Characterization of Cembranoid Interaction with the Nicotinic Acetylcholine Receptor1

  1. Richard M. Hann1,2,
  2. Oné R. Pagán1,2,
  3. Liza Gregory1,
  4. Tomas Jácome1,
  5. Abimael D. Rodríguez2,3,
  6. P. A. Ferchmin1,2,
  7. Ruiliang Lu2,1,2 and
  8. Vesna A. Eterovic1,2
  1. 1Department of Biochemistry (R.M.H., O.R.P., L.G., T.J., P.A.F., R.L., V.A.E.) and 2Center for Molecular and Behavioral Neuroscience (R.M.H., O.R.P., A.D.R., P.A.F., R.L., V.A.E.), 3Universidad Central del Caribe, Bayamón and Department of Chemistry (A.D.R.), University of Puerto Rico-Rio Piedras Campus, San Juan, Puerto Rico
     
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    Abstract

    The class of diterpenoids with a 14-carbon cembrane ring, the cembranoids, includes both competitive and noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR). All 20 coelenterate-derived cembranoids studied in this report inhibited [piperidyl-3,4-3H]-phencyclidine ([3H]-PCP) binding to its high-affinity site on the electric organ AChR, with IC50s ranging from 0.9 μM for methylpseudoplexaurate to 372 μM for lophotoxin. Inhibition was complete with all cembranoids but lophotoxin and most Hill coefficients were close to 1. Methylpseudoplexaurate and [3H]-PCP binding was competitive. Methylpseudoplexaurate and the fourth most potent cembranoid, eunicin, competed with each other for [3H]-PCP displacement, indicating that there exist one or more cembranoid sites on the AChR. Cembranoid affinity for the AChR correlated with hydrophobicity, but was also dependent on other features. Methylpseudoplexaurate and n-octanol also competed with each other for [3H]-PCP displacement, indicating that the cembranoid site is linked to the n-octanol site on the AChR. Unlike lophotoxin, the five cembranoids tested did not inhibit [125I]Tyr54-α-bungarotoxin binding to the AChR agonist sites. All seven cembranoids tested on oocyte-expressed electric organ AChR reversibly blocked acetylcholine-induced currents, although the inhibitor concentration curves were shallow and the inhibition was incomplete.

    Cembranoids are diterpenoids that contain a fourteen-carbon cembrane ring structure (fig. 1). To date more than 300 cembranoids of natural origin have been reported, the majority of which has been isolated from marine coelenterates (Wahlberg and Eklund, 1992;Rodríguez, 1995). Several coelenterate-derived cembranoids are toxic to vertebrates at low micromolar concentrations, although in most cases the basis for the toxicity is unknown (Wahlberg and Eklund, 1992). The best-characterized cembranoids are lophotoxin ([20] in fig. 1) and the structurally related bipinnatins, which act as irreversible competitive inhibitors of the peripheral AChR (Abramsonet al., 1991), a member of the ligand-gated ion channel superfamily, which also includes neuronal AChRs and the γ-aminobutyric acid type A, glycine and serotonin type 3 receptors (Karlin, 1993). These cembranoids led to identification of tyrosine 190 on the α subunit of the AChR as an important residue in the agonist binding site (Abramson et al., 1989). Three other cembranoids ([9], [12] and [13] in fig. 1) act as reversible noncompetitive inhibitors of the electric organ and mouse muscle AChRs (Eterović et al., 1993a, b). These cembranoids inhibit binding of the noncompetitive inhibitor, [3H]-PCP, to its high-affinity site (Eterović et al., 1993a) which is believed to lie in or near the ion channel of the AChR molecule (Karlin, 1993). These last findings are remarkable because, unlike most known noncompetitive inhibitors of the AChR, these cembranoids do not contain nitrogen and are uncharged at physiological pH.

    Figure 1
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    Figure 1

    Structures of the 20 cembranoids studied for this report. Molecules are all drawn in the same orientation with carbon numbering as shown in [1]. Trivial names are presented in table 1.

    This report extends earlier studies by characterizing the inhibition of [3H]-PCP binding to its high-affinity site on the electric organ AChR by 17 additional cembranoids, including three synthetic derivatives of coelenterate natural products. Evidence is presented that cembranoids bind directly to the electric organ AChR at one or more sites that are linked, either sterically or allosterically, to the binding sites for [3H]-PCP and n-octanol.

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    Methods

    Materials.

    Torpedo californica electric organ was obtained frozen (Pacific Biomarine, Venice, CA). [3H]-PCP (41–50 Ci/mmol) and [125I]-Bgt (104–128 Ci/mmol) were from Du Pont-New England Nuclear (Boston, MA). cDNAs coding for the subunits of the T. californica electric organ AChR in SP64T vectors were kindly provided by Dr. Mark G. McNamee (University of California, Davis, CA). All other supplies were from Fisher (Cayey, PR), Sigma Chemical Co. (St Louis, MO) or ICN (Costa Mesa, CA). FemaleXenopus laevis frogs were from stocks maintained in the institutional animal resources unit and were originally purchased from Nasco (Modesto, CA).

    Seventeen of the cembranoids studied were natural products, 15 of which were extracted from coelenterates collected offshore from Puerto Rico. These include the following compounds whose purification and characterization are described in the indicated references (see fig.1): 14-epi-sarcophytol-A [2] and marasol [3] from Plexaura flexuosa (Peniston and Rodrı́guez, 1991), pseudoplexaurol [4], 14-deoxycrassin [16] and crassin acetate [17] fromPseudoplexaura porosa (Rodrı́guez and Martı́nez, 1993), methylpseudoplexaurate [5], euniolide chlorohydrin [8], eupalmerin [11], eupalmerin acetate [12] and eupalmerone [15] from Eunicea mammosa (Rodrı́guezet al., 1993; Fontán and Rodrı́guez, 1991;Fontán et al., 1990) and methyluproeunioloate [6], euniolide [7], eunicin [9], 12,13-bis-epi-eupalmerin [13] and 12,13-bis-epi-eupalmerin acetate [14] from Eunicea succinea (Morales et al., 1990; Rodrı́guez and Dhasmana, 1993; Rodrı́guez and Acosta, 1997). Sarcophytol-A [1] was a kind gift from Dr. Masaru Kobayashi (Hokkaido University, Sapporo, Japan). Lophotoxin [20] was a kind gift from Dr. Robert Jacobs (University of California, Santa Barbara, CA). Samples of eunicin, eupalmerin acetate and crassin acetate were also kindly provided by Dr. Leon Ciereszko (University of Oklahoma, Norman, OK).

    Three of the cembranoids studied were synthesized from natural product cembranoids. These include eunicin acetate [10] obtained by acetylation of [9], inolide-A [18] from [13] and inolene oxide [19] from inolene (not shown) (Rodrı́guez et al., 1995).

    Purified cembranoids were dissolved and maintained below 0°C in DMSO, chloroform or methanol at stock concentrations of 20 to 100 mM. All cembranoids were analyzed for purity on HPTLC before use and showed a single component on 250-μm silica gel plates using two different solvent systems, (v:v) 70:30::hexane:ethyl acetate and 19:1::chloroform:methanol.

    Preparation and characterization of AChR-rich membranes.

    AChR-rich membranes were prepared from the total membrane fraction of homogenized T. californica electric organ as previously described (Szczawinska et al., 1992). Total membrane protein was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard. Total AChR was determined as total [125I]-Bgt binding sites in solubilized AChR using a filtration assay previously described (Schmidt and Raftery, 1973) as modified (Szczawinska et al., 1992). The specific activity of AChR in membrane preparations ranged from 0.2 to 0.5 nmol AChR/mg protein.

    Radioligand binding assays.

    Cembranoid inhibition of [125I]-Bgt binding at equilibrium both to membrane-bound and detergent-solubilized electric organ AChR was measured as previously described (Eterović et al., 1993a).

    Equilibrium binding of [3H]-PCP to membrane-bound AChR was measured using modification of a previously-described filtration assay (Eldefrawi et al., 1980). Briefly, membranes were preincubated for 30 min at 25°C in buffer A (10 mM sodium phosphate, 5 mM EDTA at pH 7.4) containing CCh to induce the desensitized (PCP-high affinity) conformation. This was followed by incubation for 60 min at 25°C in buffer A containing [3H]-PCP, 100 μM carbamoylcholine and 5% DMSO in the absence or presence of added inhibitors. AChR concentration in the incubation mixture (based on total [125I]-Bgt sites) ranged from 56 to 520 nM. Membranes were filtered onto glass fiber filters (Whatman, Hillsboro, OR) and washed quickly with 1.0 ml of buffer A. Air-dried filters were left overnight in 10 ml Scintiverse BD liquid scintillation cocktail (Fisher, Cayey, PR) and counted in a liquid scintillation counter to determine the total filter-bound PCP (B). The concentration of specific receptor-bound PCP ([RL]) was calculated from the equation:FormulaEquation 1where N represents filter-bound [3H]-PCP measured in the presence of either 30 μM unlabeled PCP or 1.3 mM tetracaine (nonspecific binding). The nonspecifically-bound [3H]-PCP was always less than 20% of total binding under these conditions. The concentration of unbound [3H]-PCP in the incubation mixture ([L]f) was calculated from the [3H]-PCP concentration measured directly in the filtrates and corrected for the total filtrate volume and the [3H]-PCP nonspecifically bound to filter. This value always agreed within 5% with the difference between total [3H]-PCP concentration in the incubation mixture and specifically bound [3H]-PCP.

    [3H]-PCP saturation curves were constructed by varying the total [3H]-PCP concentration from 10 nM to 10 μM. [3H]-PCP in samples from 10 nM to 1 μM was diluted with unlabeled PCP to 10% its initial specific activity and in samples above 1 μM with unlabeled PCP to 2% initial specific activity. For each membrane preparation, three separate experiments were performed to generate a saturation curve. Binding data were fit to the following equation:FormulaEquation 2Awhere [R]t is total AChR concentration, [L]f is the concentration of unbound [3H]-PCP, Kd is the apparent dissociation constant of PCP and n is the Hill coefficient. Because n was equal to 1, binding data were also analyzed using the Rosenthal-Scatchard linear transformation of equation 2A:FormulaEquation 2BFor inhibitor studies the final total [3H]-PCP concentration in the incubation mixture was approximately 300 nM. Binding was measured as above at different cembranoid concentrations [I] in a minimum of three experiments and the data were fit to the following equation:FormulaEquation 3Ato determine the IC50 and Hill coefficient, n, where [RL]o is the specific binding in the absence of cembranoid.

    Binding data were also fit to the normalized equation for mutually exclusive binding of L and I to a single class of noninteracting sites to estimate the dissociation constant of the cembranoid (Ki):FormulaEquation 3BFormulawhere [L]o and [L] are the unbound [3H]-PCP concentrations in the absence and presence of inhibitor, respectively, [I] is the unbound inhibitor concentration (approximated with the total inhibitor concentration) andKd is the dissociation constant of [3H]-PCP determined from PCP saturation curves (0.30 μM).

    Methylpseudoplexaurate [5] inhibition data at different [3H]-PCP concentrations were fit to the double-reciprocal transformation of the equation for mutually exclusive binding of L and I to a single class of noninteracting sites:FormulaEquation 4Data analyses were performed using the PSI-Plot program (Poly Software International, Salt Lake City, UT) and goodness-of-fit was evaluated using a normalized Akaike information criterion generated by that program (Akaike, 1976).

    Voltage-clamp measurement of oocyte-expressed Torpedo AChR.

    The procedure followed was as previously described (White et al., 1985) as modified (Eterović et al., 1990). Briefly, upon linearization with SmaI, RNA transcripts of cDNAs were obtained by in vitro transcription with the Megascript SP6 kit (Ambion, Austin, TX). Female Xenopus laevis frogs were anesthetized by hypothermia and the oocytes were removed and injected with the RNA mixture and kept in modified Barth solution. Functional AChRs were expressed within 24 hr and their numbers increased over the next 2 days.

    ACh-induced currents were measured using a two microelectrode voltage clamp (Dagan TEV-200) using VCAN/VGEN software. The voltage and current electrodes were filled with 3 M KCl and had tip resistances of 0.5 to 1.0 and 10 to 15 MΩ, respectively. Agonists and inhibitors were applied by bath perfusion at a rate of 25 ml/min in a buffer solution containing: NaCl 82.3 mM, KCl 2.5 mM, MgCl2 5 mM, Na2HPO4 5 mM, HEPES 5 mM, and CaCl20.2 mM. Atropine (0.5 μM) was present in all solutions to block endogenous muscarinic responses. The oocyte was exposed to agonist for 40 to 70 sec with recovery intervals of 5 min. The membrane potential was maintained at −60 mV. Due to their hydrophobic character, cembranoid stocks were prepared in DMSO, aliquots of which were then added to the solution. Final DMSO concentration was 0.1%; at this concentration, the effect of DMSO on ACh-induced currents was negligible. Data from cembranoid inhibition experiments were fit to the equation:FormulaEquation 5where [I] is total cembranoid concentration and n is the Hill coefficient.

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    Results

    Lack of cembranoid inhibition of [125I]-Bgt binding to the AChR agonist sites.

    Five cembranoids, [9], [12], [13], [18] and [19], were tested against [125I]-Bgt binding to electric organ AChR. None of the cembranoids had any effect on [125I]-Bgt binding to either membrane-bound or solubilized AChR, even after 24-hr incubation at cembranoid concentrations of more than 100 μM.

    Cembranoid inhibition of [3H]-PCP binding to its high-affinity site.

    In the presence of 100 μM CCh, [3H]-PCP binding to AChR-rich membranes reached equilibrium in less than 5 min and stayed constant for up to 120 min (data not shown). Saturation curve data on five different membrane preparations over a [3H]-PCP concentration range of 10 nM to 10 μM were fit to equation 2A and gave an average Hill coefficient of 1.04 ± 0.08 (mean ± S.D.) and aKd for [3H]-PCP of 0.30 ± 0.10 μM. Figure 2 shows a typical saturation curve. The molar ratio (mean ± S.D.) of [3H]-PCP binding sites-to-[125I]-Bgt binding sites was 0.54 ± 0.15.

    Figure 2
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    Figure 2

    Typical saturation curve of [3H]-PCP binding to AChR-rich membranes from T. californica in the presence of CCh. Conditions are detailed in Methods. Receptor-bound [3H]-PCP ([RL]) and unbound [3H]-PCP ([L]f) concentrations were determined from three separate experiments. The curve represents the best fit of the data to equation2A and generated the following parameters (mean ± S.D.) in this particular case: [R]t = 408 ± 41 nM,Kd = 435 ± 157 nM, Hill coefficient = 1.01 ± 0.26. Inset, Rosenthal-Scatchard transformation of the same data. The line represents the best fit of the data to equation2B.

    Figure 3 shows the inhibitor concentration curves of the cembranoids illustrated in figure 1. All twenty cembranoids completely inhibited [3H]-PCP binding to its high-affinity site, with the exception of the weakest inhibitor, lophotoxin ([20]), whose limited solubility prevented achieving complete inhibition. In four cases ([1], [4], [10] and [18]) there was an apparent loss of inhibition at the highest concentrations, which was also probably due to the limited solubility of these cembranoids. IC50 values ranged over two orders of magnitude, from 0.9 μM for methylpseudoplexaurate ([5]) to 372 μM for lophotoxin ([20]) (table 1). Eleven of the compounds displayed IC50 values of less than 10 μM. Most Hill coefficients were close to 1 (range 0.6–1.3, table 1). Based on findings outlined below, cembranoid inhibition data were also fit to equation 3B, allowing estimation of a bimolecular dissociation constant (Ki) for each cembranoid (table 1).

    Figure 3
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    Figure 3

    Cembranoid inhibition of [3H]-PCP binding to membrane-bound AChR. PCP binding at the indicated concentrations of each cembranoid was measured as described in Methods. Fraction PCP bound represents [3H]-PCP binding normalized to control (absence of cembranoid). Values shown are the means ± S.D. from three or four experiments. Where error bars are not visible, S.D. was within the range of the symbol. Curves represent the best fit of the data to equation 3A using IC50s and Hill coefficients shown in table 1. A, Cembranoids [1] (filled triangles), [2] (open triangles), [5] (filled circles) and [6] (open circles). B, Cembranoids [4] (filled circles), [7] (open squares) and [8] (filled squares). C, Cembranoids [9] (filled circles), [10] (open circles), [11] (filled squares) and [12] (open squares). D, Cembranoids [13] (filled squares), [14] (open squares) and [15] (filled circles). E, Cembranoids [3] (filled circles), [16] (filled squares) and [17] (open squares). F, Cembranoids [18] (filled circles), [19] (open circles) and [20] (filled triangles).

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    Table 1

    Cembranoid inhibition of 3H-PCP binding to AChR and cembranoid HPTLC mobility

    [3H]-PCP saturation of its high-affinity site was studied in the presence of different concentrations of methylpseudo-plexaurate ([5]). Figure 4 shows the double-reciprocal plots of bound [3H]-PCP versus unbound [3H]-PCP in the absence or presence of cembranoid [5] at six different cembranoid concentrations ranging from 0.05 to 1 μM. The plots are linear (linear correlation coefficients > 0.98) and, within experimental error, converge on a single ordinate intercept.

    Figure 4
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    Figure 4

    Double-reciprocal plots of [3H]-PCP binding to membrane-bound AChR in the absence (filled circles) or presence of methylpseudoplexaurate (cembranoid [5]) at concentrations of 50 nM (open circles), 100 nM (filled squares), 200 nM (open squares), 325 nM (filled diamonds), 500 nM (open diamonds) or 1 μM (filled triangles). Specific [3H]-PCP binding ([RL]) and unbound [3H]-PCP ([L]f) were measured as described in “Methods”. Unbound [3H]-PCP concentration ranged from 36 nM to 1 μM. Lines are the best fit of the data to equation 4.

    Figure 5 shows inhibitor concentration curves for methylpseudoplexaurate ([5]) against [3H]-PCP binding in the absence or presence of eunicin ([9]). The presence of eunicin at a concentration near its IC50 shifted the curve significantly to the right, increasing the IC50 of methylpseudoplexaurate by about 4-fold, from 1.38 ± 0.13 to 6.25 ± 2.14 μM, although the Hill coefficient was not changed significantly (1.21 ± 0.12vs. 1.02 ± 0.32).

    Figure 5
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    Figure 5

    Inhibition of [3H]-PCP binding to membrane-bound AChR by methylpseudoplexaurate [5] in the absence (open circles) or presence (filled circles) of eunicin [9] at 1 μM concentration. [3H]-PCP binding was measured as described in “Methods” and is normalized to respective controls (absence of cembranoid [5]). Values shown are means ± S.E.M. from five experiments. Curves represent the best fit of the data to equation3A.  

    The relative hydrophobicities of the cembranoids were estimated from their mobilities on silica gel HPTLC using a solvent mixture which gave cembranoid Rf values between 0.16 and 0.64 (table 1). Cembranoid affinity for the AChR and cembranoid hydrophobicity displayed direct proportionality (fig.6), with a linear correlation coefficient for the log Ki and Rf values of 0.66 (P < .001; coefficient of determination, 0.44).

    Figure 6
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    Figure 6

    Correlation between cembranoid affinity for the AChR site, estimated by the dissociation constant (Ki) from table 1, and cembranoid hydrophobicity, estimated by cembranoid mobility on HPTLC (Rf). Each point represents 1 of the 20 cembranoids shown in figure 1. The solid line is the best linear fit to the data.  

    Methylpseudoplexaurate inhibition of [3H]-PCP binding was also studied in the presence of the long-chain alkanol, n-octanol, a noncompetitive inhibitor of the AChR (Wood et al., 1991). At 1 mM concentration, n-octanol shifted the inhibitor concentration curve of methylpseudoplexaurate significantly to the right (fig.7), increasing the IC50 about 2-fold, from 0.21 ± 0.02 to 0.40 ± 0.04 μM, although the Hill coefficient was not significantly changed (0.72 ± 0.04vs. 0.83 ± 0.06). Alone, n-octanol inhibited [3H]-PCP binding to electric organ AChR with an IC50 of 1.1 ± 0.1 mM.

    Figure 7
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    Figure 7

    Inhibition of [3H]-PCP binding to membrane-bound AChR by n-octanol (filled squares) and by methylpseudoplexaurate [5] in the absence (open circles) or presence (filled circles) of 1 mM n-octanol. [3H]-PCP binding was measured as described in “Methods”. [3H]-PCP binding is normalized to control (absence of inhibitor). Values shown are means ± S.E.M. from three experiments. Curves represent the best fit of the data to equation 3A.  

    Cembranoid inhibition of ACh current in oocyte-expressed T. californica AChR.

    When added together with ACh, eupalmerin acetate ([12]) at 5 μM concentration decreased current amplitude by 42% and increased the rate of current decay (fig.8A). This effect was reversible. In the absence of ACh, eupalmerin acetate had no effect on membrane potential. Cembranoid inhibition was concentration-dependent but displayed a shallow concentration-response curve with a Hill coefficient significantly less than 1 (fig. 8B).

    Figure 8
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    Figure 8

    Inhibition of ACh-induced currents by eupalmerin acetate [12]. A, Voltage-clamp recordings from an oocyte expressing the AChR from electric organ 1 day after injection of RNA transcripts coding for receptor subunits. The lines above the recordings indicate the time during which 5 μM ACh or 5 μM ACh plus cembranoid were applied. Traces a and c are the responses to ACh before application of cembranoid and after washing, respectively, although trace b is the response in the presence of 5 μM cembranoid. B, Eupalmerin acetate concentration-response curves 1 day (circles), 2 days (triangles) and 3 days (squares) after injection of RNA transcripts. Values shown represent means and S.E. of six replicate experiments obtained with oocytes from five frogs (one concentration-response curve per oocyte); each batch was tested on all 3 days. ACh concentration was 5 μM. Solid lines represent the best fit to equation 5, which generated the following parameters: day 1, IC50 = 13 μM,n = 0.55; day 2, IC50 = 21 μM,n = 0.68; day 3, IC50 = 57 μM,n = 0.61.

    The potency of eupalmerin acetate decreased with time after injection of the RNA transcripts into the oocyte (fig. 8B). The IC50was 13, 21 and 57 μM on day 1, 2 and 3, respectively; these values were significantly different from each other (P < .05, one-way analyses of variance followed by the Bonferroni test). The EC50 for ACh did not change significantly over this same 3-day period: 6 ± 4 μM, 11 ± 3 μM and 9 ± 4 μM (mean ± S.E.M. on four oocytes).

    Six additional cembranoids, [5], [7], [9], [11], [16] and [17], were studied on the second day after injection of RNA transcripts. All six cembranoids reversibly inhibited ACh current amplitude with IC50s in the low μM range (table2). As with cembranoid [12], the concentration-inhibition curves were shallow and reached only about 60% inhibition at the highest concentration of cembranoid.

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    Table 2

    Cembranoid inhibition of ACh-induced currents in oocyte-expressed AChR

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    Discussion

    Cembranoids inhibit [3H]-PCP binding through direct interaction with the AChR

    All 20 cembranoids studied in this report interfered with the binding of the noncompetitive inhibitor, [3H]-PCP, to its high-affinity site on the electric organ AChR. Five cembranoids tested against [125I]-Bgt binding displayed no affinity for the AChR agonist sites, unlike lophotoxin ([20]) and its analogues, the bipinnatins (Abramson et al., 1991). The observed characteristics of [3H]-PCP binding in the presence of agonist agree with previous reports (Eldefrawi et al., 1980;Heidmann et al., 1983; Aronstam et al., 1985;Amitai et al., 1987; White et al., 1991): a Hill coefficient of 1, a Kd value in the submicromolar range (0.30 μM vs. 0.1–0.8 μM) and a ratio of [3H]-PCP binding: [125I]-Bgt binding of 0.5, indicating a single high-affinity binding site for [3H]-PCP per AChR molecule.

    The [3H]-PCP inhibition findings with the 20 cembranoids are consistent with cembranoid acting either directly with a homogeneous set of independent sites on the AChR molecule or indirectly through an effect on the membrane. The mutually exclusive binding displayed by [3H]-PCP and cembranoid [5] (fig. 4) conforms to the bimolecular model for competitive inhibition (equation4) and agrees with previously reported findings on cembranoids [9], [12] and [13] at single cembranoid concentrations (Eterovićet al., 1993a). This shows that the cembranoids interact directly with the AChR molecule by ruling out a membrane-mediated displacement of [3H]-PCP, which would not display such mutual exclusivity (Wood et al., 1995). Furthermore, cembranoids [5] and [9] displayed competition for [3H]-PCP displacement (fig. 5), suggesting, because of the similarity of the cembranoid structures, that these (and possibly all) cembranoids bind to the same site. Therefore, the most likely situation is that cembranoids and [3H]-PCP share common or overlapping sites on the AChR. However, a strong negative allosteric effect, where binding of cembranoid to one or more separate sites induces a conformational change that completely prevents [3H]-PCP binding, cannot be ruled out from these data. Such an allosteric effect could conceivably be mediated either through a single site or through multiple low-affinity sites, such as those located at the boundary between receptor and membrane lipids (Heidmannet al., 1983).

    Features that affect cembranoid affinity for its AChR site.

    In general, the more highly oxygenated cembranoids tended to display lower potency for inhibiting [3H]-PCP binding (fig. 1; table1), which suggests that cembranoid binding may be a function of cembranoid hydrophobicity. The correlation between cembranoid affinity for the AChR site and cembranoid mobility on HPTLC (fig. 6) indicates that cembranoid hydrophobicity is an important factor in determining its binding affinity and suggests that the cembranoid site on the AChR has hydrophobic character. However, the dispersion of values in figure6 and the coefficient of determination of 0.44 indicate that hydrophobicity accounts only partially for differences in the cembranoid Kis; therefore, other structural features must also be important.

    An indication of which features are more or less important can be seen by comparing Kis in table 1 with the structures in figure 1. Affinity was not affected significantly by changing the chirality of the alcohol groups on carbon 14 (compare [1] and [2]) or carbon 13 (compare [11] and [13]) or of the acetoxy group on carbon 13 (compare [12] and [14]). In addition, oxidation of carbon 13 from an alcohol to a ketone did not produce a significant change in affinity (compare [11] and [13] with [15]). This suggests that either the region around carbons 13 and 14 is not interacting with the AChR site or this region is interacting with a site that is not stereospecific. Because a bulkier, but more hydrophobic, acetoxy group on carbon 13 significantly increased affinity in both conformations (compare [11] with [12] and [13] with [14]), the latter possibility seems more likely. The introduction of an alcohol group at carbon 7 (compare [6] with [5]) or an epoxy at carbons 7 and 8 (compare [17] with [16]) significantly reduced affinity. Also, although a 1,14-γ-lactone is a common structural feature of these molecules, four of the five compounds with highest affinity have no such lactone group. Considered together, these observations suggest that the C7 to C14 side of the cembranoid molecule is interacting with the AChR site via hydrophobic interactions.

    In contrast, replacing the carbon 4 alcohol on the other side of the molecule with an acetoxy group did not increase affinity as was seen with carbon 13, but had the opposite effect, although to a smaller degree (compare [10] with [9]).

    The cembranoid site on the Torpedo AChR is linked to the n-octanol site.

    Long-chain n-alkanols and cycloalkane-methanols have been shown to be noncompetitive inhibitors of the peripheral AChR, binding with relatively low affinities to a specific site on the receptor molecule (Wood et al., 1991, 1993, 1995). The n-octanol site is believed to be located in the AChR central ion channel (Formanet al., 1995). The structural similarity between these compounds and cembranoids [1] and [2], which have a single alcohol group on the cembrane ring, raises the possibility that cembranoids and alkanols may bind to the same site. Results presented in figure 7 show that methylpseudoplexaurate and n-octanol competed with each other for [3H]-PCP displacement, indicating that their binding sites are not independent but either overlap or are linked allosterically.

    Effects of cembranoids on ACh currents in oocyte-expressed AChR.

    In agreement with a previous report on cembranoid [12] (Eterović et al., 1993a), all cembranoids tested in this report inhibited ACh-induced currents with shallow concentration curves and Hill coefficients well less than 1. One possible explanation for this observation is that there are two subpopulations of oocyte-expressed receptors with different affinities for cembranoids. This is in contrast with the observation that these same cembranoids completely and homogeneously inhibited [3H]-PCP binding to desensitized AChR and indicates that similar receptor subpopulations are not present in native electric organ membranes.

    The observation that cembranoid [12] potency varied with time after RNA injection was surprising, although changes related to AChR maturation in oocytes have been reported. Li et al. (1990)noticed a large increase in oocyte-expressed AChR conductance from day 2 to 3 after RNA injection. Maturation of neuronal AChR expressed in bovine adrenal chromaffin cells was also reported (Higgins and Berg, 1988).

    In summary, the cembranoids act as noncompetitive inhibitors by binding to the electric organ AChR at one or more sites that are linked, either sterically or allosterically, to the binding sites for [3H]-PCP and n-octanol. Although the affinities of these uncharged ligands for the AChR are lower than some classical noncompetitive inhibitors, such as histrionicotoxin and PCP, they are comparable to many other important noncompetitive inhibitors, including procaine, quinacrine, cocaine and QX-222.

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    Acknowledgments

    The authors thank Derick Vergne, Dinely Pérez and Myriam Rosado for their excellent technical assistance.

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    Footnotes

    • Send reprint requests to: Dr. Richard M. Hann, Department of Biochemistry and Center for Molecular and Behavioral Neuroscience, Universidad Central del Caribe, Box 60-327, Bayamón, PR 00960.

    • ↵1 This work was supported by Grants NIH-RCMI-2G12RR03035, NIH-MBRS-SO6GM50695 and NIH-RO1-GM52277. O.R.P. was a recipient of a Viets Fellowship from the Myasthenia Gravis Foundation of America.

    • ↵2 Current address: Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore, Baltimore, MD 21202.

    • Abbreviations:
      AChR
      nicotinic acetylcholine receptor
      [3H]-PCP
      [piperidyl-3,4-3H]-phencyclidine
      [125I]-Bgt
      [125I]Tyr54-α-bungarotoxin
      ACh
      acetylcholine
      CCh
      carbamoylcholine
      DMSO
      dimethylsulfoxide
      HPTLC
      high-performance thin-layer chromatography
      Rf
      ratio of sample front to solvent front on HPTLC
      • Received March 17, 1998.
      • Accepted June 3, 1998.
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