Introduction

Top Summary Introduction Experimental Procedures Results Discussion References

The muscle nicotinic acetylcholine receptor (nAChR) consists of four homologous subunits (₂) arranged pseudosymmetrically around a central axis that is a cation-selective ion channel. Each subunit has a common primary structure motif: a hydrophilic, extracellular N-terminal half containing amino acids of the agonist-binding sites, followed by three hydrophobic membrane spanning segments (M1-M3), a cytoplasmic domain, a fourth transmembrane segment (M4), and short extracellular C-terminal tail. Affinity labeling studies, site-directed mutagenesis, and low-resolution (9 Å) cryoelectron microscopy provide considerable information about nAChR structure (reviewed in Karlin and Akabas, 1995; Hucho et al., 1996; Unwin, 1998). The two agonist-binding sites, which are located extracellularly at the - and - subunit interfaces, are composed of multiple loops of primary structure from  and  (or ) subunits (reviewed in Prince and Sine, 1998). M2 domains from each subunit line the pore of the ion channel (Imoto et al., 1988; Unwin, 1995), with additional contributions from the extracellular ends of the M1 segments (Zhang and Karlin, 1997), whereas M3 and M4 segments are more peripheral and in contact with lipid (Blanton and Cohen, 1994).

Noncompetitive antagonists (NCAs) block the nAChR permeability response without preventing the binding of ACh (acetylcholine). A structurally diverse group of drugs act as NCAs, including many aromatic amines, general anesthetics, fatty acids, steroids, and neuropeptides such as Substance P (reviewed in Arias, 1998). Studies of the binding of the aromatic amines [³H]meproadifen and [³H]phencyclidine (PCP) or of the spiropiperidine [³H]histrionicotoxin ([³H]HTX) to nAChR-rich membranes from Torpedo electric organ establish that each binds with high affinity (K ~ µM) to one site per nAChR and to additional lower-affinity sites (Heidmann et al., 1983). The high-affinity site is linked allosterically to the ACh site, with most aromatic amines binding at equilibrium with highest affinity in the presence of agonist [i.e., to the desensitized state of the nAChR (Cohen et al., 1985; Arias, 1996; Lurtz and Pedersen, 1999)].

Affinity-labeling studies have identified homologous residues near the cytoplasmic end of each M2 segment that contribute to the high-affinity binding site in desensitized Torpedo nAChRs for the aromatic amine NCAs [³H]chlorpromazine (Revah et al., 1990) and [³H]trimethylphenylphosphonium (Hucho et al., 1986). Mutational analyses also implicate these amino acids as affinity determinants for the aromatic amine QX-222 (Charnet et al., 1990) and for aliphatic alcohols (Forman, 1997) acting as open channel blockers. However, in each conformational state, there may be distinct, nonoverlapping sites for positively charged NCAs, and different regions within the ion channel may contribute to the binding site for a single ligand in different conformational states. In the desensitized nAChR, [³H]meproadifen mustard reacts with Glu²⁶² at the extracellular end of M2 (Pedersen et al., 1992), and based on fluorescence energy transfer, the binding site for ethidium is located above the level of the bilayer in the vestibule of the channel (Johnson and Nuss, 1994, but see Lurtz et al., 1997). In the open channel state, [³H]quinacrine azide is photoincorporated into amino acids within M1 (DiPaola et al., 1990), and mutations within M1 affect quinacrine potency as an NCA but not chlorpromazine (Tamamizu et al., 1995). Photoaffinity labeling studies with 3-(trifluoromethyl)-3-(M-[¹²⁵I]iodophenyl) diazirine ([¹²⁵I]TID; White and Cohen, 1992) and [³H]diazofluorene (Blanton et al., 1998), uncharged, hydrophobic NCAs, have identified a binding site in the M2 domain in the absence of agonist (closed channel), as well as changes in structure of the M2 domain between resting and desensitized states. However, the TID site in the closed channel appears distinct from the binding site for PCP because PCP does not inhibit [¹²⁵I]TID photoincorporation and TID does not inhibit [³H]PCP binding (White et al., 1991).

Tetracaine (dimethylaminoethyl-p-butylaminobenzoate) is an unusual aromatic amine NCA because it is 100-fold more potent as an inhibitor of [³H]HTX binding (K  1 µM) in the absence of agonist than in the presence (Blanchard et al., 1979), and it stabilizes the resting state rather than the desensitized state of the nAChR (Boyd and Cohen, 1984). Here, we characterize the binding properties of several structural analogs of tetracaine to further define the requirements for preferential binding to the closed channel state, and we use [³H]tetracaine itself as an intrinsic photoaffinity reagent to define the structure of its high-affinity binding site in the nAChR in the absence of agonist. [³H]Tetracaine is specifically photoincorporated with similar efficiency into each nAChR subunit. In the following report (Gallagher and Cohen, 1999), we identify the homologous amino acids in the M2 segment of each subunit that contribute to this high-affinity [³H]tetracaine site.

	Experimental Procedures

Top Summary Introduction Experimental Procedures Results Discussion References

Materials. [ring-3,5-³H]Tetracaine ([³H]tetracaine, 36 Ci/mmol) and [³H]HTX (60 Ci/mmol) were prepared at New England Nuclear Research Products (Boston, MA) by tritium gas catalytic reduction of 3,5-dibromotetracaine and dl-decahydro(pentenyl)histrionicotoxin (H₁₀-HTX), respectively. For binding experiments, [³H]tetracaine was purified to >95% by silica thin-layer chromatography (5:4:1 cyclohexane/chloroform/diethylamine; R_f = 0.17). When stored in ethanol, [³H]tetracaine decomposed at ~10% per month, forming tritiated degradation products that did not partition into nAChR-rich membranes or bind to glass filters. Also, these degradation products did not appear to photoincorporate into nAChR-rich membranes because the same [³H]tetracaine photolabeling patterns were seen with [³H]tetracaine of 95 or 50% radiochemical purity (not shown).

Radioligand Binding Assays. The equilibrium binding of [³H]tetracaine to Torpedo nAChR-rich membranes in Torpedo physiological saline (TPS; 250 mM NaCl, 5 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 5 mM sodium phosphate, pH 7.0) was assayed by centrifugation. Membrane suspensions (400 nM ACh sites) were equilibrated at 4°C for 4 h with varying concentrations of [³H]tetracaine (0.5 Ci/mmol). Membranes were also preequilibrated with agonist or competitive antagonist, or with excess nonradioactive tetracaine (50-100 µM) to define nonspecific binding. Aliquots (0.1 ml) were then centrifuged in a Beckman Airfuge at 100,000g for 10 min at ~8°C. After removal of the supernatants, membrane pellets were resuspended in 0.1 ml of 10% SDS, and pellet and supernatant ³H were determined by liquid scintillation counting. This centrifugation assay was also used to quantify the concentration of high-affinity [³H]tetracaine sites compared with the concentration of [³H]ACh and/or [³H]HTX sites measured simultaneously with the same membrane suspension. For studies with [³H]ACh, membrane suspensions were pretreated with 0.3 mM diisopropylphosphofluoridate to inhibit cholinesterase activity. [³H]HTX was diluted isotopically with H₁₂-HTX to produce a final radiochemical specific activity of 2 Ci/mmol for binding assays.

Photoaffinity Labeling of nAChR-Rich Membranes with [³H]Tetracaine. Membrane suspensions (30 µl, 1.5-1.8 mg protein/ml) in TPS were equilibrated at room temperature with [³H]tetracaine (4 Ci/mmol) and cholinergic ligands and then irradiated for 30 min with a 302-nm lamp (Spectroline EB-280C, 1150 µW/cm²) at a distance of 12 cm as described (Pedersen and Cohen, 1990). Unless indicated otherwise, suspensions also included 50 mM GSSG as an aqueous photochemical scavenger. The 96-well microtiter plate containing the samples was placed in a water bath during photolysis, so the temperature increased <2°C during 30 min of irradiation. After photolysis, samples were usually prepared for SDS-polyacrylamide gel electrophoresis (PAGE) by the direct addition of 10 µl of 4× sample loading buffer to reaction mixtures. To quantify the dependence of ³H incorporation as a function of free [³H]tetracaine, after photolysis the reaction mixtures were centrifuged, with the supernatants assayed to determine free [³H]tetracaine, and the pellets dissolved in sample loading buffer. In preliminary experiments, the efficiency of [³H]tetracaine photoincorporation into nAChR-rich membranes was found to be 10 times higher for the 302-nm lamp than for lamps of 254 or 365 nm of similar radiant flux density (not shown).

Gel Electrophoresis. Polypeptides were resolved by SDS-PAGE on 8% acrylamide gels with a modified Laemmli buffer system (White and Cohen, 1988), and the incorporation of [³H]tetracaine was determined by fluorography or quantified by scintillation counting of gel slices as described previously (Middleton and Cohen, 1991). Proteolytic mapping of the [³H]tetracaine-labeled  subunit with S. aureus V8 protease was performed according to the procedure of Cleveland et al. (1977) as described by White and Cohen (1988). Membrane suspensions (540-µg aliquots) were photolabeled with [³H]tetracaine and then electrophoresed on an 8% acrylamide gel. After a brief staining with Coomassie brilliant blue and destaining, the band containing  subunit was excised and transferred to the well of a mapping gel (15% acrylamide) to which V8 protease (3 µg) was added. ³H incorporation into the proteolytic fragments of [³H]tetracaine-labeled  subunit was analyzed by fluorography and by scintillation counting of gel slices.

Data Analysis The equilibrium binding of [³H]tetracaine and the concentration dependence of ³H incorporation into nAChR subunits were fit by nonlinear least-squares (SigmaPlot; Jandel Scientific, San Rafael, CA) to the function {B = A/[1 + (K_eq/L)] + m * L}, where B is the bound [³H]tetracaine (or cpm incorporated), A is the maximum specific binding (or cpm incorporated), K_eq is the dissociation constant (or apparent dissociation constant, K_AP), L is the measured free [³H]tetracaine concentration, and m is the slope of nonspecific binding (or labeling), which was usually determined in parallel experiments performed in the presence of excess nonradioactive tetracaine or H₁₀-HTX. For labeling experiments, the measured free ³H cpm was adjusted to account for the 50% radioimpurities known to be present in the [³H]tetracaine used for photolabeling.

	Results

Top Summary Introduction Experimental Procedures Results Discussion References

Inhibition of [³H]HTX Binding by Tetracaine Analogs. Because tetracaine's preferential binding to the closed channel state of the nAChR appeared unusual for aromatic amine NCAs, we first examined other benzoic acid esters as inhibitors of [³H]HTX binding to identify structural features responsible for tetracaine's binding properties (Table 1). All drugs at high concentrations completely inhibited specific binding of [³H]HTX, with the concentration dependence of inhibition for each drug consistent with competition at a single site. With the exception of procaine (V), all bound with highest affinity in the absence of agonist. For the esters of p-butylaminobenzoic acid, replacement of the dimethylaminoethyl of tetracaine (I) by DEAE (II) resulted in a ~6-fold increase in binding affinity in both the absence and presence of agonist. Comparison of procaine (V) with compound II established that aliphatic substitution at the aryl nitrogen increased affinity for the closed channel conformation by 4000-fold but by only 300-fold for the desensitized conformation. However, high-affinity binding in the absence of agonist did not depend uniquely on the butylamino group because the p-ethoxybenzoic acid ester (IV) also bound preferentially to the resting state (K_I = 1.6 µM), and piperocaine (VI), an unsubstituted benzoic acid ester, was bound with 50-fold higher affinity in the absence of agonist. Thus, selective, high-affinity binding in the absence of agonist appeared to be the rule for many simple benzoic acid esters, with procaine a notable exception.

View this table: [in this window] [in a new window]   TABLE 1 Equilibrium binding of tetracaine analogs to Torpedo nAChR NCA site Values of KI were determined as described in the text by nonlinear regression fit (nH = 1) of the observed concentration dependence of the inhibition of equilibrium binding at 20°C of [3H]HTX (20 nM) by Torpedo nAChR-rich membranes (200 nM ACh sites) in the absence () or presence of 0.1 mM carbamylcholine (+ Carb) or d-tubocurarine (+dTC). Parameter uncertainties were 5 to 15% of KI.

Equilibrium Binding of [³H]Tetracaine. The equilibrium binding of [³H]tetracaine to nAChR-rich membranes in TPS at 4°C was determined by centrifugation and filtration assays (Fig. 1A). For [³H]tetracaine concentrations to 4 µM, in each assay the total binding was well fit by a hyperbolic binding function with a linear, nonspecific component. The nonspecific binding calculated from the fit of the total binding function was approximately the same as that measured in the presence of 50 µM nonradioactive tetracaine. [³H]Tetracaine bound to the same number of high-affinity sites in both assays, with the ratio of sites determined by filtration and centrifugation equal to 1.2 ± 0.2 in three experiments. The same value of the dissociation constant was determined by centrifugation (K_eq = 0.5 ± 0.1 µM) and filtration (K_eq = 0.6 ± 0.1 µM). The nonspecific binding determined by filtration, with a brief (5 s) wash, was only 20% that determined by centrifugation. The partition coefficient, determined as the ratio of the nonspecifically bound to the free tetracaine concentration normalized to the membrane protein concentration was 0.08 ± 0.02 (mg/ml)¹ by centrifugation and 0.014 ± 0.005 by filtration. Thus, in the absence of agonist, specific [³H]tetracaine binding to the nAChR-rich membranes was characterized by a single K_eq, and a filtration assay can be used to quantify equilibrium binding of [³H]tetracaine to its high-affinity site.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Equilibrium binding of [3H]tetracaine to nAChR-rich membranes. A, nAChR-rich membranes (0.5 µM [3H]ACh sites, 0.5 mg/ml) were incubated with [3H]tetracaine up to 4 µM for 4 h at 4°C in the absence (, ) or presence (, ) of 50 µM nonradioactive tetracaine, and binding was determined by centrifugation (, ) or filtration (, ) as described in Experimental Procedures. Dashed lines are the nonlinear least-squares fit to a hyperbolic function as a single class of sites (Bmax, Keq) and a linear nonspecific component (slope, m). For total binding by centrifugation, Bmax = 0.23 ± 0.03 µM, Keq = 0.59 ± 0.07 µM, and m = 0.064 ± 0.008. For total binding by filtration, Bmax = 0.31 ± 0.02 µM, Keq = 0.59 ± 0.07, and m = 0.005 ± 0.0006. Calculated specific binding (total binding  directly measured nonspecific binding) by centrifugation was fit by Bmax = 0.25 ± 0.01 µM and Keq = 0.61± 0.05 µM, whereas for filtration, Bmax = 0.27 ± 0.004 µM and Keq = 0.51 ± 0.03 µM. B, nAChR-rich membranes were preincubated for 30 min in the absence () or presence of 100 µM carbamylcholine () or 50 µM d-tubocurarine () or for 60 min with 4 µM -bungarotoxin () before the addition of [3H]tetracaine at concentrations to 20 µM. Parallel samples also contained 100 µM nonradioactive tetracaine to determine nonspecific binding. After an additional 4 h of incubation at 4°C, bound and free [3H]tetracaine were determined by filtration, and at each concentration, specifically bound [3H]tetracaine was calculated as the difference between total and nonspecific binding. Specific binding in the absence of cholinergic ligands was characterized by Bmax = 120 ± 2 nM, Keq = 340 ± 30 nM; with -bungarotoxin, Bmax = 110 ± 2 nM, Keq = 280 ± 40 nM; with d-tubocurarine, Bmax = 120 ± 3 nM, Keq = 560 ± 60 nM; and with carbamylcholine, Bmax = 86 ± 13 nM, Keq = 12 ± 3 µM. When the data in the presence of carbamylcholine were fit with Bmax constrained to 120 nM, Keq = 21 ± 1 µM.

Inhibition of [³H]Tetracaine Binding by NCAs. The desensitizing NCAs PCP, meproadifen, proadifen, and H₁₀-HTX were examined as inhibitors of the equilibrium binding of [³H]tetracaine at 4°C in the absence of other cholinergic ligands or in the presence of either -bungarotoxin or d-tubocurarine (Fig. 2). Although HTX binds with similar affinity (K_eq = 0.3 µM) in the absence or presence of agonist at 20°C (Blanchard et al., 1979), at 4°C it binds with high affinity (K_eq = 0.3 µM) in the presence of agonist but only weakly (K_eq = 8 µM) in the absence (Cohen et al., 1985). The four NCAs inhibited [³H]tetracaine binding in a concentration-dependent manner, with high concentrations inhibiting [³H]tetracaine binding by the same extent as 100 µM tetracaine. K_I values for inhibition of [³H]tetracaine binding were 6- to 10-fold lower in the presence of d-tubocurarine than in its absence, consistent with the preferential binding of these NCAs to desensitized nAChR (Heidmann et al., 1983; Cohen et al., 1985). The concentration dependence of inhibition by H₁₀-HTX (Fig. 2A) and PCP (Fig. 2B) was well fit by Hill coefficients of 1 in both the absence and presence of d-tubocurarine or -bungarotoxin, consistent with simple competitive inhibition of [³H]tetracaine binding. For proadifen (Fig. 2C), the Hill coefficients for inhibition were unitary except in the presence of -bungarotoxin (n_H = 1.6). For meproadifen (Fig. 2D), the dose dependence of inhibition was characterized by n_H = 1 in the presence of d-tubocurarine but by n_H = 1.5 in the absence of ACh site ligand and by n_H = 1.8 in the presence of -bungarotoxin.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]tetracaine binding to nAChR-rich membranes by NCAs. nAChR-rich membranes (0.2 µM tetracaine sites) were preincubated at 4°C for 30 min without competitive antagonist (), with 50 µM d-tubocurarine (), or with -bungarotoxin (, 4 µM, 90 min) before the addition of 10 nM [3H]tetracaine. Aliquots were immediately mixed with (A) H10-HTX, (B) PCP, (C) proadifen, or (D) meproadifen, at concentrations ranging from 10 nM to 1 mM. After 4-h incubation at 4°C, binding was determined by filtration. Parallel samples contained 100 µM nonradioactive tetracaine for determination of nonspecific binding (open symbols). Inhibition curves were fit as described in Experimental Procedures, with nH = 1 unless noted. For H10-HTX (A): in the absence of cholinergic ligands, KI() = 2.2 ± 0.2 µM; with dTC present, KI(+dTC) = 0.3 ± 0.1 µM; and with -bungarotoxin, KI(+Btx) = 4.4 ± 0.5 µM. For PCP (B): KI() = 22 ± 2 µM, KI(+dTC) = 2.4 ± 0.4 µM, and KI(+Btx) = 44 ± 2 µM. For proadifen (C): KI() = 6 ± 1 µM, KI(+dTC) = 0.5 ± 0.04 µM, KI(+Btx) = 18 ± 3 µM, and nH = 1.6 ± 0.1. For meproadifen (D): KI() = 7 ± 1 µM, nH = 1.5 ± 0.2; KI(+dTC) = 0.9 ± 0.1 µM, nH = 1; and KI(+Btx) = 56 ± 4 µM, nH = 1.8 ± 0.2.

Photoincorporation of [³H]Tetracaine into nAChR-Rich Membranes. To determine whether [³H]tetracaine could be specifically photoincorporated into its high-affinity binding site, nAChR-rich membranes were equilibrated with 5 µM [³H]tetracaine and then irradiated at 302 nm for 30 min in the presence or absence of the NCA H₁₀-HTX (30 µM). When the proteins were resolved by SDS-PAGE and the gel was processed for fluorography, it was seen (Fig. 3, lanes 2 and 3) that H₁₀-HTX decreased [³H]tetracaine incorporation not only in the nAChR subunits but also into another protein of the nicotinic postsynaptic membrane [rapsyn/43 kilodaltons (kDa) protein] and into polypeptides of contaminating membrane fragments such as the  subunit of the Na⁺,K⁺-ATPase (90 kDa; White and Cohen, 1988). Photoincorporation into nonreceptor polypeptides was also decreased by the agonist carbamylcholine, which allosterically inhibits [³H]tetracaine binding (see Figs. 6 and 8). It was improbable that HTX and carbamylcholine actually inhibited [³H]tetracaine binding to specific sites on the Na⁺,K⁺-ATPase. A more likely explanation was that a reactive [³H]tetracaine intermediate generated primarily when [³H]tetracaine was bound to its high-affinity site in the nAChR could dissociate from its NCA site and then react with other membrane proteins. This pseudospecific photoincorporation should be sensitive to aqueous compounds that could quench the free reactive species.

View larger version (64K): [in this window] [in a new window]   Fig. 3.   [3H]Tetracaine photoincorporation into nAChR-rich membranes. nAChR-rich membranes (53 µg) were suspended in 30 µl of TPS (2.7 µM ACh sites)/5 µM [3H]tetracaine with (lanes 3, 5, and 7) or without (lanes 2, 4, and 6) 30 µM H10-HTX. Membrane suspensions also contained GSSG at 0 mM (lanes 2 and 3), 1 mM (lanes 4 and 5), or 50 mM (lanes 6 and 7). Samples were irradiated for 30 min at 302 nm and polypeptides were resolved by SDS-PAGE as described in Experimental Procedures. Coomassie blue stain (lane 1) and fluorograph (lanes 2-7) exposed for 4 days are shown.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of GSSG on [3H]tetracaine photoincorporation into nAChR-rich membranes. Membranes were photolabeled as described in Fig. 3 with 5 µM [3H]tetracaine in the absence () or presence () of 30 µM H10-HTX and with GSSG from 0 to 50 mM. nAChR  and  subunits and nonreceptor polypeptides of 37 kDa (calelectrin) and 90 kDa (Na+,K+-ATPase  subunit) were excised from the stained gel, and the incorporated 3H cpm was determined by liquid scintillation counting. The H10-HTX inhibitable labeling () was calculated as the difference between 3H incorporation in the absence () and presence () of H10-HTX.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of [3H]tetracaine incorporation into the nAChR subunits. nAChR-rich membranes (53 µg) were suspended in 30 µl of TPS (2.7 µM ACh sites)/50 mM GSSG with various concentrations of [3H]tetracaine, in the absence (solid) or presence (open) of 50 µM H10-HTX. Membrane suspensions were irradiated at 302 nm, and the [3H]tetracaine incorporation into the  (, ),  (, ),  (, ), and  (, ) subunits determined as described in Experimental Procedures. Free [3H]tetracaine was measured as supernatant 3H cpm after centrifugation of irradiated samples. Lines represent nonlinear least-squares fits to the data as described in Experimental Procedures, with KAP values of 1.3 ± 0.3, 1.3 ± 0.3, 1.3 ± 0.3, and 1.5 ± 0.3 µM for the , , , and  subunits, respectively. The maximum specific incorporation was 1610, 1370, 1270, and 1520 cpm for the , , , and  subunits, respectively.

Effects of Cholinergic Ligands on [³H]Tetracaine Photoincorporation into nAChR-Rich Membranes. [³H]Tetracaine photoincorporation was examined in the presence of agonists, competitive antagonists, and NCAs (Fig. 6) for comparison with the known pharmacology of [³H]tetracaine binding to the NCA site. The NCAs proadifen, PCP, and H₁₀-HTX each inhibited the photoincorporation of [³H]tetracaine into all four nAChR subunits (Fig. 6, lanes 2 versus 3-5). PCP was the least potent, consistent with its relative potency as an inhibitor of [³H]tetracaine equilibrium binding in the absence of agonist (Fig. 2). [³H]Tetracaine photoincorporation into the nAChR subunits was not inhibited by excess -bungarotoxin (Fig. 6, lanes 2 versus 9 and 16), and the three NCAs still inhibited subunit photolabeling in the presence of -bungarotoxin (lanes 6-8). Carbamylcholine (10 and 300 µM) reduced [³H]tetracaine incorporation into all four nAChR subunits by 70 to 80% (Fig. 6, lanes 2 versus 17 and 18, and counting of excised gel bands), but this inhibition was not seen when -bungarotoxin was present, preventing carbamylcholine from binding to the agonist site (lanes 14 and 15). Although d-tubocurarine at 2 µM had little effect on [³H]tetracaine photoincorporation (lane 13), d-tubocurarine at 50 µM reduced labeling by 25 to 40% (lane 12), an inhibition not seen in the presence of -bungarotoxin (lane 10). Thus, the inhibition of [³H]tetracaine photolabeling by the NCAs was consistent with a competitive inhibition of high-affinity [³H]tetracaine binding, whereas the inhibition of photolabeling by carbamylcholine and d-tubocurarine resulted from the allosteric inhibition of [³H]tetracaine binding when the nAChR was desensitized by drugs binding to the agonist site.

View larger version (101K): [in this window] [in a new window]   Fig. 6.   Effects of cholinergic ligands on [3H]tetracaine incorporation into nAChR-rich membranes. nAChR-rich membranes (53 µg) were suspended in TPS (2.7 µM)/50 mM GSSG for preincubation (60 min) in the absence (lanes 2-5, 12, 13, 17, and 18) or presence (lanes 6-11 and 14-16) of -bungarotoxin (10 µM) and the following cholinergic ligands: lane 2, no further additions; lane 3, 30 µM H10-HTX; lane 4, 50 µM PCP; and lane 5, 100 µM proadifen. Lanes 6 to 11, 10 µM -bungarotoxin with 30 µM H10-HTX (lane 6), 50 µM PCP (lane 7), 100 µM proadifen (lane 8), no other drug (lane 9), 50 µM d-tubocurarine (lane 10), and 2 µM d-tubocurarine (lane 11). Lane 12, 50 µM d-tubocurarine; lane 13, 2 µM d-tubocurarine. Lanes 14 to 16, 10 µM -bungarotoxin with 300 µM carbamylcholine (lane 14), 10 µM carbamylcholine (lane 15), and no other drug (lane 16). Lane 17, 300 µM carbamylcholine; lane 18, 10 µM carbamylcholine. [3H]Tetracaine was added, the samples were irradiated at 302 nm, and the polypeptides were resolved by SDS-PAGE as described in Experimental Procedures. Coomassie blue stain of a gel lane (lane 1) and the fluorograph (lanes 2-17) exposed for 2 weeks are shown.

1

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Inhibition of [3H]tetracaine photoincorporation into nAChR-rich membranes by H10-HTX. nAChR-rich membranes (53 µg) were suspended in 30 µl of TPS (2.7 µM ACh sites) with 2 µM [3H]tetracaine/50 mM GSSG and various concentrations of H10-HTX. After irradiation, membrane polypeptides were fractionated by SDS-PAGE. The nAChR  (),  (),  (), and  () subunits, as well as nonreceptor polypeptides of 37 kDa (, calelectrin) and 90 kDa (, Na+,K+-ATPase  subunit), were excised from the stained gel, and the incorporated 3H cpm was determined by liquid scintillation counting. Data were fit with the inhibition function described in Experimental Procedures with KI values of 2.8 ± 0.6, 3.0 ± 0.5, 4.1 ± 0.9, and 3.1 ± 0.5 µM for , , , and  subunits, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Inhibition of [3H]tetracaine photoincorporation into nAChR-rich membranes by carbamylcholine. nAChR-rich membranes (53 µg) were suspended in 30 µl of TPS (2.7 µM ACh sites) with 2 µM [3H]tetracaine/50 mM GSSG and various concentrations of carbamylcholine. After irradiation, membrane polypeptides were fractionated by SDS-PAGE. The nAChR  (),  (),  (), and  () subunits, as well as nonreceptor polypeptides of 37 kDa (, calelectrin) and 90 kDa (, Na+,K+-ATPase  subunit), were excised from the stained gel, and the incorporated 3H cpm was determined by liquid scintillation counting. Data were fit to calculate KAP value for free carbamylcholine as described (White and Cohen, 1988), with KAP = 0.9 ± 0.2, 0.6 ± 0.2, 0.4 ± 0.1, and 0.5 ± 0.1 µM for the nAChR , , , and  subunits, respectively.

Mapping the [³H]Tetracaine-Labeled Site in nAChR  Subunit with S. aureus V8 Protease. Digestion of the nAChR  subunit by S. aureus V8 protease using the procedure of Cleveland et al. (1977) produces four nonoverlapping fragments of 20 (V8-20), 18 (V8-18), 10 (V8-10), and 4 kDa (V8-4) that are readily resolved by SDS-PAGE (Pedersen et al., 1986). The N termini of V8-20, V8-18, V8-10, and V8-4 begin at Ser¹⁷³, Val⁴⁶, Asn³³⁹, and Ser¹, respectively (Pedersen et al., 1986; White and Cohen, 1988). V8-18 contains the only Asn-linked carbohydrate  subunit, and this fragment is shifted to a band of 12 kDa (V8-12) when the carbohydrate is removed by digestion with endoglycosidase H (Pedersen et al., 1986). nAChR-rich membranes were labeled with [³H]tetracaine in the absence or presence of 50 mM GSSG and with or without 30 µM H₁₀-HTX. After treatment of the labeled membranes with endoglycosidase H,  subunits were isolated by SDS-PAGE and then digested with V8 protease as described in Experimental Procedures to determine the ³H incorporation within each  subunit proteolytic fragment (Fig. 9). In the presence of 50 mM GSSG (lanes 5-8), V8-20 was the only fragment labeled specifically. When gel pieces corresponding to the proteolytic fragments were excised and the ³H incorporation was quantified by scintillation counting, it was found that 96% of the specific incorporation was in V8-20, with specific labeling in V8-18, V8-10, and V8-4 each <2% that of V8-20. The principal effect of 50 mM GSSG was to reduce the nonspecific incorporation into V8-20 by 80%, whereas the specific labeling was not decreased at all. In contrast, H₁₀-HTX-sensitive labeling of V8-18, V8-10, and V8-4 was reduced by 78, 69, and 97%, respectively.

View larger version (63K): [in this window] [in a new window]   Fig. 9.   Proteolytic mapping of [3H]tetracaine photoincorporation into the nAChR  subunit using S. aureus V8 protease. nAChR-rich membranes (540 µg) were suspended in 310 µl of TPS (2.7 µM) with 5 µM [3H]tetracaine in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7, and 8) of 30 µM H10-HTX and with (lanes 1-4) or without (lanes 5-8) 50 mM GSSG. Membrane suspensions were irradiated for 30 min at 302 nm and then pelleted, and the pellets were resuspended in 50 mM sodium phosphate, pH 7.0/1.0% SDS (47 µl) and incubated overnight in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 4.3 mU endoglycosidase H. [3H]Tetracaine-labeled  subunit was resolved by SDS-PAGE and then excised for digestion with 3 µg of V8 protease in a proteolytic mapping gel as described in Experimental Procedures. The mapping gel was stained with Coomassie blue (A) and subjected to fluorography for 4 weeks (B). The  subunit proteolytic fragments are labeled by the nomenclature of White and Cohen (1988), and the molecular weight standards are bovine albumin (66 kDa), egg albumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), and cytochrome c (13 kDa). Based on counting of excised gel slices, in the presence of 50 mM GSSG the incorporation into V8-20 in the absence and presence of HTX was 3433 and 622 cpm, respectively, whereas incorporation in V8-18 was 63 and 30 cpm; in V8-10, 154 and 75 cpm; and in V8-4, 33 and 27 cpm. After  subunit deglycosylation with endoglycosidase H, incorporation in V8-20 was 3574 and 664 cpm. In the absence of GSSG, incorporation in V8-20 was 4824 and 2736 cpm in the absence and presence of HTX.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

The study presented here concerns the nature of the binding site for amine NCAs that bind selectively to the nAChR in the absence of agonist (i.e., to the closed channel state of the nAChR). In contrast to the selectivity for the desensitized state seen for most bulky amine NCAs containing fused aromatic rings or multiple aromatic or aliphatic rings, we found that most N,N-substituted ethanolamine esters of benzoic acid actually bind selectively in the absence of agonist (Table 1). With the exception of procaine, which binds weakly and with similar affinity in the absence and presence of agonist, 4-butylamino and 4-ethoxybenzoate esters bind preferentially to the resting state, as does piperocaine, an unsubstituted benzoate. The selectivity of tetracaine for the resting state results from contributions both from the 4-butylamino substitution and from the presence of the N,N-dimethyl rather than N,N-diethyl. With reference to procaine (compound V, Table 1), the 4-butylamino substitution (compound II) enhanced binding to the resting state by 10-fold more than to the desensitized state, with the further methyl replacements in tetracaine (compound I) weakening binding to the desensitized state by slightly more than to the resting state. In addition to the benzoic acid esters, at least one phenylacetic acid ester of N,N-diethylaminoethanol binds preferentially in the absence of agonist (Cohen et al., 1986). Although proadifen (the ester of 2,2-diphenylpropionic acid) bound with 10-fold higher affinity to the desensitized state and adiphenine (the ester of diphenylacetic acid) bound with similar affinity (K_I = 4 µM) in the presence or absence of agonist, butethemate (the 2-phenylbutyryl ester) bound with 8-fold higher affinity to the resting state. The only other drug known to have high resting state selectivity is amobarbital, which binds with 500-fold higher affinity to the resting state, whereas other barbiturates bind preferentially to the desensitized or open channel states (Cohen et al., 1986; de Armendi et al., 1993).

In the absence of agonist, [³H]tetracaine was bound at equilibrium with high affinity (K_eq = 0.5 µM) to one site per nAChR monomer, and [³H]tetracaine bound with the same high affinity when ACh sites were occupied with -bungarotoxin. [³H]Tetracaine also bound to a single site in the desensitized state of the nAChR but with 60-fold lower affinity. In contrast to other, more hydrophobic aromatic amine NCAs that bind to as many as 30 low-affinity sites as well as to the high-affinity site (Heidmann et al., 1983), there was no evidence that [³H]tetracaine (up to 20 µM) binds to additional low-affinity sites in the nAChR-rich membranes. [³H]Tetracaine bound to the same number of high-affinity sites as [³H]HTX, and as seen previously for [³H]PCP and [³H]HTX (Heidmann et al., 1983), this number was half the number of [³H]ACh sites in the nAChR-rich membranes. Because [³H]ACh binds to two sites per nAChR (Neubig and Cohen, 1979), these NCAs each bind with high affinity to one site per nAChR.

HTX, PCP, proadifen, and meproadifen each completely inhibited the specific [³H]tetracaine binding to nAChR-rich membranes in the absence as well as in the presence of the competitive antagonists d-tubocurarine and -bungarotoxin. For HTX and PCP, in each condition the concentration dependence of inhibition was well fit by a simple, single-site model (n_H = 1). In the presence of d-tubocurarine, the concentration dependence of inhibition by meproadifen or proadifen was also consistent with competition at a single site (n_H = 1). However, at the higher concentrations (>10 µM) of these drugs necessary to inhibit [³H]tetracaine binding in either the presence of -bungarotoxin or the absence of other agonist or competitive antagonists, the inhibition curves were characterized by Hill coefficients between 1.5 and 2.0. The steep concentration dependence seen in the presence of -bungarotoxin suggests that high concentrations of meproadifen and proadifen desensitize the nAChR by a mechanism unrelated to their binding to the high-affinity NCA site or to the ACh sites (Heidmann et al., 1983; Boyd and Cohen, 1984). Of the four NCAs studied, meproadifen binds with highest affinity to the ACh site (K_eq = 50 µM; Heidmann et al., 1983), so in the absence of -bungarotoxin or d-tubocurarine, it may also inhibit [³H]tetracaine binding by binding to the agonist site and desensitizing the nAChR.

To characterize the structure of the aromatic amine NCA site in the closed channel state of the nAChR, we tested whether [³H]tetracaine itself could be used as a photoaffinity probe. Irradiation of nAChR-rich membranes equilibrated with [³H]tetracaine resulted in covalent incorporation into membrane polypeptides. However, the observed photolabeling in the absence of aqueous scavenger was surprising, with photoincorporation into all polypeptides in the membrane suspensions inhibited by the presence of the NCA H₁₀-HTX (Figs. 3 and 4) or the agonist carbamylcholine, drugs that inhibit [³H]tetracaine binding to its high-affinity site in the nAChR. Because the H₁₀-HTX-sensitive [³H]tetracaine incorporation into polypeptides other than the nAChR subunits was reduced by at least 85% in the presence of the aqueous scavenger GSSG (50 mM), this labeling probably occurred after photoactivated [³H]tetracaine dissociated from its nAChR binding site. GSSG was also an effective aqueous scavenger of the nonspecific photolabeling by [³H]d-tubocurarine (Pedersen and Cohen, 1990) and [³H]nicotine (Middleton and Cohen, 1991). However, the specific component of [³H]tetracaine photolabeling was insensitive to GSSG concentrations to 50 mM, whereas for the photolabeling at the agonist site, concentrations of GSSG of more than 1 mM inhibited the specific as well as the nonspecific components. Thus, it appears that the [³H]tetracaine binding site in the closed channel state of the nAChR is substantially more protected from the aqueous environment than the ACh site. Another difference between the photolabeling by [³H]tetracaine and that by [³H]d-tubocurarine or [³H]nicotine is the wavelength dependence, with tetracaine photoincorporation more efficient for irradiation above 300 nm, whereas the other drugs required irradiation below 300 nm. Because tetracaine's long wavelength absorption maximum is 300 nm in benzene and 310 nm in water, it is tetracaine and not the nAChR that is photoactivated.

In the presence of 50 mM GSSG, nearly all [³H]tetracaine photoincorporation sensitive to H₁₀-HTX was restricted to the four nAChR subunits. The specific photolabeling of the four subunits resulted from activation of [³H]tetracaine bound to its high-affinity NCA site, as evidenced by the dependence of this specific photolabeling on free [³H]tetracaine concentration (Fig. 5) and the concentration dependence of inhibition by H₁₀-HTX (Fig. 7) or carbamylcholine (Fig. 8). Also, as expected from the equilibrium binding data, the subunit photolabeling was inhibitable by other aromatic amine NCAs but not by -bungarotoxin (Fig. 6). Thus, [³H]tetracaine bound to its single high-affinity binding site in the nAChR in the absence of agonist is photoincorporated into all four subunits with similar efficiency, which indicates that the binding site is located within the central ion channel domain at the interface of all five subunits. Within  subunit, the amino acids specifically labeled by [³H]tetracaine are restricted to a 20-kDa fragment containing the M1-M3 hydrophobic segments. As described in Gallagher and Cohen (1999), amino acids from the M2 segments of each subunit are specifically photolabeled by [³H]tetracaine, and the binding site for [³H]tetracaine in the closed state of the ion channel overlaps and extends beyond the binding site for [¹²⁵I]TID in the M2 ion channel domain (White and Cohen, 1992).