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Agonist-Induced Conformational Changes in the Extracellular Domain of 7 Nicotinic Acetylcholine Receptors

Departments of Pharmacology (L.K.L., A.D.S., D.E., J.T.M., R.L.R.) and Cell and Molecular Physiology (R.L.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received April 2, 2003; accepted June 17, 2003.

	Abstract

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The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the 9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E¹⁷² in chick 7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L²⁴⁷T 7 nAChRs. In the absence of agonist, ACh-evoked responses of E¹⁷²C/L²⁴⁷T 7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N¹⁷⁰C and did not change inhibition of G¹⁷¹C receptors. The antagonist dihydro--erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N¹⁷⁰ to a more accessible position and E¹⁷² to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

Ligand-gated ion channels containing the conserved Cys loop are formed as a rosette of five homologous or identical subunits around a central pore (Karlin, 2002). In each subunit, the N-terminal 210 residues make an extracellular domain that forms the ligand binding region and the outer vestibule of the ion-conducting pore. The extracellular domain of each subunit is connected to a transmembrane domain consisting of four helices. The transmembrane pore is lined by the second (M2) helix. The "gate" of the channel, the region that controls whether the channel is in a conducting or nonconducting state, is located at the cytoplasmic end of the M2 helix (Karlin, 2002).

The molecular mechanisms that couple agonist binding in the extracellular domain to the opening of the gate are not well understood. A wave of conformational change is believed to propagate from the ligand binding site to the gate (Grosman et al., 2000). These conformational changes could include symmetric (Unwin et al., 2002) or asymmetric (Horenstein et al., 2001) rotation of the M2 helices, perhaps driven by interactions between the extracellular ligand binding domain and the extracellular loop between the M2 and M3 transmembrane helices (Grosman et al., 2000; Kash et al., 2003).

Our understanding of ligand binding has been advanced greatly by the crystal structure of the ACh binding protein (AChBP) (Brejc et al., 2001). This soluble protein shares sequence homology with the N-terminal ecto-domain of nicotinic receptor subunits (24% sequence identity with the 7 subunit), contains the archetypal Cys loop, and forms a pentamer around a central cavity. These properties, together with functional ligand binding properties, make the AChBP the best model to date for the structure that forms the extracellular ligand binding domain of Cys-loop ionotropic receptors. Structural details from the AChBP also provide insight into possible mechanisms for the conformational changes associated with gating (Kash et al., 2003).

The structure of the AChBP contains a core of 10 strands and shows the ligand binding site at the cleft between subunits (Brejc et al., 2001). Among the residues that form the binding site are conserved tyrosines (Y¹⁸⁷ and Y¹⁹⁴ in chick 7 numbering) and a vicinal disulfide (C¹⁸⁹–C¹⁹⁰) that define a region identified previously as essential for ligand binding (Brejc et al., 2001; Karlin, 2002). This region is located at the C terminus of the 9 strand. At the other end of the 9 strand, in loop 9, is a glutamate residue (E¹⁷² in chick 7) that is conserved among nAChR receptor subunits (except 1) (Elgoyhen et al., 1994, 2001; Le Novere and Changeux, 1995). This glutamate is important for modulating the gating of 7 nAChRs by extracellular Ca²⁺. In chimeric receptors formed from the N terminus of 7 nAChRs and the transmembrane domain of 5-hydroxytryptamine-3 receptors, the mutation of E¹⁷² abolishes the enhancement of receptor activation by extracellular Ca²⁺ (Galzi et al., 1996). In nondesensitizing L²⁴⁷T 7 receptors (L9'T in the M2 numbering system) (Revah et al., 1991), mutation of E¹⁷² to Gln or Cys eliminates the enhancing effects of extracellular Ca²⁺ and other permeant divalent cations (Eddins et al., 2002a,b). Thus, E¹⁷² is part of a region that mediates a modulatory role of permeant divalent cations on 7 receptor activation.

Because ligand binding involves residues at the C terminus of the 9 strand and Ca²⁺-dependent modulation is mediated by E¹⁷² at the N terminus of 9, we reasoned that the transmission of information from the ligand binding site to the gate might involve the 9 strand and its N-terminal loop (loop 9). We tested this hypothesis using the substituted cysteine accessibility method (Karlin and Akabas, 1998) on residues in this loop. We found evidence for agonist-dependent movement of loop 9. Using a model of the 7 extracellular domain derived from the crystal structure of the AChBP, we propose three possible mechanisms that account for the observations. These mechanisms involve the rotation of subunits and/or intrasubunit movement of the 9–10 hairpin and loop 9.

	Materials and Methods

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Reagents. Dihydro--erythroidine was obtained from Sigma/RBI (Natick, MA). Methanethiosulfonate (MTS) reagents were obtained from Toronto Research Chemicals (Toronto, Canada). Gentamicin was from Invitrogen (Carlsbad, CA). All other reagents were obtained from Fluka (Buchs, Switzerland) or Sigma (St. Louis, MO).

Site-Directed Mutagenesis. L²⁴⁷T (L9'T) 7 receptors were used as the "parent" receptor in this study because of their large current amplitudes and nondesensitizing kinetics (Revah et al., 1991). The mutagenesis of E¹⁷²C in L²⁴⁷T 7 nAChR cDNA was described previously (Eddins et al., 2002b). Cysteines at other positions were introduced into chick 7 L²⁴⁷T cDNA by site-directed mutagenesis using the QuikChange method (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing.

Xenopus laevis Oocyte Maintenance and Expression. Oocytes were surgically removed from female X. laevis frogs in accordance with University of North Carolina Institutional Animal Care and Use Committee guidelines. Oocytes were treated with collagenase to remove follicular cells and were maintained using standard procedures as described previously (Eddins et al., 2002a). cRNA was prepared using the T7 RNA polymerase and mMessage mMachine kit (Ambion, Austin, TX) as described by the manufacturer. Oocytes were injected with 20 ng of cRNA and incubated at 18°C in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4) for 2 to 5 days before use.

Two-Electrode Voltage Clamp. Oocytes were superfused in normal extracellular solution containing Ca²⁺ (ES-Ca²⁺) (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, and 10 mM HEPES, pH 7.5). In some experiments where indicated, oocytes were bathed in normal extracellular solution containing EGTA (ES-EGTA), in which 2.5 mM CaCl₂ was replaced with 1 mM EGTA. Two-electrode voltage clamp was performed with a GeneClamp 500B controlled by pCLAMP6 software (Axon Instruments, Inc., Union City, CA). Electrodes were filled with 3 M KCl contacting Ag-AgCl wires and had resistances of 0.5 to 2.0 M. Currents were recorded at a constant holding potential of –60 mV. Currents were filtered at 50 or 125 Hz and sampled at 250 Hz. To prevent the activation of Ca²⁺-activated chloride channels, oocytes were injected with 46 nl of 50 mM BAPTA 15 to 60 min before recording (Lyford et al., 2002).

Dose-Response Curves. Agonist dose-response curves were obtained as described previously (Eddins et al., 2002a), and data were fit to the Hill equation using Prism software (GraphPad Software Inc., San Diego, CA). Inhibitory dose-response curves were obtained in the presence of approximate EC₅₀ concentrations of ACh.

Modification by MTS Reagents. MTS reagents (Karlin and Akabas, 1998) were prepared daily in water and stored on ice. Because these reagents have a short half-life in physiological buffer, they were diluted to the appropriate working concentration in recording buffer immediately before applying to the oocytes. MTS reagents were applied by continuous flow for 60 s in the presence or absence of agonists or antagonists followed by a 5-min wash in extracellular solution. For each mutant, we tested high concentrations of MTS reagents (1 mM) to determine the maximal effect of modification. Limiting doses were then used to evaluate differences in the modification between liganded and unliganded receptors. For coapplication experiments, we used a maximal dose of agonist or antagonist (10 x EC₅₀). Currents evoked by 75% of the maximal ACh doses (3 x EC₅₀) were compared before and after MTS treatment to assess the effects of modification. For coapplications, data were normalized to the percentage inhibition (if any) of 60-s applications of the agonist or antagonist alone. At least two oocyte batches from different frogs were tested for each experimental treatment.

Rates of Modification by MTSET. Before assessing rates of reaction, a maximal dose of ACh was applied every 3 min until consistent responses were achieved. MTSET with or without ACh (maximal dose) was repeatedly applied by continuous flow. Between treatments, oocytes were washed with ES-Ca²⁺ for 5 min, and responses to ACh were measured. Data were fit to the equation I_t = I_infinity + (I_o – I_infinity)e^(–kt), where I_t is the current at time t, I_infinity is the current after complete modification (1 mM MTSET), I_o is the initial current before MTSET modification, k is the pseudo first-order rate constant, and t is cumulative time of exposure to MTSET. The pseudo first-order rate constants were converted into second-order rate constants (M^–1s^–1) by dividing by the concentration of MTSET.

Homology Model of the Extracellular Domain of 7 Receptors Based on AChBP. A model of the chick 7 nicotinic receptor was constructed from the coordinates of the ACh binding protein (Brejc et al., 2001), as described previously (Eddins et al., 2002b).

	Results

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Most Receptors with Cys Substitutions at the N Terminus of the 9 Strand Are Functional. Table 1 shows the ACh EC₅₀ values, maximal current amplitudes, and Hill coefficients for the receptors bearing the Cys substitutions used in this study. All receptors were functional except for W¹⁷³C/L²⁴⁷T 7 receptors. Tryptophan-173 is conserved among nicotinic receptor subunits (Le Novere and Changeux, 1995) and thus may be essential for proper folding, gating, and/or ligand binding. Cys substitutions at other positions were tolerated, although the EC₅₀ values of the mutant receptors were higher than that of the parent L²⁴⁷T 7 receptors. In particular, receptors bearing the E¹⁷²C mutation had an EC₅₀ that was 100-fold greater than the parent receptors. Previous studies (Eddins et al., 2002b) have shown this site to be important for the modulation of activation, so the observed difference in EC₅₀ is not surprising. Cys mutants with similar differences in EC₅₀ from wild-type receptors have also been used to characterize functional motifs of muscle nAChRs, GABA_A receptors, and 5-hydroxytryptamine-3 receptors (Akabas et al., 1992; McLaughlin et al., 1995; Bera et al., 2002; Panicker et al., 2002).

View this table: [in this window] [in a new window]   TABLE 1 Properties of L247T 7 nAChR cysteine mutants Maximum currents were evoked by ACh at 10 x EC50 doses in ES-Ca2+ at 3 days of expression, except for L247T (2 days of expression). Data are presented as mean ± S.E.M.

ACh Reduces the Extent of Modification of E¹⁷²C by MTSET. Figure 1A shows traces of ACh-evoked currents from E¹⁷²C/L²⁴⁷T 7 nAChRs before and after MTSET treatment. MTSET inhibited 94% of the ACh-evoked current. After coapplication of 10 µM MTSET with a maximal dose of ACh, however, the ACh-evoked currents were inhibited by only 45% (Fig. 1B). A subsequent application of 10 µM MTSET inhibited 94% of the remaining current, indicating that some cysteines were unmodified during the coapplication of ACh and MTSET. MTSET had no effect on ACh-evoked currents of E¹⁷²Q/L²⁴⁷T receptors (Fig. 1C) or L²⁴⁷T 7 receptors (Eddins et al., 2002b), indicating specific reaction with Cys-172. These results suggest that agonist-dependent reduction in the effects of MTSET were caused by changes in the extent of modification of E¹⁷²C.

View larger version (17K): [in this window] [in a new window]   Fig. 1. ACh coapplication reduces the inhibition of E172C/L247T 7 nAChRs by MTSET. Currents were evoked by 10- to 20-s applications of 100 µM ACh () before and after MTSET treatment. A, E172C/L247T 7 nAChRs were inhibited 94 ± 1% (n = 8) after application of 10 µM MTSET (). B, E172C/L247T 7 nAChRs were inhibited 45 ± 4% (n = 4) after coapplication of 10 µM MTSET () and 300 µM ACh (). Subsequent application of 10 µM MTSET on the same cells resulted in 94 ± 1% (n = 4) inhibition of subsequent ACh-evoked currents. C, control E172Q/L247T 7 nAChRs were unaffected by the application of 1 mM MTSET (n = 5).

Several mechanisms could explain the reduced inhibition by MTSET during ACh coapplication. ACh could have directly shielded E¹⁷²C from reaction with MTSET by steric occlusion. However, ACh at the ligand binding site is unlikely to directly occlude MTSET access to E¹⁷²C, because this residue is located at a vestibule-lining position, and the ligand binding site is on the outer surface, 25 Å away. On the other hand, ACh could directly protect E¹⁷²C from MTSET during open-channel block (Sine and Steinbach, 1984). This block, however, occurs only at high concentrations of ACh (>300 µM). In addition, the voltage-dependence of block suggests that ACh must enter into the transmembrane electric field, which is unlikely to extend to the extracellular vestibule. Thus, this type of direct steric occlusion of MTSET is also unlikely. An alternative to steric occlusion is the possibility that receptor activation causes a conformational change at or around E¹⁷²C, moving it to a position that is less reactive or less accessible to MTSET. Similarly, the movement of residues surrounding E¹⁷²C (rather than of E¹⁷²C itself) could have hindered MTSET access or reaction to the substituted Cys. The presence of ACh could also have caused a change in charge distribution near the reaction site, slowing the diffusion of MTSET to the Cys residue, and thus slowing the reaction rate (Karlin and Akabas, 1998). These possibilities are explored below.

Dose-Responses of ACh-Dependent Protection from MTSET Modification and ACh-Dependent Activation Are Similar. To test the hypothesis that the reduced modification of E¹⁷²C correlates with receptor activation, MTSET was coapplied with varying doses of ACh. Figure 2 displays the fraction of ACh-evoked current remaining after MTSET modification as a function of the coapplied concentrations of ACh (shaded bars, left axis). In comparison, the dose-response curve for ACh-evoked currents had a similar dose-dependence profile (Fig. 2, circles and line fit, right axis). Thus, there was a good correlation between the amount of receptor activation by ACh and the amount of ACh-dependent protection from MTSET modification. These data support the interpretation that ACh-dependent protection from MTSET modification is caused by receptor activation and not by direct steric occlusion.

View larger version (31K): [in this window] [in a new window]   Fig. 2. The dose-dependence of ACh inhibition of MTSET modification correlates with the dose-dependence of ACh activation. , fraction of ACh-evoked current remaining after the coapplication of 10 µM MTSET plus ACh at the concentrations indicated (n = 4). Data were normalized to the fractional current remaining after a 60-s application of that concentration of ACh alone. The EC50 of ACh inhibition of MTSET modification was 27 ± 6 µM. , current remaining after treatment with 10 µM MTSET alone after MTSET plus ACh coapplication (n = 4). , normalized ACh dose-response of E172C/L247T 7 receptors. The solid line is a fit of the dose-response data to the Hill equation, with an EC50 of 38 ± 3 µM and a Hill coefficient of 1.8 ± 0.2 (n = 8).

Epibatidine also Inhibits the Modification of E¹⁷²C by MTSET. To further test the hypothesis that activation induces a conformational change that inhibits the modification of E¹⁷²C by MTSET, we tested epibatidine, a nicotinic agonist that is structurally dissimilar to ACh (Gerzanich et al., 1995). Epibatidine was a full agonist of E¹⁷²C/L²⁴⁷T 7 receptors with an EC₅₀ of 0.31 µM (data not shown). Coapplication of 10 µM MTSET with 3 µM epibatidine inhibited 61% of the subsequent ACh-evoked current (Fig. 3). Subsequent treatment with 10 µM MTSET alone inhibited most of the remaining current (Fig. 3). Thus, epibatidine, like ACh, reduced the extent of MTSET modification. These results with differently structured agonists are also consistent with the hypothesis that activation decreases the accessibility or reactivity of E¹⁷²C caused by a conformational change.

View larger version (13K): [in this window] [in a new window]   Fig. 3. The agonist epibatidine also reduces the inhibition by MTSET at E172C. Coapplication of 3 µM epibatidine () and 10 µM MTSET () inhibited 61 ± 3% of subsequent 100 µM ACh-evoked currents (n = 4). Application of 10 µM MTSET alone () inhibited 92 ± 1% (n = 3) of the remaining ACh-evoked currents. Epibatidine activated these receptors with an EC50 of 0.31 ± 0.03 µM (n = 3).

The Antagonist DHE Does Not Inhibit the Modification of E¹⁷²C. If the modification of E¹⁷²C by MTSET is decreased by an agonist-induced conformational change rather than by simple occupancy of the binding site, then a similar decrease should not be observed in the presence of a competitive antagonist. We confirmed that DHE was an antagonist of E¹⁷²C/L²⁴⁷T 7 receptors with an IC₅₀ of 15 µM (data not shown). As has been shown for other E¹⁷² mutations (Eddins et al., 2002b), DHE showed no agonist activity on E¹⁷²C/L²⁴⁷T 7 receptors and had no effect on subsequent ACh-evoked currents (Fig. 4). When MTSET was coapplied with 100 µM DHE, subsequent ACh-evoked currents were almost completely inhibited (99%) (Fig. 4). This inhibition was similar to the amount of inhibition obtained by MTSET alone (Fig. 1). Thus, the agonist-dependent inhibition of E¹⁷²C modification by MTSET required activation of the receptor, not just occupancy of the ligand binding site.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Coapplication of antagonist DHE did not decrease MTSET inhibition at E172C. Application of 100 µM DHE alone () had no effect on subsequent ACh-evoked currents (n = 4). Coapplication of 10 µM MTSET () plus 100 µM DHE resulted in 99 ± 1% inhibition (n = 4), similar to that observed with MTSET alone (see Fig. 1A). DHE was an antagonist of E172C/L247T 7 nAChRs with an IC50 of 15 ± 6 µM (in the presence of 100 µM ACh; n = 3, data not shown).

Rates of MTSET Modification Are Decreased in the Presence of ACh and Increased by DHE. ACh-dependent inhibition of E¹⁷²C modification should be reflected as an ACh-dependent decrease in the rate of reaction (Karlin and Akabas, 1998). Figure 5 shows that this difference in reaction rates was observed. The rate of MTSET modification of E¹⁷²C decreased from 4,000 M^–1s^–1 in the absence of ACh to 1,400 M^–1s^–1 in the presence of 100 µM ACh. In contrast, the rate of modification was increased to 6,600 M^–1s^–1 in the presence of 100 µM DHE, suggesting that the antagonist caused conformational changes that were different from those driven by agonists. The rates of E¹⁷²C modification are in the same order of magnitude as the rates of modification of accessible residues in the M2 domain (Wilson and Karlin, 2001). The difference in rates with and without ACh was smaller than agonist-dependent differences observed in the M2 domain (Karlin and Akabas, 1995), but it was similar to those observed during allosteric interactions between GABA, benzodiazepine, and pentobarbital binding sites of GABA_A receptors (Teissere and Czajkowski, 2001; Holden and Czajkowski, 2002). The modest difference in rates we observed is expected if the N terminus of 9 is important for conformational changes caused by gating but is not the gate itself.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Rates of E172C modification by MTSET are slower in the presence of ACh and faster in the presence of DHE. Data are plotted as the normalized ACh-evoked current after treatment with 10 µM MTSET ± 100 µM ACh versus the cumulative time of MTSET exposure. In the absence of ACh, the rate constant was 4,000 ± 200 M–1s–1 (, n = 9 from three batches). In the presence of 100 µM ACh, the rate of reaction decreased to 1,400 ± 200 M–1s–1 (, n = 6 from three batches). In the presence of 100 µM DHE, the rate of reaction increased to 6,600 ± 200 M–1s–1 (, n = 6 from two batches). Where not visible, error bars are smaller than the symbols. Rates of reaction were significantly different (p < 0.001).

Modification by MTSES also Inhibits E¹⁷²C/L²⁴⁷TReceptors and Is Reduced in the Presence of ACh. To determine whether ACh reduced the reactivity of E¹⁷²C to MTSET by an electrostatic mechanism, we tested the negatively charged MTSES (Karlin and Akabas, 1998). The lowest effective dose of MTSES on E¹⁷²C/L²⁴⁷T receptors was 300 µM, 30 times higher than that of MTSET, consistent with the 10-fold slower rates for reactions with MTSES (Karlin and Akabas, 1998). Treatment with MTSES alone inhibited 73% of ACh-evoked currents (Fig. 6A). The inhibition by MTSES indicates that the steric bulk of the MTS adducts, rather than their electrostatic charge, mediates the inhibitory effects of E¹⁷²C modification. Coapplication with ACh significantly reduced the inhibition by MTSES (Fig. 6B). This suggests that ACh does not alter the modification of E¹⁷²Cby changing the electrostatic environment of the cysteine (Karlin and Akabas, 1998). In addition, these results suggest that the mechanism by which MTS modification of E¹⁷²C causes a reduction of current amplitudes is not via electrostatic repulsion of permeating ions.

View larger version (24K): [in this window] [in a new window]   Fig. 6. ACh attenuates the modification at E172C by MTSES. A, treatment with 300 µM MTSES () inhibited 73 ± 1% (n = 5) of ACh-evoked currents. B, coapplication of 300 µM MTSES and 300 µM ACh () inhibited 11 ± 3% (n = 6) of ACh-evoked currents. Subsequent treatment with MTSES alone (300 µM) inhibited 74 ± 5% (n = 5) of remaining currents (data not shown).

Neighboring Residues. The data so far are consistent with the idea that agonists decrease the accessibility of E¹⁷²C by causing an activation-dependent conformational change of loop 9 at the N terminus of the 9 strand. If this is the case, activation may also change the accessibility of residues near E¹⁷². Thus, we tested the ligand-dependent changes in reactivity of cysteine substitutions of N¹⁷⁰, G¹⁷¹, and D¹⁷⁴ in the L²⁴⁷T 7 receptor background.

ACh Enhances MTSET Inhibition of N¹⁷⁰C/L²⁴⁷T 7 Receptors, but DHE Does Not. N¹⁷⁰C/L²⁴⁷T 7 receptors were modestly inhibited (14%) by 1 µM MTSET in the absence of agonist (Fig. 7A), indicating that N¹⁷⁰C was at least partially accessible. In the presence of 30 µM ACh, the inhibition by MTSET increased to 30% (Fig. 7B). Thus, the effect of MTSET on N¹⁷⁰C/L²⁴⁷T 7 receptors was greater in the presence than in the absence of ACh. The data suggest that N¹⁷⁰C/L²⁴⁷T 7 receptors, unlike E¹⁷²C/L²⁴⁷T receptors, were modified to a greater extent in the activated state because of an increase in the accessibility of N¹⁷⁰C.

View larger version (20K): [in this window] [in a new window]   Fig. 7. ACh increases the modification of N170C by MTSET. A, 1 µM MTSET () inhibited 14 ± 3% (n = 13) of ACh-evoked currents in ES-Ca2+. The dotted line indicates the rundown adjustment. , 10 µM ACh. B, 1 µM MTSET plus 30 µM ACh () inhibited 30 ± 4% (n = 9) of ACh-evoked currents. Similar effects of ACh were observed with MTSET in ES-EGTA and with MTSES (data not shown). MTSET modification rate constants (in ES-Ca2+) were approximately 8,000 ± 2,000 M–1s–1 (no ACh; n = 4) and 16,000 ± 3,000 M–1s–1 (30 µM ACh, n = 5). C, application of 100 µMDHE() in ES-EGTA had no effect on subsequent ACh-evoked currents (n = 4). Coapplication of 1 µM MTSET plus 100 µM DHE had a minimal effect on ACh-evoked currents (n = 7). DHE was an antagonist of N170C/L247T 7 nAChRs with an IC50 of 2.6 ± 0.2 µM (in ES-EGTA with 10 µM ACh; n = 3, data not shown).

To determine whether the enhanced modification by MTSET of N¹⁷⁰C/L²⁴⁷T receptors depended on receptor activation, MTSET was coapplied with the antagonist DHE. In ES-Ca²⁺, DHE was a partial agonist of N¹⁷⁰C/L²⁴⁷T 7 receptors because the receptors retain the native E¹⁷² that is necessary for the Ca²⁺-dependent activation of L²⁴⁷T receptors by antagonists (Eddins et al., 2002b). Thus, these experiments were carried out in ES-EGTA, in which DHE was an antagonist with an IC₅₀ of 2.6 µM (data not shown). In the absence of extracellular Ca²⁺, DHE evoked no responses from these receptors and had no effect on subsequent ACh-evoked currents (Fig. 7C). Modification by MTSET in the presence of DHE caused an insignificant amount of inhibition (Fig. 7C). We infer that receptor activation, not simply occupancy of the ligand binding site, moves N¹⁷⁰C into a more accessible position, whereas E¹⁷²C moves to a less accessible position. These opposing effects are consistent with a shift in the position of loop 9 (see Discussion).

Reactivity of G¹⁷¹C to MTSET and MTSES Is the Same in the Presence and Absence of ACh. Like E¹⁷²C/L²⁴⁷T and N¹⁷⁰C/L²⁴⁷T 7 receptors, modification of G¹⁷¹C/L²⁴⁷T receptors by MTSET caused a partial inhibition (56 ± 3%, n = 4, data not shown). Unlike the other receptors, modification of G¹⁷¹C/L²⁴⁷T 7 receptors was the same in the presence of ACh (50 ± 4%, n = 3, data not shown). However, MTSET was a partial agonist of G¹⁷¹C/L²⁴⁷T receptors, which could have confounded the results. Thus, MTSES, which was not an agonist of G¹⁷¹C/L²⁴⁷T 7 receptors, was used to assay the effects of agonist on modification. MTSES produced results similar to MTSET; the inhibition was the same in the presence and absence of ACh (Fig. 8). These data suggest that G¹⁷¹C is equally accessible to MTSES, regardless of receptor activation.

View larger version (20K): [in this window] [in a new window]   Fig. 8. MTSES modification of G171C is unchanged by ACh. A, 1 mM MTSES inhibited 22 ± 5% (n = 4) of ACh-evoked currents. B, MTSES plus 30 µM ACh inhibited 24 ± 5% (n = 4) of ACh-evoked currents. , 10 µM ACh. The limiting dose for this mutant was 1 mM MTSES.

D¹⁷⁴C/L²⁴⁷T 7 Receptors Are Not Inhibited by MTSET or MTSES. Treatment of D¹⁷⁴C/L²⁴⁷T 7 nAChRs with 1 mM MTSET produced no effect on ACh-evoked currents when applied either in the presence (n = 4) or absence (n = 4) of 30 µM ACh (data not shown). Similarly, MTSES (1 mM) also had no effect on D¹⁷⁴C/L²⁴⁷T receptors in the presence or absence of ACh (n = 3 each) (Fig. 9). Thus, D¹⁷⁴C was either not accessible to MTSET or MTSES, or modification by these reagents had no functional effect.

View larger version (24K): [in this window] [in a new window]   Fig. 9. Effects of ACh and DHE on MTS modification. A, data are displayed as the percentage inhibition of ACh-evoked currents after MTS and ACh cotreatment minus that of MTS alone. MTSET data are shown for N170C and E172C; MTSES data are shown for G171C and D174C because MTSET was a partial agonist of these receptors. (t test: ***, p < 0.001; **, p < 0.005). B, MTS ± DHE. For all mutants except E172C, data in B were recorded in ES-EGTA because DHE was a partial agonist in ES-Ca2+.

	Discussion

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As summarized in Fig. 9A, ACh increased the modification of N¹⁷⁰C and decreased the modification of E¹⁷²C in L²⁴⁷T 7 nicotinic receptors. These effects were not observed in the presence of the antagonist DHE (Fig. 9B), indicating that the effects of agonists required activation, not simply ligand binding. The ACh dose-dependence of inhibition of E¹⁷²C modification was virtually identical with the dose-dependence of receptor activation by ACh. These data support the idea that receptor activation involves conformational changes near the N terminus of the 9 strand.

Model for Agonist-Driven Movements of Loop 9. Figure 10, A and B, shows a model of the 7 receptor from the AChBP crystal structure that we have used as a framework to interpret our results. The model shows that both E¹⁷² and N¹⁷⁰ are in the intersubunit interface, but E¹⁷² is closer to and projects toward the vestibule, whereas N¹⁷⁰ is buried more deeply (Fig. 10A). The model and previous results (Eddins et al., 2002b) suggest that E¹⁷²C is accessible from the vestibule.

View larger version (26K): [in this window] [in a new window]   Fig. 10. Models of activation-induced conformational changes in the extracellular domain. A, homology model of the 7 nAChR extracellular domain taken from the crystal structure of the AChBP (Brejc et al., 2001). This view is from the top of the receptor. The positions of N170 and E172 are shown in blue and red, respectively. Both residues are located in the intersubunit interface, but E172 is closer to the vestibule. B, two subunits of the 7 homology model viewed from the lumen of the vestibule. N170 and E172 in loop 9 are shown in blue and red, respectively. A few of the residues in loop C that form the ligand binding site (Y187, C189, C190, and Y194) are shown in gold. The 9–10 hairpin is shown in black, and the C-terminal end of 1 is brown. Arrows indicate the hypothetical intrasubunit movement of the 9–10 hairpin and loop 9 during activation. C, diagram of symmetric rotation. Positions of N170 and E172 are indicated in blue and red, respectively. This model assumes that N170 is accessible from the outer surface, and E172 is accessible from the vestibule. In this model, one (or more) subunits rotate counterclockwise. N170 residues become more accessible, as indicated in dark blue. Upon activation, E172 becomes less accessible (pink) by moving deeper in the intersubunit interface. D, diagram of asymmetric rotation. In this model, accessibility of N170 and E172 is from the vestibule. N170 residues that would show an increase in accessibility in the open state are shown in dark blue. E172 positions that would show a decrease in accessibility are pink. The extent of rotation is exaggerated to illustrate the differences in accessibility from the vestibule. In this model, none of the residues is accessible from the outside surface.

The question of how agonists can decrease the modification of E¹⁷²C and increase the modification of N¹⁷⁰C is also worth examining. Figure 10C shows a diagram in which activation causes the counterclockwise rotation of a single subunit. This model assumes that N¹⁷⁰C becomes more accessible to MTSET from the outer surface, and E¹⁷²C becomes less accessible to MTSET from the vestibule. This model could also apply to the rotation of several subunits. This model, however, is inconsistent with the 15° clockwise rotation of subunits proposed for Torpedo californica nAChRs (Unwin et al., 2002). To explain our results, clockwise rotations would require a 60° rotational movement, which seems unlikely.

A second model in which pairs of adjacent subunits rotate asymmetrically during activation (Horenstein et al., 2001) is shown in Fig. 10D. This model assumes that MTS modifiers access both E¹⁷²C and N¹⁷⁰C from the vestibule. Clockwise rotation of one subunit of the pair would cause an increase in accessibility of N¹⁷⁰C by moving it from a poorly accessible position in the intersubunit interface toward the vestibule. This clockwise rotation would not change the accessibility of E¹⁷²C because it remains in a vestibule-lining position. The observed decrease in the accessibility of E¹⁷²C is explained by the counterclockwise rotation of the other subunit of the pair, moving E¹⁷²C away from the vestibule into the intersubunit interface. This counterclockwise rotation of this subunit would also shift N¹⁷⁰C deeper into the interface, but because this residue was poorly accessible in the closed state, this decreased accessibility would be undetectable.

Our observations are also consistent with a model in which the activation of the receptor involves an intrasubunit movement (Fig. 10B). Ligand binding could transmit the activation signal via a shift in position of the 9–10 hairpin structure, as suggested by Brejc et al. (2001). As shown in Fig. 10B, movement of the 9–10 hairpin and loop 9 could increase the accessibility of N¹⁷⁰C because of its movement from the intersubunit interface toward the vestibule. Simultaneously, the accessibility of E¹⁷²C could be decreased by movement to a more protected position closer to the 1 strand. Alternatively, it is also possible that the movements of neighboring regions alter the accessibility of positions 170 and 172. Combinations of intrasubunit movement with either symmetric or asymmetric rotational movement of subunits could constitute part of the gating machinery. As described previously, conformational changes in the extracellular ligand binding domain could transmit activation signals to the "gate" in M2 via the 10–M1 linker and/or the M2–M3 linker (Grosman et al., 2000; Horenstein et al., 2001; Kash et al., 2003). Our results indicate that loop 9, in addition to loop 2 and the signature loop (loop 7) (Kash et al., 2003), undergoes agonist-driven conformational changes and may be an important part of the mechanism coupling ligand binding with the opening of the channel.

	Acknowledgements

 
We thank Dr. Brenda Temple at the University of North Carolina at Chapel Hill R. L. Juliano Structural Bioinformatics Core Facility for assistance with molecular modeling.

	Footnotes

 
This work was supported by National Institutes of Health grant NS37317.

ABBREVIATIONS: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; DHE, dihydro--erythroidine; MTSET, 2-trimethylammonioethylmethane thiosulfonate; MTSES, 2-sulfonatoethylmethane thiosulfonate; MTS, methanethiosulfonate; AChBP, acetylcholine binding protein; ES-Ca²⁺, extracellular solution containing Ca²⁺; ES-EGTA, extracellular solution containing EGTA; BAPTA, 1,2,-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

Address correspondence to: Dr. Robert L. Rosenberg, Department of Pharmacology, CB #7365, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: bobr{at}med.unc.edu

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