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CELLULAR AND MOLECULAR
-Conotoxin-PnIA Sensitivity on the 3 Subunit of Neuronal Nicotinic Acetylcholine Receptors
Departments of Molecular and Cellular Pharmacology (D.E., A.M., C.W.L.), Biochemistry and Molecular Biology (A.M.), and Neuroscience Graduate Program (E.R.), University of Miami School of Medicine, Miami, Florida; and Departments of Psychiatry and Biology, University of Utah, Salt Lake City, Utah (J.M.M.)
Received March 28, 2003; accepted May 5, 2003.
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Abstract |
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3 and 
2 subunits are at least 1000-fold more sensitive to blockade by -conotoxin-PnIA than are 22 receptors. A series of chimeric subunits, formed from portions of 2 and 3, were coexpressed with 2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of 3. Analysis of receptors formed by a series of mutant 3 subunits, in which residues that differ between 3 and 2 were changed from what occurs in 3 to what occurs in 2, allowed identification of three determinants of -conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of -conotoxin-MII and 
-bungarotoxin sensitivity on the 3 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 -conotoxin-PnIA blockade was 25 ± 4 nM for 32, 84 ± 7 nM for 3P182T2, 700 ± 92 nM for 3I188K2, and 870 ± 61 nM for 3Q198P2. To examine the location of these residues within the receptor structure, we generated a homology model of the 32 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 3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.
). 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, 2–10 and 2-4 (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 7 receptors), as simple heteropentamers of one type of subunit and one type of subunit (such as 42 receptors) and as complex heteropentamers of three or more subunits (such as 354 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 -conotoxin family displaying various selectivities for muscle and neuronal nAChRs (McIntosh et al., 1999). The -conotoxins MII from C. magus, PnIA from C. pennaceus, and GIC from C. geographus all selectively antagonize 32 neuronal nAChRs (Cartier et al., 1996; Hogg et al., 1999; Luo et al., 1999; McIntosh et al., 2002). We previously identified residues on both 3 (K185 and I188) and 2 (T59) that confer sensitivity to MII (Harvey et al., 1997). Recently, it became clear that MII also antagonizes neuronal nAChRs containing 6, a subunit highly homologous to 3 (Gerzanich et al., 1997). Whether PnIA and GIC antagonize 6-containing receptors is not currently known. The similar ability of PnIA and MII to antagonize 32 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 32 receptor or, as has been demonstrated for antagonism of 7 receptors by the -conotoxins ImI and ImII (Ellison et al., 2003), could act at different sites. We used a series of chimeric and mutant subunits to identify residues that confer sensitivity to PnIA and to determine the relationship, if any, to the residues that determine MII sensitivity.
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Materials and Methods |
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).
Expression of Neuronal nAChRs in X. laevis Oocytes. cDNA clones encoding rat 2, 3, 4, 2, and 4 subunits, as well as 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 3 subunit sequence), and then followed by the source of the carboxyl-terminal portion. For example, the chimeric subunit 2-195-3 is composed of 2 sequence from the amino terminus until residue 195, after which it is composed of 3 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, 3I188K is an 3 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 M
. 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 32 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.
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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 32, 25 µM for 3P182T 2, 35 µM for 3I188K 2, 80 µM for 3N191D/E194A 2, and 35 µM for 3Q198P 2). 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 32 receptors by BSA (100 µg/ml).
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32 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 32 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 (2, 3, and 2) with the AChBP was performed using the ALIGNX module of VECTOR NTI 5 (InforMax, Inc., Bethesda, MD). The alignments between the AChBP and 2, 3, and 2 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 3 (or 2) and 2 subunits were modeled, using the AChBP pentamer structure (PDB ID: 1I9B
[PDB]
) to get initial coordinates for an 32 (or 22) pentamer (subunit ordering of ). 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 32 and 22 models may be obtained at http://chroma.med.miami.edu/pharm/faculty_Luetje.html.
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The orientations of the C-loops in different models were compared in LSQMAN (Kleywegt, 1999). Models were aligned using -helix 1 and strands 1 to 8 (numbered as in Brejc et al., 2001) of each monomer as a core region. Differences in the positions of 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.
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Results |
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-CTx-PnIA Is Selective for 32 Receptors. We examined the sensitivity of several heteromeric neuronal nAChRs to blockade by PnIA (Fig. 1). The current response of an 32-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 22-expressing oocyte to 10 µM ACh is unaffected by 1 µM PnIA (Fig. 1A, right). In Fig. 1B, we show that although the 32 receptor was inhibited, with an IC50 value of 68 ± 12 nM, receptors containing a different subunit (22or 42) or a different subunit (34) were insensitive to 1 µM PnIA. Even 10 µM PnIA failed to significantly inhibit 22 receptors. These results are similar to what has been reported previously (Luo et al., 1999). We used this large difference in sensitivity between the 32 and 22 receptors as a probe to identify residues on the 3 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 32 receptors but had no effect on 22 receptors.
Determinants of -CTx-PnIA Are Located within Several Regions of the 3 Extracellular Domain. To determine which regions of the 3 sequence are responsible for PnIA sensitivity, a series of chimeric subunits were expressed in combination with 2. When the amino-terminal extracellular domain of 3 is replaced by 2 sequence (2-215-3), the resulting receptor is insensitive to 1 µM PnIA (Fig. 2A). Conversely, when the entire amino-terminal extracellular domain of 2 is replaced by 3 sequence (3-215-2), the resulting receptor is as sensitive to 1 µM PnIA as wild-type 32 receptors (Fig. 2B). Thus, determinants of PnIA sensitivity are localized to the amino-terminal extracellular domain of the 3 subunit.
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To localize critical residues to smaller regions of the 3 extracellular domain, we determined the effect of replacing smaller portions of the subunit sequence. In Fig. 2A portions of the 3 subunit were replaced with 2 sequence and receptors formed by the resulting chimeras were tested for loss of toxin sensitivity. Although replacement of the first 84 residues of 3 with 2 sequence (2-84-3) had no effect on PnIA sensitivity, replacement of the first 121 residues of 3 with 2 sequence (2-121-3) 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 3 subunit. Additional significant losses of PnIA sensitivity occur upon replacement of the first 181, 195, and 215 residues of 3 with 2 sequence (2-181-3, 2-195-3, and 2-215-3, 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 3 subunit (Fig. 2A).
In Fig. 2B, portions of the 2 subunit were replaced with 3 sequence, and receptors formed by the resulting chimeras were tested for gain of toxin sensitivity. Replacement of the first 195 residues of the 2 subunit with 3 sequence resulted in a chimera (3-195-2) that formed receptors significantly more sensitive to PnIA than wild-type 22 receptors, but significantly less sensitive than receptors formed by the 3-215-2 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 2 are replaced with 3 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 3 Are Determinants of -CTx-PnIA Sensitivity. Within the identified portions of the 3 extracellular domain, the residues of interest are those that differ between 3 and 2. To test the role of each of these residues, we determined the PnIA sensitivity of receptors formed by each of a series of mutant 3 subunits, in which the residue of interest was changed to what occurs in the 2 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 -CTx-MII (Harvey et al., 1997), we found that although the 4 subunit is more homologous to 2 than to 3, receptors formed by an 4-183-3 chimera are highly sensitive to toxin block but 2-181-3-containing receptors display a loss of toxin sensitivity. This result suggested that 4 contains determinants of -CTx-MII sensitivity that 2 lacks. In Fig. 2C, we find that the same is true for PnIA sensitivity. Although the 2-181-3-containing receptors are less sensitive to PnIA block than wild-type 32, the 4–183-3 receptors are as sensitive to toxin as 32. This result suggests a strategy for identifying determinants of PnIA sensitivity. Within region 84 to 181 of 3, residues that differ in 2, but are identical in 4, will be examined. This decreases the number of mutations to be examined within this region from 28 to eight.
Each mutant 3 subunit was coexpressed with 2 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 3 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 3 subunits that formed receptors with significant changes in toxin sensitivity (3P182T, 3I188K, 3Q198P, and 3N191E/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 3P182T subunit were 3-fold less sensitive to PnIA blockade (IC50 = 84 ± 7 nM) than 32 (IC50 = 25 ± 4 nM). The 3I188K subunit formed receptors that were 28-fold less sensitive to toxin (IC50 = 700 ± 92 nM) than 32. The 3Q198P subunit formed receptors that were 35-fold less sensitive to PnIA (IC50 = 870 ± 61 nM) than 32. The toxin sensitivity of receptors formed by the 3N191E/E194A subunit (IC50 = 17 ± 4 nM) was not significantly different from the sensitivity of wild-type 32, 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 32 (0.6 ± 0.1).
Despite the high sequence homology between the 3 and 6 subunits, only one of the three residues we have identified as determinants of sensitivity (I188) is present in 6. This suggested that receptors formed by 6 might not be sensitive to blockade by PnIA. Although functional receptors containing the rat 6 subunit can be difficult to express (Gerzanich et al., 1997), functional receptors can be formed by using a chimera constructed from the 6 extracellular domain and the remainder of 3 (Kuryatov et al., 2000). We found that such a chimera of rat 6 and rat 3 expressed more efficiently with 2 and 3, than with 2 alone. We tested the PnIA sensitivity of this 6-207-323 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 6-containing receptors are highly sensitive to PnIA. We also found that -bungarotoxin (-Bgt), another "32-selective" toxin, effectively blocked the 6-207-323 receptor with an IC50 of 3.2 ± 0.7 nM (n = 3).
Location of Determinants of -CTx-PnIA Sensitivity within a Homology Structure of 32. 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 32 receptor model is shown in Fig. 5. Conserved aromatic residues at the ACh binding site (3: tyr93, trp149, tyr190, and tyr197; 2: trp57) are shown in gray. Proline 182, isoleucine 188, and glutamine 198 of 3 are shown in black. The side chains of I188 in 3, K188 in 2 and K188 in 3I188K 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 3, P198 in 2, and P198 in 3Q198P 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 3, T182 in 2, and T182 in 3P182T 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).
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3 or 2 subunits have dramatically different sensitivities to antagonists. For example, 32 receptors are at least 1000-fold more sensitive to blockade by PnIa, MII, and -Bgt than are 22 receptors (Fig. 1; Luetje et al., 1993; Cartier et al., 1996). Through analysis of receptors formed by chimeras of the 3 and 2 subunits, we found that determinants of PnIA sensitivity are widely distributed throughout the extracellular domain of the 3 subunit. By examining receptors formed by a series of mutant 3 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 2 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 32 receptor, using the atomic coordinates of the AChBP (Brejc et al., 2001) as a template. All three residues are located on the C-loop ( strands 9 and 10) of 3, with isoleucine 188 positioned nearest the ACh binding pocket.
The PnIA and MII toxins are both members of the 4/7 subfamily of -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 32 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 3 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 ( 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 -Bgt sensitivity (Luetje et al., 1993). Within region 181 to 195 ( strand 9), I188 is a determinant of sensitivity to PnIA (Figs. 3 and 4), MII, and -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 -Bgt sensitivity, whereas K185 is a determinant of MII and -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 3 subunit, they interact with the same general region (the C-loop). This contrasts with the situation for -conotoxins ImI and ImII, which seem to interact with completely distinct regions of the 7 subunit (Ellison et al., 2003).
Although in initial work MII seemed to be selective for the 32 receptor (Cartier et al., 1996), it later became clear that MII also antagonizes neuronal nAChRs containing the 6 subunit (Gerzanich et al., 1997; Kuryatov et al., 2000). This is not surprising given the high homology between the 3 and 6 subunits and in particular the conservation of both identified determinants of MII sensitivity (K185 and I188) in the 6 subunit (Lamar et al., 1990). In contrast, only one of three identified determinants of PnIA sensitivity is conserved in 6 (I188), suggesting that 6-containing receptors might not be sensitive to this toxin. However, we found the 6-207-323 receptor to be highly sensitive to PnIA blockade. This result, together with the proximity of I188 to the ACh binding site in our 32 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 32 and 22 receptor extracellular domains. We also constructed models for each of the mutant 32 receptor extracellular domains. Our models did not differ substantially from the structure of the template (AChBP). Comparison of our 2, 3 and 2 structures with the AChBP monomer structure yielded C 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 7 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 -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 3 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 3 and 2 subunits from our best models (see Materials and Methods). A comparison of the C-loops of 3 and 2 yielded a C 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 3 and 2 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 2 and 3 have similar conformations. However, we can say that the differing residues at positions 182 and 198 of 3 and 2 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 -conotoxin-PnIA sensitivity on the 3 subunit of neuronal nAChRs. Our development of a homology model of the 32 receptor extracellular domain shows these determinants to be located within the C-loop of the 3 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.
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Footnotes |
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ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine binding protein; ACh, acetylcholine; BSA, bovine serum albumin; MII, -conotoxin-MII; PnIA, -conotoxin-PnIA; -Bgt, -bungarotoxin.
1 Current address: Cornell Veterinary School, Ithaca, NY 14853. 
Address correspondence to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101. E-mail: cluetje{at}chroma.med.miami.edu
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