Introduction

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Nicotinic acetylcholine receptors are expressed throughout the central and peripheral nervous systems. Neuronal nAChRs are similar to neuromuscular nAChRs in that they are thought to be pentameric assemblies of subunits surrounding a central ion pore (Anand et al., 1991; Cooper et al., 1991). There are currently twelve known neuronal nAChR subunits (2-10, 2-4) (Corringer et al., 2000; Elgoyhen et al., 2001). Many different combinations of these subunits can assemble to form functional nAChRs when expressed in Xenopus laevis oocytes or mammalian cell lines, with each functional subunit combination displaying a distinct array of biophysical and pharmacological properties (Role, 1992). Within the pentameric structure of neuronal nAChRs, the actual subunit composition can range from simple to complex. In exogenous expression experiments, functional receptors can be formed as homopentamers of the 7 subunit, as simple heteropentamers of an  subunit (2, 3, 4, or 6), and a  subunit (2 or 4), or as various complex combinations of three or more of the 2-6 and 2-4 subunits (Corringer et al., 2000). Relating these observations to the situation in neurons has been difficult; however, examples of homopentamers (7) (Chen and Patrick, 1997; Drisdel and Green, 2000), simple heteropentamers (42) (Whiting et al., 1991; Flores et al., 1992), and complex heteropentamers (354) (Conroy and Berg, 1995) have been observed.

The ligand binding sites of neuronal nAChRs appear to be formed at the interface between two subunits. This is analogous to the situation for the neuromuscular nAChR where the two ligand binding sites are located at the interface between an  subunit and either the / subunit or the  subunit (Corringer et al., 2000). The recent publication of the crystal structure of a soluble, pentameric ACh-binding protein (AChBP) secreted from molluscan glia supports the positioning of the ligand binding site at the interface between subunits (Brejc et al., 2001). In simple heteropentameric neuronal nAChRs, the ligand binding sites would then be located at the interface between the  subunit (2, 3, 4, or 6) and the  subunit (2 or 4). This is supported by observations that the pharmacological properties of these neuronal nAChRs are determined by the identities of both the  and  subunits that form the receptor (Luetje and Patrick, 1991; Hussy et al., 1994; Parker et al., 1998). Furthermore, specific amino acid residues have been identified on both  and  subunits that play a role in determining the pharmacological properties of the receptors (Figl et al., 1992; Luetje et al., 1993, 1998; Cohen et al., 1995; Harvey and Luetje, 1996; Harvey et al., 1997).

We demonstrated previously that  subunits are important in determining the sensitivity of neuronal nAChRs to activation by agonists (Luetje and Patrick, 1991) and blockade by antagonists (Harvey and Luetje, 1996). Residue 59 of the 2 subunit was found to be particularly important in determining sensitivity to the antagonists neuronal bungarotoxin, -conotoxin-MII, and dihydro--erythroidine (Harvey and Luetje, 1996; Harvey et al., 1997). We recently examined the equilibrium agonist binding affinities of the six simple heteropentameric neuronal nAChRs that can be formed in X. laevis oocytes upon expression of 2, 3, or 4 with 2 or 4 (Parker et al., 1998). Although each subunit combination had a distinct agonist pharmacology, the most striking finding was that the  subunits had a profound effect on the equilibrium agonist binding affinity of the receptors. Receptors containing the 2 subunit consistently had much higher affinities for agonists than did 4-containing receptors.

There are several regions of the  subunit that could be involved in determining agonist binding affinity. Although the  subunit supplies the "principal component" of the binding site, which consists of the A, B, and C sequence loops, the  subunit supplies the "complementary component", consisting of the D, E, and F loops of sequence (Corringer et al., 2000; Brejc et al., 2001). The D loop is located roughly within sequence segment 50-70 and contains threonine 59 of 2 (lysine 61 in 4) that we have identified previously as a critical determinant of competitive antagonist sensitivity (Harvey and Luetje, 1996; Harvey et al., 1997). The E and F loops are located within sequence segments 100-120 and 160-180, respectively. We have now used a series of chimeric and mutant  subunits to demonstrate that several residues within the D loop sequence are important in determining the equilibrium agonist binding affinity of neuronal nAChRs.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. X. laevis frogs were purchased from Nasco (Ft. Atkinson, WI). Care and use of X. laevis frogs in this study has been approved by the University of Miami Animal Research Committee and meets the guidelines of the National Institutes of Health. RNA transcription kits were obtained from Ambion (Austin, TX). [³H]Epibatidine was purchased from PerkinElmer Life Science Products (Boston, MA). Acetylcholine, DMPP, nicotine, gentamicin, HEPES, polyethylenimine, and 3-aminobenzoic acid ethyl ester were purchased from Sigma (St. Louis, MO). Collagenase B was obtained from Roche Molecular Biochemicals (Indianapolis, IN). 934-AH glass microfiber filters were obtained from Whatman (Clifton, NJ).

Expression of Neuronal nAChRs in X. laevis Oocytes. cDNA clones encoding rat 2, 2, and 4 subunits, as well as the  chimeras and  mutants engineered into the pGEMHE expression vector (Liman et al., 1992) were used as template for cRNA production. Chimeric and mutant  subunits were constructed as described previously (Harvey and Luetje, 1996). Our notation for chimeric subunits is to list the subunit from which the N-terminal sequence is derived, followed by the position at which the chimeric joint is made, followed by the subunit from which the C-terminal sequence is derived. For example, the chimeric subunit 2-54-4 consists of 2 sequence from the N-terminal until residue 54 followed by 4 sequence from residue 54 until the C-terminal. Our notation for mutant subunits is to list the naturally occurring residue, followed by the position of that residue, followed by the change that has been made. For example, the mutant subunit 2-T59K is a 2 subunit in which threonine 59 has been changed to a lysine.

Preparation of X. laevis Oocyte Homogenates and [³H]Epibatidine Binding Assays. Crude membrane homogenates were prepared from X. laevis oocytes expressing wild-type, chimeric, and mutant receptors as described previously (Parker et al., 1998). Briefly, 0.25 to 15 oocytes (depending on expression levels) were homogenized per milliliter of buffer (140 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 25 mM HEPES, pH 7.5, containing freshly added 0.1 mM phenylmethylsulfonyl fluoride), using a model PT 10/35 homogenizer (Brinkmann, Atlanta, GA). Homogenates were centrifuged at 4°C at 2000g for 10 min. The supernatant was removed for use in experiments, avoiding both the surface lipid layer and the pellet. Receptor expression levels averaged 480 fmol/mg protein (16 fmol/oocyte). In previous work (Parker et al., 1998), we determined that the radioligand binding properties of neuronal nAChRs assayed in crude membrane homogenates or more purified membrane preparations were indistinguishable.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   The D loop region of  subunits is important in determining epibatidine affinity. A, saturation analysis of specific [3H]epibatidine binding to membrane homogenates of X. laevis oocytes expressing rat 22 (), 24 (), 22-54-4 (), or 22-63-4 (). Data are the mean ± S.E.M. of three to six separate experiments, each performed in triplicate. Data were fit as described under Experimental Procedures. B, diagram of  subunit chimeras. The location of the D, E, and F loops within the extracellular domain sequence of  subunits (extending from the N terminus to transmembrane domain 1 at approximately residue 210) is shown at the top. The location of 2 (hatched bars) and 4 (open bars) sequence within the extracellular domain of each chimera is shown below. C, [3H]epibatidine affinity of receptors formed by 2 in combination with either 2, 4, or a series of chimeric  subunits. KD values were derived from fit data as in panel A. Dashed lines are drawn to indicate the KD values for 22 and 24.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   The D loop region of  subunits is important in determining ACh, nicotine, and DMPP affinity. Affinity of receptors formed by 2 in combination with either 2, 4, or a series of chimeric  subunits for ACh (A), nicotine (B), and DMPP (C). KI values were calculated from IC50 values that were obtained in competition assays, as described under Experimental Procedures. Dashed lines are drawn to indicate the KI values for 22 and 24. Data are the mean ± S.E.M. of two to five separate experiments, each performed in sextuplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of mutations within the D loop of 2 on [3H]epibatidine affinity. A, alignment of 2 and 4 sequence within the 54-63 region. Residues that differ are indicated with asterisks. Conserved W57 is indicated with a dot. B, saturation analysis of specific [3H]epibatidine binding to membrane homogenates of X. laevis oocytes expressing rat 22 (), 24 (), 22N55S (), or 22T59K(). Data are the mean ± S.E.M. of three to eight separate experiments, each performed in triplicate. C, [3H]epibatidine affinity of receptors formed by 2 in combination with either 2, 4, or a series of mutant 2 subunits. KD values were derived from fit data as in panel B. Dashed lines are drawn to indicate the KD values for 22 and 24.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of mutations within the D loop of 2 on ACh, nicotine, and DMPP affinity. Affinity of receptors formed by 2 in combination with either 2, 4, or a series of mutant 2 subunits for ACh (A), nicotine (B), and DMPP (C). KI values were calculated from IC50 values that were obtained in competition assays, as described under Experimental Procedures. Dashed lines are drawn to indicate the KI values for 22 and 24. Data are the mean ± S.E.M. of two to four experiments, each performed in sextuplicate.

Data Analysis. Data from saturation experiments were analyzed using the equation: B = (B_max · Lⁿ)/(K_Dⁿ + Lⁿ), where B is the binding at free ligand concentration L, B_max is the maximum specific binding, K_D is the equilibrium dissociation constant, and n is the Hill coefficient. Values for B_max, K_D, and n were calculated by nonlinear regression. IC₅₀ values were derived using the equation: B = B_o/[1 + (I/IC₅₀)ⁿ], where B is ligand bound at competitor concentration I, B_o is binding in the absence of competitor, IC₅₀ is the concentration of ligand that reduces the specific binding by one-half, and n is the Hill coefficient. K_I values were calculated using the Cheng and Prusoff equation: K_I = IC₅₀/[1 + ([L]/K_D)] (Cheng and Prusoff, 1973). Because of the variation in receptor expression level from day to day after injection of the oocytes and among oocyte batches, all results were normalized as the percentage of maximum specific binding. Prism software (GraphPad, San Diego, CA) was used to fit the data and to assess statistical significance using a two-tailed unpaired t test.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Region 54-63 of  Subunits is Critical for Determining Agonist Affinity. Previously, we found that receptors containing the 2 subunit had consistently higher affinities for agonists than did receptors containing the 4 subunit (Parker et al., 1998). Amino acid residues responsible for these differences in agonist affinity might be located within one of the three loops of the complementary component (D, E, and F) or within some previously unidentified sequence segment of  subunits. To determine which region should be examined in detail, we expressed receptors in X. laevis oocytes using a series of chimeric  subunits. The chimeric subunits consisted of portions of the 2 and 4 subunits. The structure of these chimeras is presented diagrammatically in Fig. 1B. In our previous work (Parker et al., 1998), the largest differences in agonist affinity were observed when the  subunits were coexpressed with the 2 subunit. For this reason, all chimeric and mutant  subunits in the current study are coexpressed with the 2 subunit.

View this table: [in this window] [in a new window]   TABLE 1 Agonist binding affinities of receptors formed by wild-type and chimeric neuronal nAChR subunits KD values for [3H]epibatidine were taken from the fit data in Fig. 1. KI values for acetylcholine, DMPP, and nicotine were calculated from the IC50 values determined in competition assays, as described under Experimental Procedures. Wild-type KD and KI values were taken from Parker et al. (1998). All  subunits were coexpressed with 2.

Multiple Residues within Region 54-63 Determine Agonist Affinity. Our results indicate that the 54-63 region of the  subunit is a major determinant of epibatidine, ACh, nicotine, and DMPP affinity. An alignment of this region from the 2 and 4 subunits is shown in Fig. 3A. These two subunits differ at positions 55, 56, 59, and 63. One or more of these residues could be responsible for the affinity differences we have mapped to this region.

View this table: [in this window] [in a new window]   TABLE 2 Agonist binding affinities of receptors formed by wild type and mutant neuronal nAChR subunits KD values for [3H]epibatidine were taken from the fit data in Fig. 3. KI values for acetylcholine, DMPP, and nicotine were calculated from the IC50 values determined in competition assays, as described under Experimental Procedures. All mutations are made in the 2 subunit. All  subunits are coexpressed with 2.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Neuronal nAChRs display a wide range of equilibrium binding affinities for agonists. We demonstrated previously that the primary determinant of agonist affinity for simple heteromeric receptors was the identity of the  subunit, with 2-containing receptors having consistently higher affinities for agonists than 4-containing receptors (Parker et al., 1998). We have now used chimeric and mutant  subunits to demonstrate that several amino acid residues within the region known as the D loop are important in determining agonist binding affinity.

Within the D loop region, the 2 and 4 subunits differ at only four positions. Mutation at each of these positions in 2 resulted in a significant loss in both ACh and nicotine affinity. Although experiments in Fig. 2 suggest that the D loop accounts for a large fraction of the difference in affinity between 2 and 4 receptors, the loss of affinity for each mutant was relatively small. The largest effect for both ACh and nicotine affinity occurred with the T59K mutation and was only 7- to 8-fold. However, when considered together, the effects of the four mutations more than account for the differences in ACh and nicotine affinity between 2- and 4-containing receptors. Each of the four mutations had only a minimal (<2-fold) effect on epibatidine affinity. This is not surprising given the modest difference in epibatidine affinity between 2 and 4 receptors. What was surprising was the lack of effect of any of the mutations on DMPP affinity. The largest difference in agonist affinity between 2 and 4 receptors was seen with DMPP, and results in Fig. 2 clearly indicate the importance of the 54-63 region. However, Fig. 2 also indicates that region 1-54 plays an important role in determining DMPP affinity. Thus, it may be that loss of any one determinant in the D loop, although leaving intact other D loop determinants and region 1-54, is not sufficient to destabilize the high affinity binding of DMPP.

The T59K mutation had the largest effect on ACh and nicotine affinity. The most obvious change in side chain character is the change from a polar hydroxyl group to a positive charge. In previous work, we found that the 2-T59K mutation decreased the sensitivity of the 32 receptor to antagonism by neuronal bungarotoxin (Harvey and Luetje, 1996). When a negative charge was introduced at this position (T59D), sensitivity to neuronal bungarotoxin was increased, suggesting that the decreased sensitivity of the T59K mutant was indeed due to the introduction of the positive charge. To determine whether side chain charge at position 59 is also the critical factor in determining agonist affinity, we examined the agonist affinity of receptors formed by 2-T59D. In contrast to the increased neuronal bungarotoxin sensitivity, we found that the T59D mutation decreased ACh and nicotine affinity. This result suggests that some other change in side chain property, such as loss of the hydroxyl, is important in determining ACh and nicotine affinity.

All of the work presented in this study involves radioligand binding assays of mammalian neuronal nAChR expressed in X. laevis oocytes. Exogenous expression of mammalian receptors in a nonmammalian system raises concern regarding the accuracy and relevance of the results. Specifically, are the pharmacological properties of neuronal nAChRs that we measure using the oocyte expression system an accurate reflection of the properties that these receptors would have in a mammalian context? We have previously provided evidence that this is the case for several different subtypes of nAChR. The agonist pharmacology of mouse muscle nAChRs expressed in oocytes (Luetje and Patrick, 1991) was similar to the pharmacology of the same receptors natively expressed by BC3H-1 cells (Sine and Steinbach, 1986, 1987). Rat 42 receptors expressed in oocytes displayed affinities for agonists and antagonists (Parker et al., 1998) that were similar to those of 42 receptors expressed in rat brain (Pabreza et al., 1991). Rat 34 receptors expressed in oocytes (Parker et al., 1998) displayed agonist binding affinities that were similar to those of 34 receptors expressed by rat trigeminal ganglia neurons (Flores et al., 1996) and to those of rat 34 receptors exogenously expressed in HEK 293 cells (Xiao et al., 1998). Rat 22 and 24 receptors expressed in oocytes (Parker et al., 1998) displayed [³H]epibatidine binding affinities that were similar to those of rat 22 and 24 receptors exogenously expressed in HEK 293 cells (Xiao et al., 1996). These results suggest that mammalian nAChRs expressed in X. laevis oocytes display accurate pharmacological properties.

The recent publication of the crystal structure of a soluble ACh-binding protein secreted by molluscan glia provides new insight into the structure of the ligand binding site of nAChRs (Brejc et al., 2001). The AChBP is pentameric, has a nicotinic pharmacology, and possesses many of the residues thought to be critical participants in the ligand binding site of nAChRs. Thus, it seems likely that the extracellular domains of neuronal nAChR subunits will conform, at least approximately, to this structure. The "D loop" region of nAChR subunits corresponds to the second  strand of the AChBP structure. Within this region, side chains of residues in AChBP that correspond to N55, T59, and E63 of 2, all extend toward the binding site, providing a clear explanation for why alteration of these residues causes changes in agonist binding affinity. The side chain of the residue in AChBP that corresponds to V56 of 2 faces away from the binding site and into the hydrophobic core of the protein. The V56I mutation, which increases the side chain volume, might alter the position of the D loop region and thus the position of the side chains extending into the binding site.

The current model of nAChR agonist binding site structure consists of three loops of amino acid sequence (A, B, and C) from the  subunit forming a principal component and three loops of sequence (D, E, and F) from the non- subunit (, , or  in muscle nAChRs,  in heteromeric neuronal nAChRs) forming a complementary component (Corringer et al., 2000). The recently published AChBP structure confirms this binding site structure (Brejc et al., 2001). Loop E contains a sequence segment (104-120 in 2, 106-122 in 4) that affects agonist sensitivity (Cohen et al., 1995). A residue within this region (2F106, 4V108) has been identified as a determinant of substance P sensitivity (Stafford et al., 1998). Loop F contains a glutamate at position 177 of 2 (179 in 4) that is analogous to E183 of  (E189 of ), which has been shown to be a determinant of agonist affinity (Czajkowski et al., 1993; Martin et al., 1996). Our results indicate that the D loop contains critical residues that determine differences in agonist binding affinity among neuronal nAChRs.

The muscle nAChR D loop contains a tryptophan residue (55/57) that is labeled by nicotine (Chiara et al., 1998). This tryptophan is one of a series of conserved "core" residues that have been identified by affinity labeling and mutagenesis within loops A, B, C, and D (Corringer et al., 2000). These core residues are flanked by variable residues that control the pharmacological diversity of nAChRs. In muscle nAChRs, residues H60 of  and A61 of  are important in determining affinity for dimethyl-d-tubocurarine (Bren and Sine, 1997), whereas residues E57 of  and D59 of  are important in determining carbamylcholine binding affinity (Prince and Sine, 1996). In neuronal nAChRs, loop D contains T59 in 2 (K61 in 4). We previously identified this residue as a critical determinant of sensitivity to the competitive antagonists neuronal bungarotoxin, dihydro--erythroidine and -conotoxin-MII (Harvey and Luetje, 1996; Harvey et al., 1997). Our current results show that several residues in the D loop determine agonist binding affinity, with 2T59/4K61 having the largest effect on ACh and nicotine affinity. Thus, amino acid residues within the D loop play a critical role in determining the affinity of both muscle and neuronal nAChRs for agonists and competitive antagonists.