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Volume 16, Number 12, Issue of June 15, 1996 pp. 3798-3806
Copyright ©1996 Society for Neuroscience

Determinants of Competitive Antagonist Sensitivity on Neuronal Nicotinic Receptor  Subunits

Scott C. Harvey and Charles W. Luetje

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

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ABSTRACT

We constructed a series of chimeric and mutant neuronal nicotinic acetylcholine receptor  subunits to map amino acid residues that determine sensitivity to competitive antagonists. The 2 and 4 subunits form pharmacologically distinct receptors when expressed in combination with the 3 subunit in Xenopus oocytes. At equipotent acetylcholine concentrations, 32 is 56-fold more sensitive to blockade by dihydro--erythroidine than is 34. The 32 combination is also sensitive to long-term blockade by neuronal bungarotoxin, whereas 34 is not. Pharmacological analysis of receptors formed by chimeric  subunits reveals that amino acid residues that determine both dihydro--erythroidine and neuronal bungarotoxin sensitivity are located within several sequence segments. The major determinant of sensitivity to both competitive antagonists is located between residues 54 and 63. A minor determinant of sensitivity to both antagonists lies between residues 1 and 54, whereas a minor determinant of NBT sensitivity lies between residues 74 and 80. Within region 54-63 of 2, mutant 2 subunits were used to identify threonine 59 as a residue critical in determining competitive antagonist sensitivity. Changing threonine 59 to lysine, as occurs in 4, causes a 9-fold decrease in dihydro--erythroidine sensitivity and a 71-fold decrease in neuronal bungarotoxin sensitivity. Changing polar threonine 59 to negatively charged aspartate causes a 2.5-fold increase in neuronal bungarotoxin sensitivity and has no effect on dihydro--erythroidine sensitivity.

Key words: nicotinic receptor; neuronal; antagonists; mutant; chimera; neuronal bungarotoxin; dihydro--erythroidine

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INTRODUCTION

Nicotinic acetylcholine receptors (nAChRs) are found throughout the central and peripheral nervous systems, with 11 distinct genes encoding neuronal nAChR subunits (2-9, 2-4) currently identified (Sargent, 1993; Elgoyhen et al., 1994). Functional neuronal nAChRs can be formed in Xenopus oocytes by expression of various combinations of these subunits (Duvoisin et al., 1989; Papke et al., 1989; Luetje et al., 1990b; Luetje and Patrick, 1991). Although these neuronal nAChR subunits are homologous with one another, each functional subunit combination is pharmacologically distinct. This may account for the diversity of neuronal nAChRs observed in vivo (Luetje et al., 1990a; Role, 1992; Sargent, 1993).

Identification of amino acid residues that are involved in forming the ligand-binding sites of nAChRs is essential to understanding how these receptors function. Affinity labeling experiments have identified several critical amino acid residues of the muscle-type  subunit (Kao et al., 1984; Dennis et al., 1988; Abramson et al., 1989; Galzi et al., 1990; Middleton and Cohen, 1991). Non- subunits are also involved in forming the ligand-binding site. The ligand-binding sites of muscle nAChRs appear to be located at the interface between the  and  subunits and between the  and  subunits (Blount and Merlie, 1989; Pederson and Cohen, 1990; Czajkowski and Karlin, 1991; Middleton and Cohen, 1991). The ligand-binding sites of neuronal nAChRs appear to be formed in a similar manner, because both  and  subunits influence the pharmacological properties of these receptors (Luetje and Patrick, 1991). The residues identified by affinity labeling experiments, using Torpedo electric organ nAChRs, are highly conserved among muscle and neuronal nAChR subunits. Thus, these residues may form parts of the ligand-binding site common to all nAChRs, but cannot be responsible for the pharmacological diversity observed among nAChR subtypes.

An approach to identification of the amino acid residues of receptor subunits that confer differential pharmacological properties is to construct chimeras of pharmacologically distinct subunits. This approach has been used to identify several sequence segments of neuronal nAChR  subunits that affect sensitivity to agonists and the competitive antagonist neuronal bungarotoxin (NBT) (Luetje et al., 1993). Chimeric subunits have been used to identify regions of 2 and 4 that determine sensitivity to agonists (Figl et al., 1992; Cohen et al., 1995). This technique has also been used to localize the  subunit contribution to NBT sensitivity to the N-terminal 119 (Papke et al., 1993) or 80 (Wheeler et al., 1993) residues of 2.

We constructed a series of chimeric  subunits to more precisely identify regions of  subunits that determine competitive antagonist sensitivity. We used the structurally distinct competitive antagonists dihydro--erythroidine (DHE) and NBT, which can distinguish between the 32 and 34 subunit combinations. Having identified residues 54-63 of 2 as containing the major determinant of competitive antagonist sensitivity, we then used a series of mutant  subunits to identify threonine 59 as the critical residue within this region.

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MATERIALS AND METHODS

Materials. Xenopus laevis frogs were purchased from Nasco. RNA transcription kits were from Ambion. ACh, atropine, and 3-aminobenzoic acid ethyl ester were from Sigma (St. Louis, MO). Collagenase B was from Boehringer Mannheim (Indianapolis, IN). Sequenase 2.0 kits were from United States Biochemicals (Cleveland, OH). NBT was from Biotoxins. CloneAmp kits were from Gibco (Gaithersburg, MD). DHE was a gift from Merck (Rahway, NJ).

Mutagenesis and construction of chimeric receptors. Chimeric and mutant subunits were constructed using PCR (Higuchi, 1990). Our notation for these subunits is to list the source of the N-terminal portion, followed by the residue number in the amino acid sequence in which the chimeric joint is made (numbering taken from the mature 2 subunit sequence), followed by the source of the C-terminal portion. For example, the chimeric subunit 4-204-2 is composed of 4 sequence from the N terminus until residue 204, after which it is composed of 2 sequence. The 2 and 4 cDNAs in the Bluescript SK vector were used as templates for PCR reactions. PCR products were subcloned into the pAMP1 vector using a CloneAmp kit (Gibco) or into the pCR-Script SK⁺ vector (Stratagene, La Jolla, CA). To minimize the amount of PCR product in the final construct that would have to be sequenced, as much PCR product as possible was replaced with wild-type 2 or 4 sequence using existing restriction sites. Remaining sequence derived from PCR product was sequenced using Sequenase 2.0 (United States Biochemicals).

Injection of in vitro synthesized RNA into Xenopus oocytes. m⁷G(5)ppp(5)G-capped cRNA was synthesized in vitro from linearized template DNA encoding the 3, 2, and 4 subunits, as well as the various chimeric and mutant subunits, using an Ambion mMessage mMachine kit. 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 hr at room temperature. Each oocyte was injected with 5-50 ng of cRNA in 50 nl of water and incubated at 19°C in modified 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 2-7 d. RNA transcripts encoding each subunit were injected into oocytes at a molar ratio of 1:1.

Electrophysiological recordings. Oocytes were perfused at room temperature (20-25°C), in a 300 µl chamber with perfusion solution (115 mM NaCl, 1.8 mM CaCl₂, 2.5 mM KCl, 10 mM HEPES, pH 7.2, and 1.0 µM atropine). Perfusion was continuous at a rate of ~20 ml/min. ACh was diluted in perfusion solution, and the oocytes were exposed to ACh for ~10 sec using a solenoid valve. NBT sensitivity was tested by comparing ACh-induced current responses before and after the oocytes were incubated for 30 min in perfusion solution containing various concentrations of NBT and 100 µg/ml bovine serum albumin. Preincubation with NBT results in a slowly reversible competitive blockade of 32 but not 34 (Boulter et al., 1987; Duvoisin et al., 1989; Luetje et al., 1990b). DHE sensitivity was tested by measuring the reduction of ACh-induced current responses when DHE was coapplied with ACh. The response to ACh alone, before treatment with either NBT or DHE, is taken as the control response. The ACh-induced response, after treatment with NBT or during coapplication with DHE, is reported as a percent of the control response.

Current responses to agonist application were measured under two-electrode voltage clamp, at a holding potential of 70 mV, using a Knight Industrial Technologies voltage clamp unit. Micropipettes were filled with 3 M KCl and had resistances of 0.5-1.0 M. Agonist-induced responses were captured, stored, and analyzed on a Macintosh IIci computer using a data acquisition program written with LabVIEW (National Instruments) and LIBI (University of Arizona) software (Luetje et al., 1993).

Dose-response and dose-inhibition data were fit with Passage II software by the nonlinear least-squares method. For dose-response data, we used the equation: current = maximum current/[1 + (EC₅₀/[agonist])ⁿ], where n and EC₅₀ represent the Hill coefficient and the agonist concentration producing half-maximal response, respectively. Rapid desensitization of these receptors can affect the accuracy of dose-response curves. However, this was found to account for only a small fraction of the difference in EC₅₀ between 32 and 34 (Cohen et al., 1995). Rapid desensitization also makes the maximal response an unreliable standard with which to normalize data. For this reason, we normalized each response to the response to a low concentration of agonist. To compare and display results for different receptors, we then renormalized each value to the fit maximal response. For DHE dose-inhibition data, we used the equation: current = maximum current/[1 + ([antagonist]/IC₅₀)ⁿ], where n and IC₅₀ represent the Hill coefficient and the antagonist concentration producing half-maximal inhibition, respectively. Fold differences between NBT dose-inhibition data for various receptors were determined by visual inspection of Figure 5B. Statistical significance was determined by using a two-sample t test after an F test to ensure equality of variance. For samples with unequal variance (p > 0.05), statistical significance was determined by using a two-sample t test for samples with unequal variance (Cochran's method).

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Fig. 5. Effect of mutations of threonine 59 on DHE and NBT sensitivity. A, DHE sensitivity of 32,T59K (filled squares) and 32,T59D (filled circles). Current in response to an EC₂₀ concentration of ACh in the presence of various concentrations of DHE is presented as a percent of the response to ACh alone (mean ± SD of 3-6 oocytes). The lines are fits to a Hill equation (see Materials and Methods). IC₅₀ values are 3.8 ± 0.9 µM for 32,T59K and 0.30 ± 0.07 µM for 32,T59D. Inhibition curves for 32 and 34 from Figure 2B are shown as dashed lines for reference. B, NBT sensitivity of 32,T59K (filled squares), 32,T59D (filled circles), 32 (open circles), and 34 (open squares). Current in response to an ACh concentration at or below the EC₅₀ after 30 min incubation with various concentrations of NBT is presented as a percentage of the response to ACh alone (mean ± SD of 3 separate oocytes). Significant differences from 2 are denoted by asterisks (*p < 0.02; ***p < 0.001). Significant differences from 4 are denoted by daggers (p < 0.01). Some error bars are obscured by symbols.
[View Larger Version of this Image (17K GIF file)]

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RESULTS

The 2 and 4 subunits form receptors that are differentially sensitive to competitive antagonists

The 2 and 4 subunits each can form functional neuronal nAChRs when expressed in Xenopus oocytes in combination with 3 (Fig. 1). These two subunit combinations differ in their sensitivity to both DHE and NBT. At equipotent agonist concentrations (see below), the 32 receptor is almost completely blocked by coapplication of 3 µM DHE, whereas the 34 receptor is blocked only slightly. In addition, the 32 combination is completely blocked by 100 nM NBT, whereas the 34 is insensitive to this concentration of NBT. Because these competitive antagonists distinguish between the receptors based on the identity of the  subunits, they are useful probes to identify the structural basis for the contribution of the  subunit to competitive antagonist sensitivity.

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Fig. 1. 32 and 34 are pharmacologically distinct. Top traces, Current responses of an 32-expressing oocyte to 10 µM ACh alone and in combination with 3 µM DHE (left), and current responses of a different 32-expressing oocyte to 1 µM ACh before and after 30 min incubation with 100 nM NBT (right). Bottom traces, Current responses of an 34-expressing oocyte to 100 µM ACh alone or in combination with 3 µM DHE (left), and current responses of a different 34-expressing oocyte to 10 µM ACh before and after 30 min incubation with 100 nM NBT (right). ACh application of ~10 sec is indicated by arrowheads. Scale bars: 150 nA, 10 sec.
[View Larger Version of this Image (12K GIF file)]

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Amino acid residues involved in determining competitive antagonist sensitivity are of particular interest because the competitive antagonist-binding sites of receptors are thought to overlap, at least partially, with the agonist-binding sites. DHE has been shown to act competitively with ACh in ligand-binding experiments on rat brain homogenates (Williams and Robinson, 1984), and essential atomic groups of DHE can superimpose with those of the agonist nicotine, suggesting that the two compounds can share a similar conformation (Sheridan et al., 1986). In addition, DHE has been shown to be a purely competitive antagonist of the 7 neuronal nAChR exogenously expressed in Xenopus oocytes (Bertrand et al., 1992). We find that DHE antagonism of both 32 and 34 can be overcome by increasing the ACh concentration, a result indicative of competitive antagonism (Table 1). NBT has also been shown to antagonize nAChRs in a competitive manner (Halvorsen and Berg, 1987).

Table 1. DHE antagonism can be overcome by increasing the acetylcholine concentration Receptor [DHE] (µM) [ACh] (µM) Percent of control  32 1 10 21.5  ± 5.0  32 1 1000 96.1  ± 1.1  34 30 100 40.4  ± 9.9  34 30 10,000 95.9  ± 1.9  32,T59K 10 30 17.2  ± 5.8  32,T59K 10 3000 85.5  ± 1.8 ACh-induced current in the presence of DHE is presented as a percentage of the control response to ACh alone (mean ± SD of 3-6 oocytes).

To evaluate accurately the degree of blockade by a competitive antagonist such as DHE on 32 and 34, it is necessary that the ACh concentrations used be equipotent (i.e., at the same point on the dose-response curve) (Craig et al., 1993). For this reason, full dose-response curves for each subunit combination were constructed (Fig. 2A). The EC₅₀ values for 32 and 34 were 70.8 ± 19.6 and 209.7 ± 40.7 µM, respectively (Table 2). The EC₂₀ was chosen as an ACh test dose, because this is high enough to reliably yield useful current responses, but low enough to avoid extensive desensitization. In contrast to DHE, incubation with NBT results in pseudoirreversible blockade; NBT slowly dissociates over a period of several hours. Because the postincubation ACh application only lasts for 10 sec, the ACh and NBT are not in direct competition. Determination of the percent blockade by NBT, therefore, is unrelated to the level of receptor activation by ACh.

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Fig. 2. A, Acetylcholine dose-response curves for 32 (circles) and 34 (squares). Symbols are the mean normalized responses ± SEM of three separate sets of oocytes, each set consisting of three to four separate oocytes. The lines are fits to a Hill equation (see Materials and Methods). EC₅₀ and n values are 70.8 ± 19.6 µM and 0.74 ± 0.11 for 32, respectively, and 209.7 ± 40.7 µM and 1.56 ± 0.02 for 34, respectively. B, DHE inhibition curves for 32 (circles) and 34 (squares). Increasing DHE concentrations were coapplied with an EC₂₀ ACh concentration of 100 µM for 34 and 10 µM for 32. The response in the presence of DHE is reported as a percent of the response to ACh alone (mean ± SD of 3 oocytes). The lines are fits to a Hill equation (see Materials and Methods). IC₅₀ values are 0.41 ± 0.17 µM for 32 and 23.1 ± 10.2 µM for 34. Some error bars are obscured by symbols.
[View Larger Version of this Image (21K GIF file)]

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Table 2. EC50, Hill coefficient, and IC50 values for receptors formed by wild-type, chimeric, and mutant  subunits Subunit EC50 (µM) n IC50 (µM)  2 70.8  ± 19.6 0.74  ± 0.11 0.41  ± 0.17  4 209.7  ± 40.7 1.56  ± 0.02 23.1  ± 10.2  4-54-2 314.3  ± 108.8 0.90  ± 0.20 -  4-103-2 433.3  ± 139.3 1.04  ± 0.16 -  4-133-2 199.1  ± 72.4 1.66  ± 0.45 -  4-204-2 518.6  ± 155.7 2.23  ± 0.30 -  2-54-4 47.8  ± 45.3 0.84  ± 0.26 -  2-63-4 72.7  ± 17.1 0.89  ± 0.02 -  2-74-4 124.3  ± 96.8 0.87  ± 0.22 -  2-80-4 40.0  ± 30.5 0.85  ± 0.12 -  2,N55S 86.3  ± 47.5 0.57  ± 0.05 -  2,V56I 67.9  ± 47.3 0.76  ± 0.10 -  2,T59K 158.5  ± 114.1 0.92  ± 0.03 3.8  ± 0.9  2,E63T 94.8  ± 26.1 0.76  ± 0.11 -  2,T59D 76.2  ± 45.5 0.78  ± 0.07 0.30  ± 0.07 All subunits were functionally expressed in combination with 3. EC50 and n values, determined by fitting to a Hill equation (see Materials and Methods), are the mean ± SD of results from three to four separate oocytes, with the exception of 2 and 4, which are the mean ± SEM of results from three separate sets of oocytes, each consisting of three to four separate oocytes. IC50 values, determined by fitting to a Hill equation (see Materials and Methods), are the mean ± SD of results from three separate oocytes.

At the EC₂₀ (10 µM for 32, 100 µM for 34) for each receptor, responses were measured in the presence of increasing DHE concentrations (Fig. 2B). The IC₅₀ values for 32 and 34 were 0.41 ± 0.17 and 23.1 ± 10.2 µM, respectively. These inhibition curves were used to select 3 µM DHE as a concentration that differentiates between the receptors. At 3 µM DHE, there is relatively little blockade of the 34 receptor (86.9 ± 5.1% of control), whereas most of the 32 response is eliminated (10.0 ± 4.6% of control). An NBT concentration of 100 nM was chosen to differentiate between the receptors, because this concentration blocks 32 almost completely (3.4 ± 1.6% of control) and has little effect of 34 (96.3 ± 8.4% of control).

Sequence segment 54-63 of  subunits contains a major determinant of DHE and NBT sensitivity

We constructed a series of chimeric  subunits to determine which sequence segments are responsible for differences in competitive antagonist sensitivity. Sections of one  subunit were substituted with homologous sections from the other  subunit. In Figure 3, A and B, the chimeras contain an N-terminal section of 4 connected to a C-terminal section of 2. In Figure 3, C and D, the chimeras contain an N-terminal section of 2 connected to a C-terminal section of 4. Each of these chimeras was then expressed in Xenopus oocytes, in combination with 3, and full dose-response curves were obtained to determine the EC₂₀ for ACh. The degree of blockade of an EC₂₀ ACh response by 3 µM DHE was then determined (Fig. 3A,C). The degree of blockade by 100 nM NBT was also determined (Fig. 3B,D).

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Fig. 3. DHE and NBT sensitivity of receptors formed by chimeric  subunits. A, DHE sensitivity of receptors formed by each of a series of chimeric subunits in which increasingly larger portions of the N-terminal end of 2 were replaced by the corresponding portion of 4. Current in response to an EC₂₀ concentration of ACh in the presence of 3 µM DHE is presented as a percent of the response to ACh alone (mean ± SD of 3-4 separate oocytes). B, NBT sensitivity of receptors formed by the chimeras in A. Current in response to an ACh concentration at or below the EC₅₀ after 30 min incubation with 100 nM NBT is presented as a percentage of the response to ACh alone (mean ± SD of 3-4 separate oocytes, except for 4, which is mean ± SEM of 3 separate sets of oocytes, each set consisting of 3-4 separate oocytes). C, DHE sensitivity of receptors formed by each of a series of chimeric subunits in which increasingly larger portions of 4 were replaced by the corresponding portion of 2. Current in response to an EC₂₀ concentration of ACh in the presence of 3 µM DHE is presented as a percent of the response to ACh alone (mean ± SD of 3-4 separate oocytes). D, NBT sensitivity of receptors formed by the chimeras in C. Current in response to an ACh concentration at or below the EC₅₀ after 30 min incubation with 100 nM NBT is presented as a percentage of the response to ACh alone (mean ± SD of 3-4 separate oocytes, except for 2-54-4, which is mean ± SEM of 3 separate sets of oocytes, each set consisting of 3-4 separate oocytes). Significant differences from 2 are denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). Significant differences from 4 are denoted by daggers (p < 0.05; p < 0.001). Some error bars are too small to appear.
[View Larger Version of this Image (29K GIF file)]

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Substitution of the first 54 N-terminal residues of 2 with the corresponding section of 4 (4-54-2) resulted in a subunit that formed receptors that were slightly, but significantly, less sensitive to 3 µM DHE and 100 nM NBT than were receptors formed by wild-type 2. This intermediate sensitivity between that of wild-type 2- and 4-containing receptors suggests that a minor determinant of DHE and NBT sensitivity is located within the first 54 N-terminal amino acids of the  subunit. A chimeric subunit in which the first 103 N-terminal residues of 2 were replaced with 4 sequence (4-103-2) formed receptors as insensitive to 3 µM DHE and 100 nM NBT as were receptors formed by wild-type 4. This suggests that the section of the  subunit responsible for DHE and NBT sensitivity is located within the first 103 N-terminal amino acids, with the segment 54-103 containing the major determinant. Chimeric subunits in which the first 133 or 204 N-terminal residues of 2 were replaced with 4 sequence (4-133-2, 4-204-2) also formed receptors with DHE and NBT sensitivities comparable to that of wild-type 4-containing receptors.

Chimeric subunits were also constructed containing an N-terminal section of 2 connected to a C-terminal section of 4 (Fig. 3C,D). Substituting the first 54 N-terminal residues of 2 into 4 (2-54-4) failed to increase the DHE or NBT sensitivity of receptors formed by this chimera beyond that of receptors formed by wild-type 4. This result is consistent with the minor nature of the determinant between residues 1 and 54 (Fig. 3A,B). Substituting the first 63 N-terminal residues of 2 into 4 (2-63-4) resulted in a chimera that formed receptors as sensitive to blockade by 3 µM DHE as receptors formed by 2. Substitution of the first 74 or 80 N-terminal residues of 2 into 4 also resulted in blockade by 3 µM DHE that was not significantly different than blockade of 32. Receptors formed by 2-63-4 and 2-74-4 were slightly, but significantly, less sensitive to blockade by 100 nM NBT than were receptors formed by 2, whereas the 2-80-4 chimera formed receptors with a sensitivity to 100 nM NBT indistinguishable from that of receptors formed by 2.

Taken together, the DHE and NBT sensitivities of receptors formed by these chimeras (Fig. 3A-D) indicate that the major determinant for sensitivity to both competitive antagonists is within the amino acid segment from 54 to 63. This can be seen most clearly by considering the chimeras 4-54-2 and 2-63-4. Both 4-54-2, which contains 2 sequence from residue 54 to the C terminus, and 2-63-4, which contains 2 sequence from the N terminus to residue 63, form receptors nearly as sensitive to DHE and NBT blockade as receptors formed by wild-type 2. The only 2 sequence common to these two chimeras is segment 54-63. In addition to this major determinant, a minor determinant of sensitivity to both antagonists is between 1 and 54, whereas a minor determinant of NBT sensitivity only may be within segment 74-80.

Threonine 59 of 2 is critical to both DHE and NBT sensitivity

We examined sequence segment 54-63 in more detail by changing individual amino acid residues. The 2 sequence differs from 4 at only four residues within this region (Fig. 4A). We changed each of these residues individually from what occurs in 2 to what occurs in 4 and then determined the DHE and NBT sensitivity of receptors formed by these mutants (Fig. 4B,C). Changing threonine 59 of 2 to lysine (T59K) resulted in a significant loss in sensitivity to DHE and NBT when compared to wild-type 2-containing receptors. Mutation V56I had a small, but significant, effect on both DHE and NBT sensitivity. Mutation N55S had a small, but significant, effect on DHE sensitivity, but no effect on NBT sensitivity, whereas mutation E63T had no effect on sensitivity to either antagonist.

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Fig. 4. Threonine 59 of 2 is critical to both DHE and NBT sensitivity. A, Alignment of 2 and 4 sequences within segment 54-63. Residues that differ are denoted by solid circles. Tryptophan 57 is starred. B, DHE sensitivity of receptors formed by each of a series of mutant 2 subunits. Current in response to an EC₂₀ concentration of ACh in the presence of 3 µM DHE is presented as a percent of the response to ACh alone (mean ± SD of 3 separate oocytes). C, NBT sensitivity of receptors formed by the 2 mutants in B. Current in response to an ACh concentration at or below the EC₅₀ after 30 min incubation with 100 nM NBT is presented as a percentage of the response to ACh alone (mean ± SD of 3 separate oocytes, except for 4, which is mean ± SEM of three separate sets of oocytes, each set consisting of 3-4 separate oocytes). Significant differences from 2 are denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). Significant differences from 4 are denoted by daggers (p < 0.01; p < 0.001). Some error bars are too small to appear.
[View Larger Version of this Image (20K GIF file)]

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In Figure 5 we examined in more detail the degree to which mutation T59K affected DHE and NBT sensitivity. The DHE inhibition curve for receptors formed by 2,T59K is shifted to the right of the wild-type 2 curve approximately ninefold, accounting for about half of the difference between 2- and 4-containing receptors (Fig. 5A). Block of 2,T59K-containing receptors by DHE remains competitive because an increased ACh concentration is able to overcome blockade (Table 1). Residue 59, together with residues 55 and 56, is responsible for most of the difference in DHE sensitivity between 2 and 4. This can be seen most clearly by considering that blockade of 2- and 4-containing receptors by 3 µM DHE differs by ~77 percentage points. Block of mutants 2,N55S, 2,V56I, and 2,T59K by DHE differs from block of 2 by 10, 9, and 41 percentage points, respectively, accounting for 60 percentage points. The remaining difference between block of 2- and 4-containing receptors (17 percentage points) is completely accounted for by segment 1-54, because block of 4-54-2 receptors differs from that of 2 receptors by 19 percentage points. Threonine 59 also accounts for a substantial portion of the NBT sensitivity difference between 2- and 4-containing receptors (Fig. 5B). Receptors formed by 2,T59K were ~71-fold less sensitive to NBT than 2-containing receptors. Although we know that 34 is at least 100-fold less sensitive to NBT than 32 (Fig. 5B), we are unable to determine exactly how much less sensitive 34 is attributable to a lack of a sufficient quantity of NBT. The remaining difference in NBT sensitivity between 2- and 4-containing receptors can be accounted for by the slight contribution from V56 and the minor determinants within regions 1-54 and 74-80.

The loss of DHE and NBT sensitivity caused by the mutation T59K could be attributable to the change from the polar side chain of threonine to the positively charged side chain of lysine, or it could be attributable to the change in side chain volume. The identification of arginine 34 of NBT as a critical residue for neuronal nAChR blockade (Dewan et al., 1994; Fiordalisi et al., 1994) suggests that it is the introduction of the positively charged lysine that interferes NBT sensitivity. To explore this idea, we introduced a negative charge by changing this residue from threonine to aspartate (T59D). The DHE and NBT sensitivity of receptors formed by 2,T59D is shown in Figure 5, A and B. The T59D mutation resulted in an increase in NBT sensitivity of ~2.5 fold. The T59D mutation had no effect on DHE sensitivity.

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DISCUSSION

The neuronal nAChRs 32 and 34 differ in their sensitivity to the antagonists DHE and NBT. Pharmacological analysis of a series of chimeric  subunits has allowed us to identify areas of the  subunits that determine sensitivity to these competitive antagonists. The major determinant of both DHE and NBT sensitivity lies in sequence segment 54-63, with a minor determinant of sensitivity to both antagonists in region 1-54 and a minor determinant of NBT sensitivity in region 74-80. Within sequence segment 54-63, we identified threonine 59 of 2 as the critical residue. Changing this residue to lysine, as in 4, results in a 9-fold loss in DHE sensitivity and a 71-fold loss in NBT sensitivity. Changing threonine 59 of 2 to aspartate, thus introducing a negative charge, caused a 2.5-fold increase in NBT sensitivity.

It has become clear recently that non- subunits are involved in determining both the physical structure and pharmacological properties of the ligand-binding sites of nAChRs (Blount and Merlie, 1989; Duvoisin et al., 1989; Pederson and Cohen, 1990; Czajkowski and Karlin, 1991; Luetje and Patrick, 1991; Middleton and Cohen, 1991). Affinity labeling and mutagenesis studies of Torpedo electric organ and mammalian muscle nAChRs have identified amino acid residues of the  and  subunits that are associated with ligand binding (Cohen et al., 1992; Czajkowski et al., 1993; Sine, 1993; Fu and Sine, 1994). In covalent labeling experiments involving the competitive antagonist d-tubocurarine, Cohen et al. (1992) demonstrated incorporation of label onto a tryptophan residue of the  and  subunits (residue 55 and 57, respectively). This residue is conserved in the rat neuronal 2 and 4 subunits (position 57, Fig. 4A), and thus appears to be a common feature of the ligand-binding sites of nAChRs. Interestingly, the homologous residue of neuronal 7 (tryptophan 54) is involved in determining sensitivity to both agonists and antagonists, leading to the proposal that 7 contributes both an `` component'' and a ``non- component'' when forming homo-oligomeric receptors (Corringer et al., 1995).

The conservation of this tryptophan among muscle and neuronal nAChRs means that this residue cannot be responsible for pharmacological differences between nAChR subtypes. It is the amino acid residues that differ among subunits that must be responsible for this diversity. We have identified such a residue, separated by only one residue from the conserved tryptophan, as the major determinant of differences in competitive antagonist sensitivity between 2- and 4-containing receptors. Changing this residue in 2 from threonine to what occurs in 4 (lysine) results in a substantial loss of both DHE and NBT sensitivity. The change from threonine to lysine is a change in both the character (polar to negatively charged) and the size (55.7-101.5 ų) of the side chain. Either or both of these properties might be responsible for the effect of changing this residue. Considering that arginine 34 of NBT has been identified as a critical residue involved in neuronal nAChR blockade (Dewan et al., 1994), we hypothesized that insertion of lysine at position 59 in 2 might be decreasing NBT sensitivity by electrostatic repulsion. If this were true, then introduction of a negative charge at this position might be expected to increase NBT sensitivity. Changing threonine 59 to aspartate does result, in fact, in an increase in NBT sensitivity (Fig. 5B).

Construction and functional analysis of chimeric receptor subunits allows identification of structural differences that confer unique pharmacological properties. This methodology has been used to map determinants of both agonist and antagonist sensitivity on neuronal nAChR  subunits. Chimeric subunits have been used to identify the general region on  subunits that contributes to NBT sensitivity. Papke et al. (1993) showed that substitution of the first 119 amino acids of the 4 subunit with the corresponding section of 2 can confer NBT sensitivity onto the 4 subunit. The identity of the  subunit also influences the NBT sensitivity of the receptors 42 and 44. Wheeler et al. (1993) showed that a chimeric subunit composed of the N-terminal 80 residues of 2 followed by 4 sequence, formed receptors with 4 that were sensitive to NBT blockade. A series of  subunit chimeras, expressed in combination with the 3 subunit, has been used to identify the sequence segment 104-120 of 2 as important in determining sensitivity to the agonist cytisine (Figl et al., 1992; Cohen et al., 1995). Consistent with these reports, we find that receptors formed by 3 and the chimera 4-103-2 have a cytisine sensitivity similar to that of receptors formed by wild-type 32 (data not shown). Cohen et al. (1995) also show that region 104-120 is responsible for part of the difference in EC₅₀ for ACh, as well as for part of the difference in Hill slope, between 32 and 34.

Differential sensitivity to agonists may result from differences in affinity or efficacy, making it difficult to infer conclusions about the functional role of the sequence segment being mapped. Competitive antagonists are ideal probes, because they compete with agonist for a common binding site but do not activate the receptors; they have no efficacy. Because our determination of the DHE sensitivity of each receptor is dependent on use of equipotent concentrations of ACh, it is important that differences in DHE sensitivity not be an artifact of differences in ACh dose-response curve characteristics. The 32 and 34 ACh dose-response curves clearly differ in both EC₅₀ and apparent Hill coefficient (Table 2). However, these differences can be taken into consideration when DHE dissociation constants (K_i) for each receptor are calculated (Leff and Dougall, 1993). The resulting DHE K_i values of 0.21 and 32.4 µM (for 32 and 34, respectively) differ by ~154-fold. Additional arguments against differences in DHE sensitivity being artifactual are that DHE sensitivity maps differently than the ACh EC₅₀ and the apparent Hill coefficient (compare Fig. 3 and Table 2), and that use of both DHE and NBT as probes has identified the same residue as a major determinant of competitive antagonist sensitivity. If DHE antagonism is competitive, why do determinants of DHE sensitivity and ACh EC₅₀ differ? Although competitive antagonists do compete for a common binding site with agonists, their interactions with the binding site would not necessarily be coextensive with those of agonists. In fact, this would not be expected, because competitive antagonists differ from agonists by lacking efficacy.

Although the pseudoirreversibility of NBT blockade makes concerns over ACh dose-response curves irrelevant, NBT is a large peptide toxin and may block receptor activation by adventitiously occluding the ligand-binding site after binding elsewhere on the receptor. Therefore, mapping the areas of the receptor responsible for differential sensitivity to NBT may identify regions of uncertain significance. However, both DHE and NBT sensitivity map similarly, identifying threonine 59 as a major determinant and region 1-54 as containing a minor determinant, supporting the view that NBT is a useful probe. The significance of region 74-80, containing an additional minor determinant of NBT sensitivity, is unclear. Another example of determinants of NBT sensitivity overlapping with those of small ligand sensitivity occurs on  subunits. The 2 subunit forms receptors with 2 that are insensitive to NBT and are more sensitive to nicotine than to ACh, whereas 32 receptors are blocked by NBT and are much less sensitive to nicotine than to ACh. Region 195-215 contains determinants of both properties. Within this region, the glutamine residue at position 198 of 3 (proline in 2) was shown to be an important determinant of both NBT and nicotine sensitivity (Luetje et al., 1993). Similar results regarding the role of glutamine 198 in determining nicotine sensitivity have been obtained recently using chicken neuronal nAChR  subunits (Hussy et al., 1994).

The exact physical role of residues identified in this and previous studies, that confer pharmacological differences among nAChR subtypes, remains unclear. In this study, we provide data consistent with a direct interaction between residue 59 of 2/4 and NBT. Thus, these residues may be structural features of the binding site and may participate in the binding of ligand. Alternatively, these residues may not actually participate in binding of ligand, but impinge upon those that do, reshaping the site enough to alter ligand sensitivity. More extensive mutagenesis of identified residues will be required to distinguish between these possibilities. Particularly promising is the potential for incorporating unnatural amino acids at these sites (Nowak et al., 1995).

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FOOTNOTES

Received Nov. 6, 1995; revised Feb. 15, 1996; accepted April 2, 1996.

  

This work was supported by grants to C.W.L. from the National Institute on Drug Abuse (DA08102), the American Heart Association Florida Affiliate, and the Pharmaceutical Research and Manufacturers of America Foundation. C.W.L. is an Initial Investigator of the American Heart Association, Florida Affiliate. We thank Floyd Maddox for technical assistance.

Correspondence should be addressed to Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101.

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