Contributions of Torpedo Nicotinic Acetylcholine Receptor gamma Trp-55 and delta Trp-57 to Agonist and Competitive Antagonist Function*

Yu XieDagger and Jonathan B. Cohen§

From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, October 4, 2000, and in revised form, October 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha -gamma and alpha -delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[3H]tubocurarine (dTC) identified two tryptophans, gamma Trp-55 and delta Trp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gamma Trp-55 and delta Trp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the delta W57L and gamma W55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gamma W55L, and delta W57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gamma W55L mutant, dTC binding at the alpha -gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma  Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nicotinic acetylcholine receptor (nAChR)1 from Torpedo electric organ and vertebrate skeletal muscle is a pentameric transmembrane protein composed of four homologous subunits with a stoichiometry of alpha 2beta gamma delta (reviewed in Refs. 1-3). The nAChR contains two binding sites for agonists and competitive antagonists, located at the alpha -gamma and alpha -delta subunit interfaces (4, 5). The two sites are nonequivalent, and many competitive antagonists bind with high affinity to only one of the sites (6, 7). Affinity labeling and mutational analyses provide evidence that amino acids from three discrete regions of alpha  subunit primary structure and from three (or more) regions of the gamma  (or delta ) subunit contribute to the structure of the binding sites (reviewed in Refs. 8 and 9).

Several experimental approaches have identified residues in both gamma  and delta  subunits that contribute to the binding sites for agonists or competitive antagonists. Photoaffinity labeling using d-[3H]tubocurarine (dTC), a competitive antagonist, and [3H]nicotine, an agonist, established that gamma Trp-55 is located near the agonist binding site in Torpedo nAChR (10, 11), and [3H]dTC also reacted with delta Trp-57, the corresponding position in the delta  subunit, as well as with gamma Tyr-111/gamma Tyr-117 (12). Substitution of gamma Trp-55 by leucine (gamma W55L) caused a 10-fold decrease in dTC potency as an inhibitor of ACh-induced currents for Torpedo nAChRs expressed in Xenopus oocytes (13). A heterobifunctional cross-linker ~9 Å in length cross-linked alpha Cys-192/alpha Cys-193 in Torpedo nAChR to a residue in the delta  subunit identified as delta Asp-180 (14, 15), and the mutation delta D180N in the delta  subunit of mouse muscle nAChR caused substantial decreases in agonist, but not antagonist, binding affinities (16-18). Analysis of binding properties of embryonic mouse nAChRs containing chimeras between gamma  and delta  subunits led to the identification of three positions in the gamma  subunit (Ile-116, Tyr-117, and Ser-161) and the corresponding residues in the delta  subunit (Ser-118, Thr-119, and Lys-163), which can account for the binding selectivity of the competitive antagonist dimethyl-d-tubocurarine (metocurine (19)), whereas for the agonist carbamylcholine, the primary determinants of site selectivity were gamma Lys-34/delta Ser-36 and gamma Phe-172/delta Ile-178 (20). For the adult mouse nAChR (alpha 2beta epsilon delta ), a similar analysis of chimeric epsilon -delta subunits identified amino acids in another region of subunit primary structure (epsilon Ile-58/delta His-60 and epsilon Asp-59/delta Ala-61) as determinants of metocurine site selectivity (21).

In this report we examine the contributions of gamma Trp-55 and delta Trp-57 as determinants of agonist binding and channel gating as well as determinants of competitive antagonist function. gamma Trp-55 and delta Trp-57 were mutated to leucine, and the interaction of agonists and antagonists were examined using both binding assays and electrophysiological recording. Concentration-dependent inhibition of 125I-alpha -bungarotoxin (alpha -BgTx) binding by agonists and antagonists was studied using wild-type and mutant Torpedo nAChRs in membranes isolated from Xenopus oocyte homogenates. In parallel experiments, we studied the activation of wild-type and mutant nAChRs by ACh using a two-electrode voltage clamp. Our results establish that the mutation gamma W55L has no effect on dTC equilibrium binding affinity. However, the mutation has a profound effect on ACh binding and on the gating of the ion channel. Furthermore, for the gamma W55L mutant nAChR, dTC acts not as an antagonist when bound to its high affinity site but as a coactivator with ACh.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- ACh, dTC, tetramethylammonium, phenyltrimethylammonium, suberyldicholine, nicotine, and gallamine were obtained from Sigma. Epibatidine and dihydro-beta -erythroidine were obtained from Sigma-RBI (Natick, MA), and pancuronium was from Organon Inc. (W. Orange, NJ). alpha -BgTx was from Biotoxins Inc. (St. Cloud, FL). 125I-alpha -BgTx (400-600 Ci/mmol) was prepared using iodogen (Pierce) and Na[125I] as described (22). Na[125I] and [35S]methionine/cysteine were from PerkinElmer Life Sciences. Affinity-purified rabbit antibodies against alpha -BgTx were kindly provided by Dr. Robert Sealock (University of North Carolina), and metocurine and 13'-iodo-dTC were donated by Dr. Steen E. Pedersen (23).

In Vitro Transcription and Expression in Xenopus Oocytes-- SP64-based plasmids (pMXT) with cDNAs encoding wild-type alpha , gamma , delta , and the gamma W55L subunits were gifts from Dr. Michael M. White, and the cDNA (in plasmid SP64) encoding the wild-type beta  subunit was from Dr. Henry Lester. Sequence analysis of the delta W57L mutant cDNA (from Dr. White) revealed a point deletion 353 bases 3' to the delta W57L mutation point that resulted in a truncated form of the subunit. Therefore, the full-length delta W57L mutant was prepared by subcloning a NheI (present in the vector) and BstXI fragment that included the desired mutation (delta W57L) but excluded the point deletion into the wild-type delta  subunit cDNA from which the corresponding region had been excised. cDNAs were linearized with either XbaI (for wild-type alpha , gamma , delta , gamma W55L, and delta W57L mutant subunits) or FspI (for the wild-type beta  subunit). In vitro transcription reactions were carried out in transcription buffer (Promega) containing 40 mM Tris (pH 7.5), 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl. Linear cDNAs (5-10 µg) were incubated with 10 mM dithiothreitol, NTPs (1 mM each except GTP, which was 0.2 mM), 0.6 mM diguanosine triphosphate (Amersham Pharmacia Biotech), 100 units of RNasin (Promega), and 40 units of SP6 RNA polymerase (Promega) in transcription buffer at 37 °C for 1 h. An additional 40 units of SP6 RNA polymerase was added after 1 h, and the incubation was continued for another hour. In some experiments, [3H]UTP was included in the reaction mixture as a tracer for quantitation of reaction yields. RNAs were extracted with phenol/chloroform and chloroform, precipitated from isopropanol, and then resuspended in water at a concentration of approx 3 µg/µl. Isolated, follicle-free oocytes were microinjected with 0.5-10 ng of subunit-specific RNAs in a molar stoichiometry of (alpha 2beta gamma delta ) for wild-type and mutant receptors. For gamma -less and delta -less receptors, nAChR subunit RNAs were mixed in a molar ratio of (alpha 2beta delta 2) and (alpha 2beta gamma 2), respectively. Oocytes were injected with 10 ng of subunit RNA for assays of 125I-alpha -BgTx binding in intact oocytes or in membrane fractions and for all electrophysiological assays with the exception of full agonist-dose response relations for wild-type receptors. Because of the high currents produced by activation of wild-type receptors, maximal responses were determined for oocytes injected with 0.5 ng of RNA. Oocytes were maintained in ND96 buffer containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES (pH 7.6), and 50 µg/ml gentamicin for at least 48 h before use.

Binding of 125I-alpha -BgTx to Intact Oocytes and to Oocyte Membranes-- To measure binding to nAChRs expressed on the surface, oocytes were incubated with 2.5 nM 125I-alpha -BgTx for 2 h in a final volume of 100 µl of low Ca2+ ND96 buffer containing 96 mM NaCl, 2 mM KCl, 0.3 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.6). Oocytes were washed 3 times with 1 ml of ice-cold low Ca2+ ND96 buffer containing 1% bovine serum albumin and counted in a gamma  counter. Nonspecific binding was determined using uninjected oocytes. Oocyte membranes were prepared by homogenizing oocytes in ice-cold homogenization buffer (HB, 0.1 ml/oocyte) containing 140 mM NaCl, 20 mM sodium phosphate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethysulfonylfluoride, and 0.1 units of aprotinin/ml (pH 7.6). Membranes were isolated as described (12) by differential and sucrose density centrifugation. Membranes were resuspended in HB at 5 µl per oocyte, frozen in liquid nitrogen, and stored at -80 °C. A centrifugation assay (12) was used to measure the initial rate of 125I-alpha -BgTx binding to oocyte membranes in low Ca2+ ND96 assay buffer (supplemented with the cholinesterase inhibitor diisopropylphosphofluoridate (0.1 mM) for ACh binding experiments). Binding assays were initiated by adding 125I-alpha -BgTx (a final concentration of 2.5 nM) to triplicate samples (2-3 oocytes/sample) in a total volume of 100 µl. Membranes were preequilibrated for 15 min with appropriate concentrations of dTC, ACh, or other ligands before the addition of 125I-alpha -BgTx, and then after a 20-min incubation, the reactions were stopped by adding 1 µM unlabeled alpha -BgTx. Under these conditions, in the absence of competing ligands 125I-alpha -BgTx binding was approx 30% of equilibrium. Nonspecific binding of 125I-alpha -BgTx to wild-type and mutant receptors was determined either in the presence of 100 µM dTC (24 ± 6% of total binding, n = 8 experiments) or 100 mM ACh (27 ± 15% of total binding, n = 8 experiments) for dTC and ACh inhibition, respectively.

Biosynthetic Labeling and Immunoprecipitation of nAChRs-- 15-30 injected oocytes were incubated in 2 ml of low Ca2+ ND96 (plus 50 µg/ml gentamicin) containing 0.1-0.5 mCi/ml [35S]methonine/cysteine (PerkinElmer Life Sciences) in 24-well plates at 18 °C for at least 48 h before use. Ten to fifteen healthy oocytes were selected and washed five times with 2 ml of low Ca2+ ND96 to remove excess [35S]Met/Cys before the addition of alpha -BgTx (2.5-5 nM) to label surface receptors in a final volume of 1 ml. After a 2-h incubation, unbound alpha -BgTx was removed by washing oocytes three times with 1 ml of low Ca2+ ND96. The oocytes were then used to prepare membranes containing alpha -BgTx-labeled surface receptors. To characterize internal receptors, membranes containing nonradioactive alpha -BgTx-prelabeled surface nAChRs were incubated with alpha -BgTx (2.5-5 nM) for another 2 h followed by washing. The membranes prepared in this manner contain both alpha -BgTx-labeled surface and internal nAChRs and are designated as "total receptors." The membranes containing either surface or total receptors were then resuspended in HB buffer supplemented with 1% Triton X-100. After solubilization for 30 min, the extracts were treated with 1% Immunoprecipitin (Life Technologies, Inc.) at 4 °C for 20 min and centrifuged for 1 min in an Eppendorf microcentrifuge. Immunoprecipitin-pretreated supernatants were then collected and incubated with an excess amount of rabbit anti-alpha -BgTx antibody at 4 °C overnight. After overnight incubation, Immunoprecipitin (1%) was added to the nAChR-antibody complexes and incubated at 4 °C for 30 min. The samples were then spun in a microcentrifuge for 1 min, and the supernatants were discarded. The immunoprecipitate was washed four times with HB buffer containing 1% Triton X-100, 0.1% SDS, and 0.5% bovine serum albumin and once with HB buffer containing 0.1% SDS and 0.05% Triton X-100 (without bovine serum albumin). After the last wash, the pellets were resuspended in 50 µl of SDS-polyacrylamide gel electrophoresis sample buffer and incubated at room temperature for 20-30 min. The samples were electrophoresed on an 8% SDS-polyacrylamide gel. After staining with Coomassie Blue and destaining, the gels were soaked in Amplify (Amersham Pharmacia Biotech) for 30 min, dried at 65 °C for 2 h, and exposed to x-ray film at -80 °C for 24 h.

Sucrose Density Gradient Analysis of nAChRs in Oocytes-- To characterize surface nAChRs, 15-30 injected intact oocytes were incubated with 2.5 nM 125I-alpha -BgTx for 2 h followed by a wash, and oocyte membranes were then prepared as described above. The membranes were then solubilized with 1% Triton X-100 in a final volume of 200 µl of HB buffer and centrifuged in a Ti 42.2 rotor at 35,000 rpm for 20 min. The supernatants (approx 180 µl) were saved and used for sedimentation analysis. For total nAChRs, oocyte membranes containing surface nAChRs prelabeled by 125I-alpha -BgTx were then incubated with 2.5 nM 125I-alpha -BgTx for another 2 h. After a 2-h incubation, membranes were washed and solubilized as described above. 180-µl extracts from 10-15 oocytes containing either surface or total receptors were sedimented on a 10-ml 3-30% sucrose gradient (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 7.6, 1% Triton X-100, and 1 mM dithiothreitol) at 40,000 rpm in a SW 45 rotor (Beckman) for 22 h at 4 °C. 200-µl extracts from Torpedo membranes prelabeled with 125I-alpha -BgTx were used as the control for monomeric (9.5 S) and dimeric nAChR (13 S), and free 125I-alpha -BgTx (1.7 S), alkaline phosphatase (6.1 S), and beta -galactosidase (15.9 S) were used as standards for sedimentation coefficients. Fractions (200 µl) were collected and counted on a gamma  counter.

Electrophysiology-- Currents elicited by ACh were measured using a standard two-electrode voltage clamp (Oocyte Clamp OC-725B, Warner Instrument Corp.) at a holding potential of -70 mV. Electrodes were filled with 3 M KCl and had resistances of 0.5-1.5 megaohms. The recording chamber (about 150 µl in volume) was perfused continually by gravity with low Ca2+ ND96 (plus 1 µM atropine, pH 7.6). Appropriate concentrations of ACh (or other agonists) in the absence or presence of antagonists were applied through solenoid valves into the recording chamber for 3-5 s. For some experiments, oocytes were preincubated with dTC by perfusing the oocytes for ~1 min with dTC in low Ca2+ ND96 before application for 5 s of solution containing ACh with the same concentration of dTC.

Data Analysis-- The concentration-dependent inhibition of 125I-alpha -BgTx binding by agonists and antagonists was fit according to two models as follows.


f=100/(1+([<UP>X</UP>]<UP>/IC</UP><SUB>50</SUB>)<SUP>n</SUP>) (Eq. 1)
where [X] is the concentration of inhibitor, n is the Hill coefficient, and IC50 is the inhibitor concentration reducing the initial rate of 125I-alpha -BgTx binding by 50% and
f={50/(1+([<UP>X</UP>]<UP>/K</UP><SUB><UP>H</UP></SUB>))}+{50/(1+([<UP>X</UP>]<UP>/K<SUB>L</SUB></UP>))} (Eq. 2)
where [X] is the concentration of competing ligand, KH and KL are the ligand affinities for the high and low affinity binding sites, respectively. This equation is based on the assumption that alpha -BgTx binds at equal rates to the two sites.

For receptor activation, concentration-response curves for ACh and other agonists were fit to the following equation.
I/I<SUB><UP>max</UP></SUB>={1+(K<SUB><UP>ap</UP></SUB><UP>/</UP>[<UP>ACh</UP>])<SUP>n</SUP>}<SUP>−1</SUP> (Eq. 3)
where I and Imax are the currents at a given concentration of ACh and the maximal value, respectively, and Kap is the concentration of ACh required for half-maximal current. Because high concentrations of ACh do not result in a concentration-independent maximal response (due to desensitization and/or channel block), the Hill coefficients (nH) for the agonist dose-response relations were estimated from the slope of plots of log I versus log[agonist] at currents less than 20% of the maximal response for each agonist.

The dose-dependent inhibition of ACh-induced currents by antagonists was fit according to Equation 1 above. SigmaPlot (Jandel Scientific) was used for nonlinear least squares fit of the data, and the S.E. of the parameter fits are indicated in the Tables.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Influence of gamma W55L and delta W57L on the Binding of d-Tubocurarine and Acetylcholine-- Torpedo nAChRs were expressed in Xenopus oocytes by injection of cRNAs encoding alpha , beta , gamma , and delta  subunits. We characterized the equilibrium binding of antagonists and agonists by their inhibition of 125I-alpha -BgTx binding to Torpedo nAChRs in membranes isolated from oocyte homogenates. dTC inhibited 125I-alpha -BgTx binding to wild-type (alpha 2beta gamma delta ) nAChRs in a concentration-dependent manner with an IC50 of 440 ± 30 nM (Hill coefficient, nH = 0.50 ± 0.01) (Fig. 1A, see "Experimental Procedures"). These data were well fit by a two-site model with KH = 50 nM (high affinity site) and KL = 4 µM (low affinity site) (see Table I), and the data were consistent with the equilibrium binding of [3H]dTC to nAChRs in Torpedo membranes (5, 6) and with the assumption that 125I-alpha -BgTx binds to the two agonist sites with equal association rate constants. dTC binds to delta -less (alpha 2beta gamma 2) receptors with an IC50 of 140 nM and to gamma -less (alpha 2beta delta 2) receptors with an IC50 of 8 µM, consistent with the notion that for dTC the high and low affinity sites are formed at the alpha -gamma and alpha -delta interfaces, respectively (Fig. 1A, inset, and Table I). We determined the effects of the gamma W55L and delta W57L mutations on 125I-alpha -BgTx binding and on dTC competition with 125I-alpha -BgTx binding. Control experiments indicated that gamma W55L and delta W57L had no effect on 125I-alpha -BgTx binding affinity (data not shown). Despite the decrease of dTC potency as an inhibitor of nAChRs containing gamma W55L (Ref. 13 and see below), nAChRs containing either a gamma W55L (gamma m), a delta W57L (delta m) subunit, or both mutant subunits (alpha 2beta gamma mdelta m) had the same binding affinities for dTC as wild-type receptors (Fig. 1A). The concentration dependence for dTC inhibition of 125I-alpha -BgTx binding to mutant receptors was clearly different from that observed for either gamma -less or delta -less nAChRs (Fig. 1, inset), which indicates that the lack of effect of the mutations on dTC binding is not due to omission of either gamma m or delta m subunit. Thus, substitution of gamma Trp-55 or delta Trp-57 by leucine has no effect on dTC binding affinity. These tryptophan residues are, however, located near the agonist binding sites because they can be affinity-photolabeled by [3H]dTC and [3H]nicotine (10, 11). The discrepancy between binding and functional antagonism will be explained below.



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Fig. 1.   Effects of gamma W55L and delta W57L mutations on the equilibrium binding of dTC (A) and ACh (B) to Torpedo nAChRs. Equilibrium binding was determined by the inhibition of 125I-alpha -BgTx binding to Torpedo nAChRs in membranes isolated from oocyte homogenates for wild-type nAChR (, alpha 2beta gamma delta ) or mutant receptors containing gamma W55L (black-square, alpha 2beta gamma mdelta ), delta W57L (black-triangle alpha 2beta gamma delta m), or both mutant subunits (black-diamond  alpha 2beta gamma mdelta m). For ACh, binding was also determined for alpha 2beta (delta m)2 (triangle ) and alpha 2beta (gamma m)2 (down-triangle) mutant nAChRs as well as for native Torpedo membranes (diamond ). Assay aliquots (100 µl) of Torpedo membranes contained 50 fmol of nAChR and membranes from 3 uninjected oocytes and were treated with 100 µM diisopropylphosphofluoridate to inactivate cholinesterase. Insets, dTC (A) and ACh (B) binding to gamma -less (, alpha 2beta delta 2) and delta -less (triangle , alpha 2beta gamma 2) nAChRs compared with wild-type (open circle ). For each data set, the data points represent the mean ± S.D. of triplicate samples from a single experiment representative of 2-4 experiments. Solid curves are calculated from the parameters of Table I for the two-site model ("Experimental Procedures," Equation 2).


                              
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Table I
Equilibrium binding parameters for dTC and ACh
Non-linear least squares fit of data in Figs. 1, A and B according to Equations 1 and 2 (see "Experimental Procedures").

In contrast to what was observed for dTC binding, gamma W55L and delta W57L dramatically increased the IC50 for ACh competition against 125I-alpha -BgTx binding to nAChRs. ACh inhibited 125I-alpha -BgTx binding to the wild-type Torpedo nAChR expressed in oocytes in a concentration-dependent manner with an IC50 of 340 nM and a Hill coefficient of 0.6 (Fig. 1B). gamma W55L and delta W57L increased the IC50 for ACh binding by 62- and 8-fold, respectively, and for the double mutant (alpha 2beta gamma m delta m), the IC50 was 530-fold higher than for wild-type receptor (Fig. 1B). The IC50 values for ACh binding to either alpha 2beta (gamma m)2 or alpha 2beta (delta m)2 receptors were each increased by about 1,300-fold (Fig. 1B). The concentration dependence of ACh inhibition of 125I-alpha -BgTx binding reflects the binding of both ACh and 125I-alpha -BgTx to two agonist binding sites. For the wild-type receptor this concentration dependence was well fit by a two-site model with KH of 55 nM and KL of 2.7 µM (Table I). ACh bound to the gamma W55L-containing receptor with KH of 750 nM and KL of 360 µM. For the delta W57L-containing receptor, ACh bound with KH of 140 nM and KL of 66 µM.

The effects of the gamma W55L and delta W57L mutations on ACh binding were clearly different than that observed for nAChRs lacking either gamma  subunit (gamma -less, alpha 2beta delta 2, IC50 = 0.8 µM) or delta  subunit (delta -less, alpha 2beta gamma 2, IC50 = 0.3 µM), which were characterized by inhibition curves similar to wild-type (Fig. 1B, inset and Table I). As judged by inhibition of 125I-alpha -BgTx binding, ACh binding to Torpedo nAChRs expressed in oocytes also differed from the binding to native nAChRs in Torpedo membranes (IC50 = 25 nM and a Hill coefficient of 0.9. (Fig. 1B)).

The observed equilibrium binding reflects the binding affinity of the ACh sites in the desensitized nAChR and the conformational equilibrium between resting and desensitized states. To test whether the leucine substitution had a predominant effect on the latter parameter, we examined the effect of proadifen, a desensitizing noncompetitive antagonist (24), on the ACh equilibrium binding function. For wild-type and alpha 2beta gamma mdelta m, proadifen produced only a modest (less than 3-fold) left shift of the ACh equilibrium binding function (data not shown), as it does for nAChRs in membranes from Torpedo electric organ (24), but in contrast to the 100-fold enhancement of ACh affinity seen for mouse muscle nAChR in the presence of proadifen (20).

Composition and Assembly of Subunits in Mutant nAChRs-- To rule out the possibility that the perturbation of ACh binding resulted from nAChRs of altered subunit composition, we examined both the size and subunit composition of the wild-type and mutant nAChRs formed in our expression system. nAChR biosynthetic assembly intermediates can form high affinity binding sites for 125I-alpha -BgTx (alpha  subunit alone) or agonists and competitive antagonists (alpha /gamma or alpha /delta subunit pairs) (4). The binding studies described above were performed with membranes isolated from oocyte homogenates that contain both surface receptors and receptors from intracellular membranes. This internal pool may contain agonist binding sites that are very different than those found in the pentameric nAChRs, since the internal alpha -BgTx binding sites may include nAChR assembly intermediates. We therefore compared surface and internal receptors in oocyte membranes by examining both the size(s) of the 125I-alpha -BgTx binding components by sedimentation analysis and the subunit composition by biosynthetic labeling and immunoprecipitation.

Sucrose density gradient analysis was carried out to determine the size(s) of the receptors expressed in oocytes. To label surface nAChRs, intact oocytes were incubated with 125I-alpha -BgTx before isolation of oocyte membranes, and isolated membranes were reincubated in 125I-alpha -BgTx to label all sites made accessible after homogenization of the oocytes (total receptors). Membranes were extracted in 1% Triton X-100, and the sedimentation properties of these nAChRs were compared (Fig. 2) with 125I-alpha -BgTx-labeled native Torpedo nAChRs (monomer, 9.5 S; dimer, 13 S) (Fig. 2A). In the membranes isolated from oocyte homogenates, there were 3-5 times more total 125I-alpha -BgTx sites (Fig. 2, B-E, closed circles) than surface sites (Fig. 2, B-E, open circles). However, the major populations of both surface and total 125I-alpha -BgTx binding sites in oocyte membranes were pentameric nAChRs, as revealed by a characteristic large peak of 125I at 9.5S for wild-type as well as mutant nAChRs (gamma m, delta m, gamma mdelta m, Fig. 2, B-E). In some experiments (Figs. 2, B and D), total receptors in oocyte membranes also yielded a much smaller peak of 125I at 5.0 S, which has been shown previously to be subunit pairs of either alpha gamma or alpha delta subunits (25). The peak at 1.7 S in all our experiments represented free 125I-alpha -BgTx. Thus, as for wild-type nAChRs, binding of 125I-alpha -BgTx to each of these mutants in membrane homogenates will reflect binding to assembled, pentameric nAChRs, and partial assembly cannot account for the altered ACh binding seen for the mutant nAChRs.



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Fig. 2.   Sucrose density gradient characterization of nAChRs containing gamma W55L and/or delta W57L mutant subunits. Membranes were prepared from oocytes preincubated with 125I-alpha -BgTx to label surface nAChRs (open circle ), and isolated membranes were further incubated with 125I-alpha -BgTx to label surface and internal alpha -BgTx binding sites (, total receptors). Triton X-100 extracts of labeled membranes prepared from pools of 10-15 injected oocytes were sedimented on 3-30% sucrose gradients. Fractions (200 µl) were collected from the top of the gradient and counted in a gamma  counter. 125I-alpha -BgTx-labeled native Torpedo nAChRs in Triton X-100 extracts, which sedimented primarily as pentameric monomer (9.5 S) along with 13 S dimers, were used as controls (Panel A). For nAChRs expressed in oocytes (panels B-H), the total receptors () were 3-5 times more abundant than surface receptors (open circle ) for alpha 2beta gamma delta (B), alpha 2beta gamma mdelta (C), alpha 2beta gamma delta m (D), alpha 2beta gamma mdelta m (E), alpha 2beta delta 2 (G), or alpha 2beta (delta m)2 (H). When the mutant gamma  subunit was coexpressed with wild-type alpha  and beta  subunits without the delta  subunit (alpha 2beta (gamma m)2, panel F), as seen for wild-type delta -less receptor (alpha 2beta gamma 2) (data not shown), no stable subunit complexes were detected in Triton X-100. For nAChRs containing four different subunits, including either gamma m, delta m, or gamma mdelta m, total as well as surface receptors sedimented as 9.5 S pentamers (panels C-E), whereas for receptors lacking the gamma  subunit but containing either delta  or delta m, a 5.0 S peak of 125I-alpha -BgTx binding was also prominent (panels G and H), consistent with the expected sedimentation of dimeric forms containing alpha -delta subunits. The peak of 125I found at 1.7 S in all experiments resulted from the presence of free 125I-alpha -BgTx.

When the mutant gamma  subunit was coexpressed with wild-type alpha  and beta  subunits without the delta  subunit (alpha 2beta (gamma m)2, Fig. 2F), stable assembly of nAChR subunits was not observed in Triton X-100. This result, which was also seen for wild-type delta -less receptor (alpha 2beta gamma 2, data not shown), is consistent with previous observations (25, 26). For the mutant delta  subunit coexpressed with wild-type alpha  and beta  subunits without the gamma  subunit alpha 2beta (delta m)2, in addition to the 9.5 S peak of 125I, there was a prominent 5 S component in the total homogenate, as was seen for wild-type gamma -less receptor (alpha 2beta delta 2) (Figs. 2, G and H). These results indicated that partial assembly intermediates are not prominent for the mutant nAChRs containing all four subunits, but they may be more significant for wild-type or mutant nAChRs lacking the gamma  or delta  subunit.

Subunit compositions of expressed wild-type and mutant nAChRs were characterized by immunoprecipitation of alpha -BgTx-labeled receptors (surface and total) extracted from oocyte homogenates after biosynthetic labeling with [35S]Met/Cys. Fig. 3 shows that for both surface (A) and total receptors (B), the presence of gamma W55L, delta W57L, or both mutant subunits had no effect on subunit assembly, and the mutations had no effect on subunit glycosylation as judged by the mobilities of mutant compared with wild-type subunits. Furthermore, the mobilities of these subunits in nAChRs expressed in Xenopus oocytes were the same as the native Torpedo nAChR subunits (not shown). However, for nAChRs lacking the gamma  subunit, mutant delta  subunit had the same mobility as wild type, but both had slightly higher mobility than delta  subunits in nAChRs also containing the gamma  subunit. Similarly, omission of the delta  subunit resulted in gamma  subunits of enhanced mobility (data not shown).



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Fig. 3.   Biosynthetic labeling and immunoprecipitation analyses of the subunit composition of nAChRs containing gamma W55L and/or delta W57L mutant subunits. 10-15 injected oocytes were incubated in ND96 (plus 50 µg/ml gentamicin) containing [35S]methionine/cysteine (0.1-0.5 mCi/ml) for 48 h. Surface (A) and total (B) receptors were labeled with nonradioactive alpha -BgTx (see "Experimental Procedures"). Triton X-100 extracts containing either surface or total receptors labeled by alpha -BgTx were incubated with affinity-purified rabbit antibody against alpha -BgTx and immunoprecipitated with 1% Immunoprecipitin. Immunoprecipitates were extracted in sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis. Gels were stained with Coomassie Blue, destained, and prepared for fluorography (36-h exposure). For both surface (A) and total (B) receptors (1, alpha 2beta gamma delta ; 2, alpha 2beta gamma mdelta ; 3, alpha 2beta gamma delta m; 4, alpha 2beta gamma mdelta m; 5, uninjected oocytes) gamma W55L and delta W57L mutant subunits were expressed and assembled with other wild-type subunits as efficiently as seen for wild-type subunits.

gamma W55L and delta W57L Alter nAChR Activation-- We examined the effects of gamma W55L and delta W57L on the activation of nAChRs expressed in oocytes using a two-electrode voltage clamp. When holding the oocyte membrane potential at -70 mV, there was a concentration-dependent activation of inward current upon 5-s application of ACh for wild-type (Fig. 4A) and mutant nAChRs (alpha 2beta gamma mdelta , Fig. 4B). For wild-type nAChR, preincubation with dTC produced a dose-dependent inhibition of ACh currents characterized by an IC50 of 40 nM (Figs. 4, C and D), as expected when binding to its high affinity site results in functional antagonism. However, as reported previously (13) for nAChRs containing gamma W55L, dTC inhibited ACh currents only at higher concentrations (IC50 = 4 µM, Fig. 4D), despite the presence of the high affinity dTC binding site (KH = 40 nM, Fig. 1A). Based upon the peak transient current observed for each ACh concentration, the ACh concentration for half-maximal response (Kap) for wild-type nAChRs was 20 ± 2 µM, whereas the Kap values for nAChRs containing gamma W55L or delta W57L subunits were 170 ± 30 µM and 86 ± 2 µM, respectively (Fig. 5A). The double mutant receptor (alpha 2beta gamma mdelta m) was activated by ACh with a Kap of 340 ± 40 µM, about 15-fold higher than the wild-type receptor. The consequences of the leucine substitutions were clearly different from that seen for gamma  or delta  subunit omission, since for gamma -less (alpha 2beta delta 2), Kap = 21 ± 4 µM and for delta -less (alpha 2beta gamma 2), Kap = 14 ± 1 µM (not shown).



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Fig. 4.   Electrophysiological recordings from wild-type and mutant nAChRs expressed in oocytes. A, inward currents evoked by 5 s applications of ACh to an oocyte expressing wild-type nAChRs. The oocyte was injected with diluted RNA (2.5 ng, a molar ratio of 2:1:1:1 alpha :beta :gamma :delta ) to limit the current amplitudes at high ACh concentrations. The recordings were made 48 h after injection, and the oocyte membrane potential was held at -70 mV. B, concentration-dependent activation of gamma W55L mutant by ACh. The oocyte was injected with 100 ng RNA (2:1:1:1 alpha :beta :gamma m:delta ), which resulted in currents of similar magnitude as seen for wild-type nAChR after injection of lower amounts of RNA. C, dTC inhibition of ACh-induced currents for wild-type nAChR. Currents evoked by 3 µM ACh (3 s) from an oocyte injected with 10 ng of RNA were inhibited by preincubation with various concentrations of dTC. D, upon preincubation, dTC inhibited 3 µM ACh-induced currents from wild-type (WT) nAChRs () with an IC50 of 40 ± 4 nM (nH = 0.9 ± 0.1). For gamma W55L mutant receptors (black-square) after preincubation, dTC inhibited 100 µM ACh-induced currents with an IC50 of 3.7 ± 0.6 µM (nH = 0.9 ± 0.1). The data represent the mean ± S.D. of three measurements.



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Fig. 5.   ACh dose-response relations for gamma W55L and delta W57L mutant nAChRs. A, for wild-type (W.T.) nAChRs (, alpha 2beta gamma delta ) the ACh dose-response was fit by Kap = 20 ± 2 µM (n = 6 oocytes). For the gamma W55L mutant (black-square alpha 2beta gamma mdelta ) Kap = 165 ± 30 µM (n = 4 oocytes), and for the delta W57L mutant (black-triangle, alpha 2beta gamma delta m) Kap = 86 ± 2 µM (n = 3 oocytes). For the double mutant (black-diamond , alpha 2beta gamma mdelta m), Kap = 340 ± 40 µM (n = 4 oocytes). Dose-response relations for wild-type receptors were determined for oocytes injected with 0.4 ng of subunit RNAs, whereas for mutant receptors, 10 ng were injected. The data represent the means ± S.D. B, oocytes were injected with 10 ng of subunit cRNAs. Mutant receptors were expressed on the oocyte surface at the same level as wild-type receptors as determined by 125I-alpha -BgTx binding (open bars, n = 10 oocytes). The maximal currents for the delta W57L mutant were twice as large as the currents seen for wild-type receptors at 3 µM ACh (hatched bar), a concentration producing only 5% of maximal currents. The maximal currents (closed bars) induced by saturating concentrations of ACh for gamma W55L (alpha 2beta gamma mdelta , 1.5 ± 0.3 µA, n = 6 oocytes) and for gamma W55L/delta W57L (alpha 2beta gamma mdelta m, 0.16 ± 0.04 µA, n = 6 oocytes) mutants were only 6 and 0.6% that seen for the delta W57L mutant (alpha 2beta gamma delta m, 25 ± 4 µA, n = 5 oocytes). The data represent the means ± S.D. C, Hill coefficients for ACh activation of wild-type and mutant nAChRs, determined from the concentration-response relationship at concentrations of ACh producing less than 20% maximal currents. ACh-induced currents (µA) recorded from wild-type (), gamma W55L (black-square), and delta W57L (black-triangle) receptors were plotted logarithmically against the concentration of ACh, with each data point the mean ± S.D. of three recordings. The data for a single oocyte were fit by linear regression, and the slope of the line (nH) for the wild-type receptor () was 1.85, with the same analysis for data from three oocytes characterized by a slope of 1.8 ± 0.1. For the gamma W55L mutant (black-square), the slope for this representative experiment was 1.04, and the average slope from three experiments was 1.10 ± 0.05. The delta W57L mutation, however, had no effect on the slope of the concentration-response relationship of ACh for receptor activation. The slope of the line for the delta W57L mutant (black-triangle) presented here was 1.71, and the average value of the slope from three experiments was 1.66 ± 0.14.

Fig. 5B shows a comparison of the number of 125I-alpha -BgTx binding sites on the oocyte surface and the current amplitudes for mutant AChRs activated by saturating concentrations of ACh. nAChRs containing either gamma W55L, delta W57L, or both mutant subunits were expressed at nearly the same levels as the wild-type nAChR, as indicated by the number of surface 125I-alpha -BgTx binding sites. In contrast, the maximal currents for the mutant nAChRs were much lower than for wild type. When equal amounts of subunit cRNAs were injected for wild-type and mutant nAChRs, the maximal currents for the delta W57L mutant were twice as large as the currents seen for wild-type nAChR at 3 µM ACh, a concentration producing ~5% of maximal currents. Thus, for nAChRs containing the delta W57L subunit, the maximal currents were ~10% that of wild-type, whereas for receptors containing gamma W55L or both mutant subunits, the maximal currents were only 6 ± 1 and 0.6 ± 0.2% that seen for delta W57L. These results indicate that mutation of gamma Trp-55 and delta Trp-57 alters the activation of these channels by ACh.

The Hill coefficients (nH) characterizing the ACh dose-response relations were estimated from log-log plots of current amplitude (I) versus [ACh] at concentrations of ACh producing currents less than 20% of maximal responses. This is a reliable method of determining nH for ACh responses without reference to the experimentally determined maximal currents, which are limited by desensitization and/or channel block. For the wild-type and delta W57L mutant receptors, the concentration-response relationship had slope values (nH) of 1.8 ± 0.1 and 1.7 ± 0.1, respectively (Fig. 5C). In contrast, the slope for the gamma W55L mutant was one (nH = 1.1 ± 0.1) (Fig. 5C).

dTC Potentiation of Agonist-induced Activation in gamma W55L nAChRs-- For the wild-type nAChR, upon preincubation, dTC acted as a competitive antagonist characterized by an IC50 of 40 nM (Fig. 4D), and without preincubation, when coapplied with 3 µM ACh for 5 s, dTC inhibited with an IC50 of 250 nM (Fig. 6C). Similar results were obtained for nAChRs containing delta W57L (data not shown). For nAChRs containing gamma W55L, despite the fact that dTC binds with high affinity (KH = 40 nM) to one of the sites (Fig. 1A), upon preincubation, dTC inhibited ACh currents only at high concentrations (IC50 = 4 µM, Fig. 4D), as reported by O'Leary et al. (13). We therefore examined in greater detail the interactions of dTC with nAChRs containing gamma W55L. When applied alone, dTC produced no detectable whole cell currents (<10 nA, data not shown). However, we observed that at low concentrations dTC (10 nM to 1 µM) actually potentiated currents activated by ACh (10-100 µM) when both ligands were applied simultaneously to individual oocytes (Fig. 6). The magnitude of the potentiation was dependent on the concentration of both dTC and ACh (Fig. 6C), with the concentration dependence of potentiation by dTC for 10 µM ACh consistent with dTC binding to its high affinity binding site. The largest dTC potentiation was observed at low concentrations of ACh, and the magnitude of the potentiation decreased with higher concentrations of dTC. Concentrations above 10 µM dTC only inhibited currents activated by any concentration of ACh (Fig. 6C). For gamma W55L nAChRs preincubated with dTC, no potentiation was ever seen for responses to ACh at concentrations between 0.3 and 100 µM.



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Fig. 6.   For the gamma W55L mutant, dTC potentiates responses to ACh. For an oocyte expressing gamma W55L mutant receptors, responses to 10 µM (A) and 30 µM (B) ACh were recorded alone or with the simultaneous application of 1 µM dTC. C, concentration-dependent potentiation of ACh-induced currents by dTC. Concentrations of dTC were applied to oocytes simultaneously with fixed concentrations of ACh for 5 s. Control responses (100%) to different concentrations of ACh were obtained in the absence of dTC. For wild-type nAChR (open circle ), dTC inhibited the response to 3 µM ACh with an IC50 of 250 nM (the data represent the mean ± S.D. of three measurements). In contrast, for gamma W55L mutant receptors, low concentrations of dTC (30 nM-1 µM) potentiated currents evoked by ACh at 10 (black-diamond ) and 30 (black-triangle) µM. For 10 µM ACh, dTC at 40 nM produced half-maximal potentiation, whereas at higher concentrations dTC inhibited the response with an IC50 approx  3 µM. At 100 µM ACh (black-square), low concentrations of dTC caused little or no potentiation, and at 300 µM ACh (), dTC inhibited responses with an IC50 of 3 µM. The data represent the mean ± S.D. from 3-5 experiments.

For the mutant receptor containing gamma W55L, potentiation by dTC was also observed for responses to two other agonists, carbamylcholine and suberyldicholine (Fig. 7). As for ACh, for these agonists the Hill coefficient (nH) characterizing the dose-response relation was reduced from 1.6 for wild-type nAChRs to ~1 for the mutant receptor (Fig. 7A). dTC at concentrations between 10 nM and 1 µM produced a dose-dependent enhancement of responses for agonist concentrations producing submaximal responses. Higher dTC concentrations produced a progressive inhibition of the currents. For carbamylcholine, dTC enhanced currents as much as 3-fold (Fig. 7B), whereas for suberyldicholine, currents were increased by about 2-fold (Fig. 7C). For alpha 2beta gamma mdelta , potentiation of agonist-induced currents was not limited to dTC, since metocurine, the 7',12'-dimethoxy-2-methyl dTC analog, also potentiated activation of the mutant receptor by ACh (Fig. 7D), However, neither 13'-iodo-dTC nor two other competitive antagonists, pancuronium and gallamine, potentiated ACh responses (Fig. 7D). These results establish that when dTC (or metocurine) first binds to nAChRs containing gamma W55L, it does not act as an antagonist and, in contrast, acts as a coactivator or weak partial agonist, as evidenced by the potentiation of responses seen when agonists bind to the site at the alpha -delta subunit interface.



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Fig. 7.   For the gamma W55L mutant nAChR, dTC potentiates currents elicited by other agonists, and other antagonists potentiate ACh-induced currents. Panel A, the gamma W55L mutation alters the cooperativity of nAChR activation by the agonists suberyldicholine (Sub; open circle , ) and carbamylcholine (Carb; , black-square). In the individual experiments presented here, the Hill coefficient (nH) of suberyldicholine for wild-type nAChRs was 1.70 (open circle ), whereas for gamma W55L, nH = 1.05 (). For carbamylcholine and wild-type nAChR (), nH = 1.60, and for gamma W55L, nH = 1.02 (black-square). For each case the data are for responses determined in a single oocyte and are means ± S.D. for three measurements at each concentration. For activation of the mutant receptor by suberyldicholine or by carbamylcholine, the average value of nH from three experiments was 1.1 ± 0.1. Suberyldicholine activated wild-type and gamma W55L nAChRs with Kap values = 1.9 ± 0.1 µM and 7 ± 2 µM, respectively, and Carb activated these receptors with Kap values = 350 ± 10 µM and 750 ± 125 µM, respectively (3 oocytes each). Panels B and C, for gamma W55L mutant nAChR, dTC potentiates currents induced by carbamylcholine (Carb, B) and suberyldicholine (Sub, C). Panel D, effects of competitive antagonists on responses to 30 µM ACh for gamma W55L. The dTC analog metocurine (black-diamond ) at concentrations between 0.1 and 10 µM potentiated responses, whereas no potentiation was seen for 13'-iodo-dTC (black-triangle), gallamine (black-square), or pancuronium (). The IC50 values for iodo-dTC (IdTC) , gallamine, and pancuronium (Pan) were 44 µM, 5 µM, and 27 nM, respectively. The data represent the means ± S.D. of three recordings made in single oocytes, and the experiments were repeated with at least three oocytes.

Influence of gamma W55L/delta W57L on the Binding of Nicotinic Agonists and Antagonists-- We also examined the effects of the double mutation (alpha 2beta gamma mdelta m) on the binding affinities of several agonists and antagonists to learn more about the effects of these substitutions on the binding of structurally diverse agonists and antagonists (Fig. 8). Most of the agonists tested, including tetramethylammonium, phenyltrimethylammonium, suberyldicholine, and epibatidine, inhibited 125I-alpha -BgTx binding to double mutant receptors with IC50 values that were ~50-500-fold larger than for the wild-type receptor. Thus, mutation of gamma Trp-55 and delta Trp-57 influences the binding of most nicotinic agonists, even very small agonists like tetramethylammonium. It is interesting that the agonists with highest affinity for wild-type receptors were affected the most by the double mutation. One exception to the general pattern was nicotine, which bound to the wild-type and double mutant receptors with very similar affinity (Fig. 8F).



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Fig. 8.   The effects of the double mutation (alpha 2beta gamma mdelta m) on the equilibrium binding affinities of nicotinic agonists and antagonists. Inhibition of 125I-alpha -BgTx binding to wild-type receptors (, alpha 2beta gamma delta ) and to gamma W55L/delta W57L (open circle , alpha 2beta gamma mdelta m) was determined for the agonists ACh (A), suberyldicholine (Sub, B), tetramethylammonium (TMA, C), phenyltrimethylammonium (PTA, D), epibatidine (Epi, E), and nicotine (Nic, F) and for the competitive antagonists gallamine (Gal, G) and pancuronium (Pan, H). The double mutation gamma W55L/delta W57L (open circle , alpha 2beta gamma mdelta m) had no effect on nicotine binding (F) but increased the IC50 of the other agonists by 50-500 fold (see Table II). The double mutation had no effect on gallamine (G) binding but decreased the binding affinity of pancuronium (H) at the high affinity site. The data represent the mean ± S.D. (three samples/point).

The effects of the double mutation (alpha 2beta gamma mdelta m) on the binding affinities of competitive antagonists are more diverse and complex than that seen for the agonists. The double mutation had no effect on the binding affinity of gallamine (Fig. 8G), similar to what was observed with dTC (Fig. 1). Pancuronium was bound with 10,000-fold selectivity by wild-type nAChRs (KH = 3 nM, KL = 20 µM), with binding at the high affinity site weakened by 100-fold in the double mutant and binding at the low affinity site weakened by less than 3-fold (Table II). The binding affinity of dihydro-beta -erythroidine was decreased by about 10-fold at each site by the double mutation. The results of the binding experiments shown in Fig. 8 are summarized in Table II.


                              
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Table II
Equilibrium binding parameters for agonists and antagonists
Non-linear least squares fit of data in Figs. 1 and 8 according to Equations 1 and 2 (see "Experimental Procedures"). TMA, tetramethylammonium; PTA, phenyltrimethylammonium; Epi, epibatidine; Sub, suberlydicholine; DHbeta E, dihydro-beta -erythroidine.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A wide body of evidence establishes that the two agonist binding sites in muscle-type nAChRs are positioned at the interfaces of alpha -gamma and alpha -delta subunits, with amino acids contributed from three distinct regions of alpha  subunit primary structure and at least three regions of gamma  (or delta ) subunit (reviewed in Refs. 3, 8, and 9). We set out to study the functional contribution of two tryptophans, gamma Trp-55 and delta Trp-57, at homologous positions of the gamma  and delta  subunits. Tryptophans gamma Trp-55 and delta Trp-57 are within or near the agonist/competitive antagonist binding sites, since they are the principle sites of specific photoincorporation by [3H]dTC within those subunits (10), and gamma Trp-55 is the amino acid in the gamma  subunit specifically photolabeled by [3H]nicotine (11). In addition, they appeared likely to contribute to dTC binding affinity, because replacement of gamma Trp-55 by leucine resulted in a decrease of dTC potency as an inhibitor of ACh-induced currents for Torpedo nAChRs expressed in Xenopus oocytes (13). Although we confirmed the observation that for the gamma W55L mutant nAChR, the IC50 for dTC inhibition was increased 100-fold compared with wild type (Fig. 4), we were surprised to find that the mutation of either or both tryptophans had no effect on dTC equilibrium binding affinity, based upon the inhibition of binding of 125I-alpha -BgTx (Fig. 1). Instead, our results indicate that the shift in dTC potency as an inhibitor of ACh-induced currents was a secondary consequence of a dramatic reduction in the affinity of ACh binding at the alpha -gamma site.

Although the leucine substitutions had no effect on dTC binding, on nAChR subunit assembly as judged by sucrose density gradient velocity sedimentation (Fig. 2) and immunoprecipitation (Fig. 3), or on the level of surface expression of nAChRs (Fig. 5B), the substitutions clearly altered agonist interactions as evidenced by the perturbation of the ACh-induced currents (Figs. 4 and 5). The ACh concentrations producing half-maximal currents (Kap) were shifted to the right 8- and 20-fold for the gamma W55L and for gamma W55L/delta W57L mutant nAChRs, and although there were no significant changes in the levels of surface expression, the maximal currents were only 0.5 and 0.05%, respectively, that seen for wild-type nAChR. In addition, for the gamma W55L mutant, there was a clear shift in the concentration dependence of the response compared with the wild-type or the delta W57L mutant receptor. For the gamma W55L mutant the observed Hill coefficient (nH), determined at ACh concentrations producing <20% maximal responses, was close to 1 (nH = 1.1 ± 0.1), whereas it was close to 2 for wild-type (nH = 1.8 ± 0.1) and for the delta W57L mutant (nH = 1.7 ± 0.1).

Although dTC binding affinity at the alpha -gamma site was unaltered by the replacement of gamma Trp-55 by leucine, concentrations of dTC sufficient to occupy the alpha -gamma site did not inhibit ACh-induced currents. After pre-equilibration with nAChRs, dTC did not inhibit the ACh response until present at concentrations sufficient to occupy the alpha -delta site (IC50 = 4 µM, Fig. 4D). When applied simultaneously with ACh, dTC at concentrations sufficient to occupy the alpha -gamma site actually potentiated the ACh responses seen at concentrations less than 100 µM (Fig. 6). Therefore, replacement of gamma Trp-55 by leucine allows dTC to function as a partial agonist or coactivator; dTC binding to the alpha -gamma site in the absence of ACh did not produce measurable currents, but when coapplied with ACh, dTC potentiated the ACh response by as much as 5-fold. These functional consequences of the replacement of gamma Trp-55 by leucine were not limited to ACh, since the concentration dependence for the current responses seen for carbamylcholine and suberyldicholine were also characterized by Hill coefficients of 1, and those responses were potentiated by dTC (Fig. 7). Not all competitive antagonists act as ACh coactivators of the gamma W55L mutant nAChR. Although potentiation was seen for dTC and its close structural analog metocurine, pancuronium and gallamine remained antagonists (Fig. 7D).

The leucine substitutions, especially the replacement of gamma Trp-55, caused a major perturbation of the equilibrium binding of ACh, as deduced from the inhibition of the initial rate of 125I-alpha -BgTx binding (Fig. 1B). The equilibrium binding function reflects both the affinity of binding to the desensitized state of the nAChR and the conformational equilibrium between resting and desensitized states. Before considering the effects of the substitutions, it is important to note several aspects of the observed binding to wild-type Torpedo nAChRs expressed in oocytes. The equilibrium binding of ACh by wild-type Torpedo nAChRs expressed in oocytes was well fit by a two-site model with an equal number of high and low affinity sites (KH = 55 nM, KL = 3 µM). The parameters for ACh binding Torpedo alpha 2beta gamma delta are similar to those seen for embryonic mouse nAChR expressed in oocytes (16), but they were quite different than those seen for ACh binding to Torpedo nAChR-rich membranes, which was characterized by high affinity (K = 25 nM) binding to a single site (Fig. 1B). We do not know the source of this difference, but it does not appear to result from a shift of the preexisting equilibrium between resting and desensitized states, since the desensitizing noncompetitive antagonist proadifen had similar effects on ACh binding to either the native or expressed Torpedo nAChR. A noteworthy distinction between ACh interactions with Torpedo and embryonic mouse nAChR (alpha 2beta gamma delta ) is that our data indicate that in the Torpedo nAChR, ACh binds with higher affinity to the alpha -gamma than to the alpha -delta site, whereas it binds with higher affinity at the alpha -delta site for the mouse nAChR (16, 27). The latter conclusion was based upon the observation that mouse alpha 2beta delta 2 binds ACh with 15-fold higher affinity than alpha 2beta gamma 2. For Torpedo nAChRs, ACh binds nonequivalently to the two sites even for receptors lacking the gamma  or delta  subunit, but ACh binds with higher affinity to alpha 2beta gamma 2 than to alpha 2beta delta 2 (Fig. 1B, Table I). For mouse nAChRs, preferential agonist binding to the alpha -delta site is not a general rule. For the adult nAChR containing an epsilon  subunit in place of the gamma  subunit, ACh binds nonselectively at the two sites, whereas epibatidine binds with higher affinity at the alpha -gamma (or alpha -epsilon ) site than at the alpha -delta site, and for carbamylcholine the rank order is alpha -delta  ~ alpha -epsilon  > alpha -gamma (28, 29).

Replacement of gamma Trp-55 by leucine had a much larger effect on ACh equilibrium binding than did replacement of delta Trp-57 (Fig. 1 and Table I). Although the high and low affinity sites characteristic of the ACh equilibrium binding to wild-type nAChRs cannot be assigned unambiguously to binding at alpha -gamma and alpha -delta sites, the observed binding by wild-type and mutant nAChRs is consistent with a simple interpretation if the high affinity ACh binding is at the alpha -gamma site in wild-type nAChR, and the effects of the mutations are greatest at the binding site containing the mutation. Then ACh binds with high affinity (KH = 50 nM) to the alpha -gamma site in wild-type nAChR, and that binding is 7,000-20,000-fold weaker in alpha 2beta gamma mdelta (KL = 360 µM) and alpha 2beta gamma mdelta m (KL = 1 mM) but shifted less than 3-fold in alpha 2beta gamma delta m (KH = 140 nM). ACh binds with low affinity (KL = 3 µM) at the alpha -delta site in wild-type nAChR, and that binding is preserved in alpha 2beta gamma mdelta (KH = 0.8 µM) but weakened by 10-20 fold in alpha 2beta gamma delta m (KL = 66 µM) and alpha 2beta gamma mdelta m (KH = 23 µM). Thus the gamma W55L mutation disrupts ACh binding at the alpha -gamma m site without causing a structural change that substantially perturbs ACh binding at the alpha -delta site. Within the alpha -gamma m site, the change in structure must be limited, because the mutation does not alter dTC binding. If the selective disruption of ACh binding at equilibrium seen at the alpha -gamma site (and not the alpha -delta site) in alpha 2beta gamma mdelta also occurs in the resting state, then the alpha -delta site will become the higher affinity site in the alpha 2beta gamma mdelta nAChR. Further studies at the level of single channel analysis will be required to determine the extent to which the leucine substitution alters the initial binding step or the conformational equilibria related to channel gating.

Since the leucine substitutions had no effect on the equilibrium binding of dTC, it was very surprising to observe for the gamma W55L nAChR that dTC binding to the high affinity (alpha -gamma ) site no longer acted as an antagonist. Rather, inhibition of ACh responses was seen only when dTC was bound to the alpha -delta site, and dTC actually acted as a partial agonist or coagonist when it bound to the mutated alpha -gamma site. These observations appear to suggest paradoxically that the gamma W55L substitution converted dTC from an antagonist into a partial agonist without having any effect on the energetics of its binding. However, an alternative explanation is that the mutation alters ACh binding only, with a secondary consequence that this mutation reveals the functional consequences of dTC occupancy of its high affinity site when ACh is binding to the alpha -delta site. Two lines of reasoning support this interpretation. First, the experimental results can be accounted for by a three-state allosteric model (resting, open, desensitized) that assumes that ACh binding to the alpha -delta site as well as dTC binding to both sites is unaltered between wild-type and gamma W55L mutant AChR. By adjusting parameters only for ACh binding at the alpha -gamma site in the three states, it is possible to account for the observed shift of ACh Kap and the Hill coefficient, the equilibrium binding of ACh and dTC to wild-type and mutant nAChRs, and the observed concentration dependence of dTC potentiation of the ACh responses in the mutant nAChR.2 Secondly, although we have not found experimental conditions where dTC acts as a coactivator for wild-type Torpedo nAChR, dTC and metocurine (but not pancuronium) act as weak partial agonists potentiating the responses seen at low ACh concentrations for rat and mouse muscle embryonic nAChRs (alpha 2beta gamma delta ) (30-33). In contrast, they all act as competitive antagonists at adult nAChRs (alpha 2beta epsilon delta ). Despite the difference in pharmacology, dTC binds with the same high affinity to the mouse alpha -gamma site as to the alpha -epsilon site, but as we discussed above, ACh binds with higher affinity to the alpha -delta site than to the alpha -gamma site in the embryonic nAChR, whereas it binds nonselectively to the alpha -epsilon and alpha -delta sites (29).

Replacement of gamma Trp-55 by leucine weakens ACh equilibrium binding at the alpha -gamma site by 7,000-fold, a decrease of affinity substantially larger than the ~20-500-fold shift of affinity seen for substitutions of the conserved tyrosines within the alpha  subunit binding loops (34) or for charge-neutralizing mutations (alpha D152Q, gamma D174N, delta D180N) in mouse nAChR (16, 18, 35). For nAChRs containing gamma D174N/delta D180N as well as for nAChRs containing alpha D152Q the mutations appear to affect ligand binding directly rather than the conformational equilibria between resting and desensitized states (18). However, for the simplest agonist, tetramethylammonium, equilibrium binding is weakened by 100-fold for the gamma W55L mutant, for gamma D174N (16), and also by substitutions at alpha Tyr-93, alpha Tyr-190, and alpha Tyr-198 (34). It is unlikely that tetramethylammonium is simultaneously in contact with all of these side chains.

Although the presence of gamma Trp-55 within the agonist binding site was identified on the basis of its photolabeling by [3H]dTC, leucine substitution has no effect on dTC binding affinity. It is likely that replacement by other amino acids will alter dTC binding affinity, because even the leucine substitution weakens the binding of the competitive antagonists dihydro-beta -erythroidine and pancuronium by 10- and 70-fold, respectively. In addition, substitution of the corresponding tryptophan in homooligomeric alpha 7 nAChRs (alpha 7Trp54) by histidine weakened dihydro-beta -erythroidine binding by 10-fold (36).

Our results establish the importance of gamma Trp-55 for agonist binding and channel gating, but they do not establish that ACh interacts directly with this tryptophan. In studies of mutant Torpedo nAChRs containing cysteines within the binding site, there was no evidence that gamma W55C was accessible for reaction with cationic methylthiosulfonates, whereas an adjacent position (gamma E57C) as well as alpha Y93C and alpha Y198C were all accessible for modification (37). This result, in conjunction with the fact that gamma Trp-55 is photolabeled by [3H]dTC and [3H]nicotine, suggests that gamma Trp-55 may be within a hydrophobic subdomain of the binding site.

Substitutions at positions equivalent to nAChR gamma Trp-55 also have important functional consequences for other members of the superfamily of ligand-gated ion channels related to the nAChRs. For alpha 1beta 2gamma 2 gamma -aminobutyric acid type A receptors, replacement of alpha 1Phe-64 by leucine results in 200-fold decrease of gamma -aminobutyric acid potency, whereas substitution of the equivalent positions in the other subunits had no effect on agonist potency (38). In contrast, substitution at the equivalent position in gamma 2 subunit (gamma 2Phe-77) selectively alters binding of ligands at the benzodiazepine site (39). The serotonin 5-HT3 receptor also contains a tryptophan (Trp-89) at the equivalent position, and substitutions at the position reduce antagonist but not agonist affinity (40).

The results presented here demonstrate that for the Torpedo nAChR a single mutation (gamma W55L) within a part of the agonist binding site contributed from the gamma  subunit can effectively prevent ACh binding at that site without altering ACh binding at the alpha -delta site. In addition, for this mutation, dTC binding to the alpha -gamma site acts as a partial agonist or coactivator of ACh responses. Since dTC binds at equilibrium to the alpha -gamma site in the mutant nAChR with the same high affinity as in wild type, it is most likely that the mutation does not alter dTC binding to any conformational state. Instead, by altering the interactions of ACh with the alpha -gamma site, the mutation reveals an aspect of dTC interaction with wild-type Torpedo nAChR that is not normally seen because of the relative affinities of dTC and ACh for the binding sites.


    ACKNOWLEDGEMENTS

We thank Dr. Stuart J. Edelstein for developing the three-state allosteric model used to interpret the data and Drs. Kenton J. Swartz, Deirdre Sullivan Stewart, and Gerald D. Fischbach for many helpful comments concerning this manuscript.


    FOOTNOTES

* This research was supported in part by United States Public Health Service Grant NS 19522.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A Harvard Mahoney Neuroscience Institute Fellow. Present address: Millenium Pharmaceuticals, Inc., Cambridge, MA 02139.

§ To whom correspondence should be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen@hms.harvard.edu.

Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M009085200

2 S. J. Edelstein and J. B. Cohen, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; alpha -BgTx, alpha -bungarotoxin; dTC, d-tubocurarine; HB, homogenization buffer.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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