Effects of an Agonist, Allosteric Modulator, and Antagonist on Guanosine-γ-[35S]thiotriphosphate Binding to Liposomes with Varying Muscarinic Receptor/Go Protein Stoichiometry

  1. Jan Jakubík1,1,
  2. Tatsuya Haga2 and
  3. Stanislav Tuček1
  1. 1Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague, Czechia (J.J., S.T.), and 2Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan (T.H.)
     
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    Abstract

    We investigated whether alcuronium, an allosteric modulator of muscarinic acetylcholine receptors, can induce receptor-mediated activation of Go proteins in liposomal membranes incorporating purified M2 receptors and Goproteins and whether its action is affected by the receptor/Go protein (R/Go) ratio. The binding of guanosine-γ-[35S]thiotriphosphate ([35S]GTPγS) served as the indicator of G protein activation. It was stimulated by empty receptors at high receptor densities, and the dose-response curve was shifted to the left by the agonist carbachol and to the right by the antagonist atropine. At an R/Go ratio of 300:100, the rate of [35S]GTPγS binding was the same in the presence or absence of 0.1 mm carbachol. Alcuronium increased the binding of [35S]GTPγS at R/Go ratios of <3:100 and diminished it at R/Go ratios of >10:100, similar to previous observations on intact cells expressing muscarinic receptors at different densities. The apparent biphasicity of alcuronium action indicates that the allosteric modulator has at least two effects on muscarinic receptor/G protein interaction but its mechanistic basis is unclear. The “active state” of muscarinic receptors induced by alcuronium probably is different from that induced by carbachol. Changes in the densities of receptors and Goproteins had little effect on the kinetics of [35S]GTPγS binding and on receptor affinity for carbachol, provided the R/Go ratio was kept constant. This suggests that the receptors and G proteins are located in microdomains in which their concentrations remain constant, despite variations in the amounts of lipidic membranes in the system.

    It has been known for some time that the binding properties of muscarinic acetylcholine receptors may be modulated by agents acting allosterically (for a review, see Tuček and Proška, 1995) and that the allosteric modulators not only diminish (Clark and Mitchelson, 1976; Stockton et al., 1983), but also enhance the affinity of the receptors for their orthosteric ligands (Tuček et al., 1990; Proška and Tuček, 1994; Guo et al., 1995; Jakubı́k et al., 1995b, 1997; Lazareno and Birdsall, 1995). We found recently that several allosteric modulators of muscarinic receptors (i.e., alcuronium, gallamine, and strychnine) not only modify the affinity of the receptors for the agonists and antagonists but also influence the interaction between the receptors and the G proteins (Jakubı́ket al., 1996). In experiments on CHO cell lines stably transfected with individual subtypes of muscarinic receptors, allosteric modulators had agonist-like effects on the synthesis of cAMP and of inositol phosphates in the cells. These effects could not be blocked by the orthosteric muscarinic antagonist QNB and could not be induced in cells that had not been transfected with the genes for muscarinic receptors. In certain cases, the direction of the effect of allosteric modulators on the synthesis of second messengers varied depending on the density of muscarinic receptors in the cell line used.

    In the current work, we investigated whether the interaction between muscarinic receptors and a G protein also can be affected by the allosteric modulator alcuronium in a simplified system consisting of artificial lipid membranes incorporating purified M2 receptors and purified Go proteins and whether it depends on the stoichiometric ratio between the densities of the M2 receptors and the Goproteins (R/Go ratio). The effects of carbachol (an orthosteric agonist) and atropine (an orthosteric antagonist) were investigated in parallel with those of alcuronium. Receptor-mediated increases in the binding of [35S]GTPγS served as the indicator of G protein activation. The results that we obtained indicate that empty receptors can themselves activate the Go proteins and that there are substantial differences among the effects of the agonist, the allosteric modulator, and the antagonist. The effects of the allosteric modulator vary depending on the R/Go ratio. The “active state” of muscarinic receptors induced by the allosteric modulator is different from the “active state” induced by the orthosteric agonist.

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    Experimental Procedures

    Materials.

    Agarose (Sepharose 4-B) and DEAE-Sephadex were from Pharmacia Fine Chemicals (Uppsala, Sweden), Ultrogel AcA-34 was from Sepracor/IBF (Villeneuve la Garenne, France). Hydroxylapatite was from Sigma Chemical (St. Louis, MO). DEAE-Toyopearl 650S was from Toyo Soda MFG (Tokyo, Japan). Digitonin, phosphatidylcholine (from egg yolk), and phosphatidylinositol (l-α-phosphatidyl-d-myo-inositol-4-phosphate from bovine brain) were from Wako Pure Chemicals (Osaka, Japan). [3H]QNB (79 Ci/mmol) and [35S]GTPγS (1 Ci/μmol) were from DuPont-NEN (Boston, MA). ABT was synthesized (Haga and Haga, 1983), and ABT-agarose gel (Haga and Haga, 1985) and heptylamine Sepharose (Shaltiel, 1974) were prepared as described.

    Purification of muscarinic M2 receptors.

    Receptors were obtained by solubilization of membranes of Sf9 cells transiently expressing human m2 gene (Rinken et al., 1994;Guo et al., 1995). The membranes were treated with 1% digitonin and 0.1% sodium cholate, and muscarinic receptors were isolated by single-step affinity chromatography on ABT-agarose. After elution with atropine, they were concentrated on hydroxylapatite columns as described previously (Haga and Haga, 1985). The purity of the final preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    Isolation of G proteins.

    Crude membranes were prepared from porcine brains and their integral proteins were solubilized with 1% sodium cholate. Go and Giproteins were isolated together by chromatography on DEAE-Sephacel, Ultrogel AcA 34, and heptylamine-Sepharose (Haga et al., 1986). DEAE-Toyopearl columns were used to separate the Go from the Gi protein as described previously (Haga et al., 1986), except that 0.7% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate was substituted for Lubrol PX in all solutions applied. The purity of Go protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots (negative reaction with monoclonal antibodies against Gi, Gq, G11, G16, and Gs, kindly provided by Dr. J. Novotný).

    Reconstitution of M2 receptors and G proteins in lipid vesicles.

    The procedure was modified from that described byShiozaki and Haga (1992). The lipid mixture was obtained by mixing 4 volumes of a solution of cholesterol hemisuccinate (10 mg/ml in methanol), 48 volumes of a solution of phosphatidyl choline (20 mg/ml in chloroform), and 48 volumes of a solution of phosphatidyl inositol (20 mg/ml in chloroform). The solvents were evaporated with a stream of nitrogen so as to obtain a thin layer of lipids on the wall of the test tube. Then, the lipids were emulsified to form vesicles in a solution of 20 mm K-HEPES, pH 8.0, 1 mm EDTA, 160 mm NaCl, and 1% sodium cholate by 30-min sonication at 4°. Vesicles were mixed with a suitable amount of purified M2 receptors and separated on a column with 2 ml of Sephadex G-50. A suitable amount of Go protein was added to them, and the mixture was incubated on ice for 60 min, after which it was diluted as requested.

    The number of receptors incorporated into vesicles was determined in saturation binding experiments with increasing concentrations of [3H]QNB, using Whatman glass fiber filters (GF/B) for the separation of the radioligand bound to vesicles. Data from the [3H]QNB saturation experiments indicated the presence of a homogeneous population of binding sites, with Kd values in the range of 330–400 pm for all vesicle preparations. The amount of G protein incorporated into vesicles was determined according to the binding of [35S]GTPγS at its single saturating concentration of 1 μm, again using Whatman GF/B filters for the separation of vesicle-bound radioactivity. These determinations were performed in the absence of GDP and in the presence of Mg2+, ensuring virtually irreversible [35S]GTPγS binding to all guanyl nucleotide binding sites (Kd = 11–26 nm; Shiozaki and Haga, 1992).

    Three kinds of reconstituted vesicles were used in most experiments, in which the ratio between the molar concentration of muscarinic receptors (as determined by [3H]QNB binding) and of Go protein (as determined by [35S]GTPγS binding) (i.e., the R/Go ratio), was close to 1:100, 10:100, or 50:100. A fourth kind of vesicles also was used when required that contained only muscarinic receptors but no G proteins (10:0), and the fifth kind of vesicles contained only G proteins but no receptors (0:100). An even broader scale of R/Go ratios was applied in experiments described in Fig. 2.

    Figure 2
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    Figure 2

    Initial velocity of [35S]GTPγS binding to reconstituted vesicles with different R/Goratios. Vesicles were preincubated in the control medium (○) or in the presence of 100 μm carbachol (▪), 10 μm alcuronium (▵), or 0.1 μm atropine (▿). After 30 min, [35S]GTPgS (50 nm) was added simultaneously with 1 μm GDP, and [35S]GTPγS binding was determined at time 0 and at three time points within the linear phase of radioligand association.Abscissa, log R/Go ratio.Ordinate, rate of [35S]GTPγS association (fmol/tube/min).

    Kinetics of [35S]GTPγS binding.

    For measurements of [35S]GTPγS binding to G proteins in reconstituted vesicles, the vesicles were preincubated at 30° in 150 μl of a medium consisting of 10 mmMgCl2, 1 mm dithiothreitol, and the investigated muscarinic ligand. After 15 min (or 30 min, where indicated), [35S]GTPγS and GDP were added to final concentrations of 50 nm and 1 μm, respectively, and the incubation was continued in a final volume of 200 μl as needed. The binding was arrested by the addition of 500 μl of a stopping solution consisting of 50 mm Tris·HCl, pH 8.0, 100 mm NaCl, 5 mm MgCl2, and 0.1 mm GTP, and the radioactivity bound to vesicles was separated on Whatman GF/B filters. In experiments designed to compare the effect of various concentrations of the same muscarinic ligand on [35S]GTPγS binding (i.e., to obtain concentration-response curves), [35S]GTPγS association was arrested at a suitable time during the phase of linear association, namely after 10 min in experiments with most ligands, after 5 min in experiments with carbachol at the 50:100 R/Go ratio, or after 30 min in experiments with atropine and alcuronium at the 1:100 and 10:100 R/Go ratios.

    Treatment of data.

    Data in figures and tables are mean ± standard error of three experiments performed with incubation in duplicate. Values of Kd (equilibrium dissociation constant for the binding of a ligand to a single population of binding sites or to an undefined number of populations),Klow, and Khigh(equilibrium dissociation constants for the binding of a ligand to two populations of binding sites displaying low and high affinity, respectively), fhigh (fraction of binding sites displaying high affinity and expressed as percentage of the total number of the binding sites), Ki(equilibrium dissociation constant of an inhibitor),Bmax (maximum binding capacity), EC50 (concentration of a ligand producing one half of maximum effect), and kobs (apparent association rate constant) were computed by nonlinear regression according to accepted conventions (Limbird, 1986; Hulme, 1992;Jakubı́k and Tuček, 1994a, 1994b; Jakubı́k et al., 1995a, 1995b). Apparent association rate constants (kobs) for the binding of [35S]GTPγS to G proteins in liposomes were obtained by fitting the equationFormulawhere Bt is binding at timet, and Beq is binding at equilibrium.

    In concentration-response curves of Figs. 3 and 5, data on the binding of [35S]GTPγS in the presence of muscarinic ligands were expressed as multiples of the binding in their absence, and the concentration-response curves were obtained by fitting the equationFormulawhere B0 describes the binding in the absence of a muscarinic ligand,BL indicates the binding in the presence of a muscarinic ligand at concentration [L], andBmax corresponds to the extrapolated value for the binding in the presence of an infinitely high concentration of the muscarinic ligand. The maximum change of [35S]GTPγS binding induced by a ligand (Emax in Tables1 and 2) corresponded toBmax/B0.

    Figure 3
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    Figure 3

    Concentration-response curves for the effects of carbachol, alcuronium, and atropine on the binding of [35S]GTPγS to lipid vesicles containing the M2 receptors and the Go protein at the ratios of 1:100, 10:100, and 50:100. Abscissa, log10 of the concentration of the muscarinic ligand (m). Ordinate, fold change in the amount of [35S]GTPγS bound, compared with the situation in the absence of any muscarinic ligand. See the text for the duration of incubation and Table 1 for numerical characteristics of the incubation conditions and of the results.

    Figure 5
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    Figure 5

    Concentration-response curves for the effects of carbachol, alcuronium, and atropine on the binding of [35S]GTPγS to lipid vesicles reconstituted with the M2 receptors and the Go protein at a fixed ratio of 10:100 and at three different receptor and Goprotein concentrations. Abscissa, log10 of the concentration (m) of muscarinic ligand.Ordinate, fold change of the amount of [35S]GTPγS bound compared with the situation in the absence of the muscarinic ligand. The arrangement of experiments was the same as in Fig. 4. See the text for the duration of incubation and Table 2 for numerical characteristics of the incubation conditions and of the results.

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    Table 1

    [35S]GTPγS binding to reconstituted vesicles containing M2 receptors and Go proteins at different concentration ratios, with the concentration of Go held constant

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    Table 2

    [35S]GTPγS binding to reconstituted vesicles containing M2 receptors and Go proteins at a fixed molar ratio of 10:100 but at different final concentrations in liposomal membranes

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    Results

    Effects of carbachol, atropine, and alcuronium on [35S]GTPγS association with reconstituted vesicles at different R/Go.

    The time course of the association of [35S]GTPγS with G proteins in vesicles with three different R/Go ratios is shown in Fig.1. Characteristics of the vesicles used and the numerical values computed from experiments in Fig. 1 are summarized in Table 1. The effects of muscarinic ligands on the rates of [35S]GTPγS association were investigated in additional separate experiments on a broader scale of R/Go ratios (Fig.2).

    Figure 1
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    Figure 1

    Effects of 1 mm carbachol, 10 μm alcuronium, and 100 nm atropine on the time course of [35S]GTPγS binding to lipid vesicles containing the M2 receptors and the Goprotein at nominal ratios of 1:100, 10:100, and 50:100.Abscissa, time after the addition of [35S]GTPγS. Ordinate, fmol of [35S]GTPγS bound/incubation tube. Note that the content of Go/incubation tube was the same in all three graphs, whereas the content of receptors increased (left graph< middle graph < right graph). See Table 1 for numerical characteristics of the incubation conditions and of the results.

    It can be seen from Table 1 that the binding of [35S]GTPγS occurred not only to vesicles reconstituted with receptors and Go proteins, but also to vesicles that did not contain any receptors (0:100 ratio). In the absence of receptors, carbachol, alcuronium, and atropine had no effect on the rate of [35S]GTPγS binding. The inclusion of receptors (with no accompanying ligand) into the membranes enhanced the rate of [35S]GTPγS binding, withkobs values changing from 0.026 min−1 at an R/Go ratio of 0:100 to 0.128 min−1 at an R/Go ratio of 50:100 (Table 1). The rate of [35S]GTPγS association was regularly accelerated (compared with control samples) by carbachol. However, at the highest R/Go ratio of 300:100, the rate of [35S]GTPγS association in the absence of the agonist reached the same high value as that which could be maximally induced by carbachol (Fig. 2).

    The rate of [35S]GTPγS association was not affected by 100 nm atropine and, as long as atropine was present, remained virtually the same at all R/Goratios between 0:100 and 50:100, although it became augmented at R/Go ratios of 100:100 and 300:100. Because the rate of [35S]GTPγS association was increased by increases in the R/Go ratios in control experiments (i.e., in the absence of any muscarinic ligand), it seemed that atropine decelerated [35S]GTPγS association at R/Go ratios of 30:100 and higher (Figs. 1 and 2 and Table 1).

    Compared with control samples, alcuronium enhanced the rate of [35S]GTPγS association at R/Go ratios of 0.3:100 and 1:100 (Figs. 1 and 2and Table 1). Increasing R/Go ratios to higher values had much less enhancing effect on the rates of [35S]GTPγS association in the presence of alcuronium than it had in the absence of muscarinic ligands. As a result, the rate of [35S]GTPγS association was faster in the presence of alcuronium than in control samples at low R/Go ratios and slower than in control samples at high R/Go ratios.

    The concentration dependence of the effects of carbachol, alcuronium, and atropine on the rates of [35S]GTPγS binding to reconstituted vesicles with different R/Go ratios is shown in Fig.3 and relevant numerical data are summarized in Table 1. The notable features of Fig. 3 include the opposite effects of alcuronium at the 1:100 and 50:100 R/Go ratios and the higher relative inhibition of [35S]GTPγS association rate by atropine at the higher R/Go ratio.

    Effects of carbachol, alcuronium, and atropine on [35S]GTPγS association with reconstituted vesicles at different densities of receptors and Go proteins.

    Data in the previous section were obtained in experiments in which the concentration of the Go proteins in the reconstituted vesicles was kept constant and the concentration of receptors was varied. The observed variations in the binding rates were presented as a consequence of changes in the ratio of receptors to G proteins. It might be argued that the rates of [35S]GTPγS association were influenced by changes in receptor densities in the membranes rather than by changes in R/Go ratio. To check such an alternative interpretation, experiments were performed in which the R/Go ratio was kept at a constant level of 10:100 and the total amounts of receptors and Goproteins per incubation tube also were kept constant, but the amounts of the lipid mixture (and thus the numbers of vesicles and the areas of the membranes available for receptor and G protein incorporation) used for reconstitution were different. Three different sets of tubes were used. In those with the “1 × Basic” densities, the densities of receptors and Go proteins in liposomal membranes were the same as they had been in the experiments described in Figs. 1-3 and in Table 1 using the 10:100 R/Go ratio. In the tubes designated as “0.1 × Basic” or “5 × Basic,” the densities of receptors and Go proteins were 10-fold lower or 5-fold higher, respectively, than for the “1 × Basic” concentrations. The R/Go ratio always remained at the 10:100 level.

    Within the range of three different receptor and G protein densities used at the constant R/Go ratio of 10:100, the rates of [35S]GTPγS association were stable both in the absence of muscarinic ligands and in the presence of 100 nm atropine or 10 μm alcuronium (Fig.4 and Table 2). Thekobs values computed from experiments with 1 mm carbachol (Table 2) indicate that the rate of [35S]GTPγS association was slightly but significantly faster under the “5 × Basic” conditions (i.e., if the densities of receptors and G proteins were high). Concentration-response curves shown in Fig.5 confirm that the inhibitory effect of atropine on [35S]GTPγS association was virtually the same at the three concentrations of receptors and G proteins examined and that various concentrations of alcuronium had little effect under the conditions used in this set of experiments.

    Figure 4
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    Figure 4

    Effects of 1 mm carbachol, 10 μm alcuronium, and 100 nm atropine on the time course of [35S]GTPγS binding to lipid vesicles containing the M2 receptors and the Go protein at the same ratio of 10:100 but at three different concentrations. Reconstituted vesicles had been obtained in such a way that the same amounts of receptors and 10-fold higher amounts of Goprotein had been incorporated into different volumes of the vesicle emulsion. Consequently, the densities of both receptors and Go were 10- and 50-fold higher (middle and right, respectively, compared with the left). Under the “1 Basic” conditions (middle), the densities of receptors and Go proteins corresponded to those in the 10:100 samples used in Figs. 1 and 3. See Table 2 for numerical characteristics of the incubation conditions and of the results.

    Inhibition of [3H]QNB binding to reconstituted vesicles by carbachol.

    It generally is assumed that receptors that are associated with a G protein display higher affinities for their agonists and that this can be seen in agonist-versus-antagonist competition binding curves. To obtain information about this aspect of our system, we performed competition binding experiments (carbachol versus [3H]QNB) on vesicles in which the R/Go ratios were 1:100, 10:100, 50:100, or 10:0. As shown in Fig. 6 (left), the curves describing the inhibition of [3H]QNB binding by carbachol differed in their steepness and their left-to-right position depending on the R/Goratio. The higher the relative density of receptors (compared with that of G proteins), the more to the right and the steeper were the binding curves, suggesting that the proportion of receptors coupled to Go ([RG]/[Rtotal]) was the highest in the vesicles with the 1:100 R/Goratio and the lowest in the vesicles without G proteins. The fraction of the high affinity binding sites (presumably reflecting the proportion of receptor/G protein complexes) computed from the binding data diminished from 73% at the 1:100 R/Go ratio to 16% at the 50:100 R/Go ratio.

    Figure 6
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    Figure 6

    Inhibition of [3H]QNB binding to reconstituted vesicles by carbachol. Abscissa, log10 of the concentration (m) of carbachol. Ordinate, [3H]QNB binding in the presence of carbachol, expressed as percentage of the binding in its absence. Left, inhibition of [3H]QNB binding to vesicles with different R/Go ratios. These vesicles corresponded to those used in Figs. 1 and 3 and Table 1. Right, inhibition of [3H]QNB binding to vesicles with a constant R/Go ratio of 10:100 but with three different concentrations of receptors and Goproteins, corresponding to those used in Figs. 3 and 4 and Table 2. The “1 Basic” concentrations of receptors and Go proteins (right) corresponded to the concentrations in the samples denoted as 10:100 (left). See Table 3 for computed characteristics of carbachol binding.

    As shown in the Fig. 6 (right), changes in the density of receptors in the liposomes had no effect on the shape of the competition binding curves of carbachol versus [3H]QNB when the R/G ratio was kept constant.

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    Discussion

    Artificial membranes reconstituted with purified G protein-coupled receptors and G proteins have been fruitfully used for investigations of receptor/G protein interactions (for a review, see Birnbaumer and Birnbaumer, 1995). Although many aspects of these interactions remain controversial (Chidiac and Wells, 1992), two points have been generally accepted:

    First, agonist-liganded receptors diminish the affinity of G proteins for GDP and stimulate the dissociation of GDP from them (Brandt and Ross, 1986; Tota et al., 1987; Florio and Sternweis, 1989;Haga et al., 1989; Shiozaki and Haga, 1992).

    Second, agonist-liganded receptors stimulate the binding of GTP or its unhydrolyzable analogue [35S]GTPγS to G proteins, and this is mainly due to the receptor-induced decrease in the binding of GDP. In studies of reconstituted systems containing Gi and Go, the addition of GDP was necessary to reveal the effect of agonists on [35S]GTPγS binding (Florio and Sternweis, 1989; Ikegaya et al., 1990; Shiozaki and Haga, 1992; but seeKurose et al., 1986, and Freissmuth et al., 1991a), whereas the addition of GDP was not necessary in systems containing Gs (Cerione et al., 1985;Brandt and Ross, 1986; Freissmuth et al., 1991b) or Gq (Nakamura et al., 1995). The meaning of these differences is not clear, and their analysis is difficult in view of the tight binding of GDP to G proteins (Fergusonet al., 1986).

    Our data offer several new insights with regard to Go protein activation by unliganded muscarinic receptors and by receptors associated with an agonist, antagonist, or allosteric modulator, to the importance of the R/Go ratio, and to the likely spacial organization of receptors and G proteins in liposomal membranes. The following are the most important findings.

    Empty muscarinic receptors increase the apparent rate of [35S]GTPγS binding to Go proteins.

    When present at a high concentration (R/Goratio = 300:100), empty receptors were able to activate the Go proteins to the same degree as the agonist-liganded receptors (Fig. 2). Similar effects of empty A1 adenosine and β-adrenergic receptors have been noted (Schütz and Freissmuth, 1992).

    Atropine prevents or diminishes the agonist-independent stimulatory effects of muscarinic receptors on [35S]GTPγS binding.

    However, increases in the rate of [35S]GTPγS binding could be observed even in the presence of atropine if the R/Go ratio was raised above 50:100 (Fig. 2). It seems that the effect of atropine can be viewed as an increase of the EC50concentration of receptors required for Goactivation.

    Carbachol increases the apparent rate of [35S]GTPγS binding to Go proteins and its effect varies with the R/Go ratio.

    Although the enhancement of [35S]GTPγS binding by muscarinic agonist was expected (Kurose et al., 1986; Florio and Sternweis, 1989; Hilf et al., 1989; Shiozaki and Haga, 1992; Lazareno and Birdsall, 1993; Nakamura et al., 1995), several features of the current data merit attention:

    First, the apparent rate of carbachol-stimulated [35S]GTPγS binding approaches a maximum when the R:Go ratio reaches 1:100 and is changed little by further increases in the relative density of receptors (Figs.1 and 2, Table 1). Although the ceiling in the amount of [35S]GTPγS bound is given by the quantity of Go available for binding, it is not clear what determines the ceiling in the rate of [35S]GTPγS binding at increasing receptor concentrations.

    Second, carbachol brings about a leftward shift of the curve describing how the rate of [35S]GTPγS association depends on the concentration of receptors, and its effect can be viewed as a decrease in the EC50 concentration of receptors required for Go activation.

    Third, the concentration of carbachol necessary for its half-maximal effect on [35S]GTPγS binding (EC50 of carbachol) increases with increasing R/Go ratios (Table 1). This can be explained by changes in the proportion of receptors that are physically associated with G proteins (Fig. 6 and Table 3). Although the absolute number of RG complexes increases with increasing R/Go ratios, the proportion of coupled receptors (RG as a percentage of Rtotal) decreases, and this is the likely cause of the increases in the EC50 values. A very small increase of the EC50 values for carbachol also was observed when the R/Go ratios were kept constant, but the densities of receptors and G proteins were augmented (Table 2). In this case, the above explanation cannot be applied, and we are uncertain as to the likely reason.

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    Table 3

    Inhibition by carbachol of [3H]QNB binding to reconstituted vesicles containing M2 receptors and Go proteins

    Alcuronium affects the binding of [35S]GTPγS to Go proteins in two directions.

    Compared with the binding that occurs in the absence of alcuronium, the binding of [35S]GTPγS in the presence of alcuronium is enhanced at low R/Go ratios (<10:100) and diminished at high R/Go ratios (>10:100; Figs.1-3). This point is discussed further.

    The effects of carbachol, atropine, and alcuronium on [35S]GTPγS binding are all receptor mediated.

    The three ligands had no effect when receptors were absent from the system (Table 1).

    The kinetics of [35S]GTPγS association with the vesicles are affected very little by changes in the density of receptors and Go proteins as long as the R/Goratio remains constant.

    The most likely explanation of relevant observations (Fig. 4 and Table 2) is that muscarinic receptors and Go proteins are not uniformly dispersed in liposomal membranes but rather are clustered in microdomains in which their concentrations remain nearly constant despite variations in the amounts of lipidic membranes in the system. The agglomeration of signaling proteins in microdomains of cell surface membranes has been highlighted by Neubig (1994; see also Huang et al., 1997).

    Increases in R/Go ratios bring about decreases of the fraction of receptors displaying high affinity for the agonist carbachol.

    The higher the R/Go ratio, the lower proportion of receptor molecules could be expected to be associated with a G protein. Data in Fig. 6 (left) and Table3 thus support the notion that receptors associated with G proteins (RG complexes) have a higher affinity for the agonist than free receptors. When, however, the densities of receptors and G proteins in liposomal membranes were manipulated by altering the amount of lipid membranes (Fig. 6, right; Table 3), no change occurred in the fraction of receptors with a high affinity for the agonist. As pointed out, such behavior of the system can be explained on the assumption that the receptors and G proteins are clustered within microdomains in which their densities remain constant.

    Judging from its effects on [35S]GTPγS binding, the allosteric modulator alcuronium can either increase or diminish the activating influence of muscarinic M2 receptors on Goproteins, depending on the R/Go ratio. This corresponds to what had been observed on intact CHO cells expressing the M1 muscarinic receptors. In cells with a low density of receptors, alcuronium enhanced the production of inositol phosphates, whereas in cells with a high density of receptors (and presumably a high R/G ratio), the production of inositol phosphates was diminished by alcuronium (Jakubı́k et al., 1996).

    Data on the effects of alcuronium are of principal interest. They demonstrate (1) that the interaction between muscarinic receptors and Go proteins may be influenced by their allosteric modulator alcuronium not only in intact cells but also in a reconstituted system and (2) that the activation of receptors induced by alcuronium is not identical with that induced by the classic agonist carbachol.

    These observations raise the question of the mechanistic background of the difference between the effects of alcuronium at low and high R/Go ratios. Presumably, the binding of alcuronium to free receptors has two opposite effects on the ability of the receptors to stimulate [35S]GTPγS binding by G proteins, which occur simultaneously but are differently pronounced at different R/Go ratios: one supporting [35S]GTPγS binding to G proteins, which prevails at low R/Go ratios, and another inhibiting [35S]GTPγS binding, which prevails at high R/Go ratios. Among the functional parameters that might be affected by alcuronium binding to receptors in opposite ways are (1) the rates of receptor/G protein association and dissociation and the resulting affinity of the receptor for the G protein, (2) the velocity of receptor-catalyzed guanine nucleotide exchange, (3) the size and direction of receptor-induced changes in the rates of GDP and, independently, GTP association to and dissociation from the G protein and of the resulting affinities for GDP and for GTP, and others. At least some of the conceivable effects of alcuronium are likely to carry different weight depending on whether the receptor (acting as the catalyst) is saturated with the G protein as its substrate (at a low R/G ratio) or undersaturated (at a high R/G ratio). This consideration applies to situations in which receptors and G proteins interact as independent monomers and bind in a 1:1 ratio. Alternative interpretations can be based on the assumption of an oligomeric arrangement of receptors and G proteins (Chidiac and Wells, 1992; Wreggett and Wells, 1995). Unfortunately, the data we have do not permit identification of the processes involved.

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    Acknowledgments

    We thank Hoffmann-La Roche (Basel, Switzerland) for providing alcuronium and Prof. E. E. El-Fakahany (Minneapolis, MN) for collaboration and extensive help.

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    Footnotes

    • Send reprint requests to: Dr. S. Tuček, Institute of Physiology AV CR, Vı́deňská 1083, 14220 Prague, Czech Republic. E-mail: tucek{at}biomed.cas.cz

    • ↵1 Current affiliation: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.

    • This work was supported by Grant 309/96/1287 from the Grant Agency of the Czech Republic (S.T.), National Institutes of Health Fogarty International Research Collaboration Award 2-R03-TW00171 (E.E.El-Fakahany), and a Travel Grant of the Japan Society for the Promotion of Science (J.J.).

    • Abbreviations:
      ABT
      3-[2′-aminobenzhydryl-oxy]tropane
      CHO
      Chinese hamster ovary
      GTPγS
      guanosine-γ-thiotriphosphate
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      QNB
      quinuclidinyl benzilate
      R/Go ratio
      ratio between the densities of muscarinic receptors and Go proteins
      • Received January 30, 1998.
      • Accepted July 7, 1998.
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    1. Molecular Pharmacology November 1, 1998 vol. 54 no. 5 899-906
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