug 2003
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Research ArticleCELLULAR AND MOLECULAR

Identification of Residues That Confer α-Conotoxin-PnIA Sensitivity on the α3 Subunit of Neuronal Nicotinic Acetylcholine Receptors

Drew Everhart, Edward Reiller, Armen Mirzoian, J. Michael McIntosh, Arun Malhotra and Charles W. Luetje
Journal of Pharmacology and Experimental Therapeutics August 1, 2003, 306 (2) 664-670; DOI: https://doi.org/10.1124/jpet.103.051656

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Research ArticleCELLULAR AND MOLECULAR

Identification of Residues That Confer α-Conotoxin-PnIA Sensitivity on the α3 Subunit of Neuronal Nicotinic Acetylcholine Receptors

Drew Everhart, Edward Reiller, Armen Mirzoian, J. Michael McIntosh, Arun Malhotra and Charles W. Luetje
Journal of Pharmacology and Experimental Therapeutics August 1, 2003, 306 (2) 664-670; DOI: https://doi.org/10.1124/jpet.103.051656
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