Vol. 287, Issue 1, 253-260, October 1998
Characterization of Cembranoid Interaction with the Nicotinic
Acetylcholine Receptor1
Richard M.
Hann ,
Oné R.
Pagán ,
Liza
Gregory,
Tomas
Jácome,
Abimael D.
Rodríguez ,
P. A.
Ferchmin ,
Ruiliang
Lu2 and
Vesna A.
Eterovi
Department of Biochemistry (R.M.H., O.R.P., L.G., T.J., P.A.F.,
R.L., V.A.E.) and
Center for Molecular and Behavioral Neuroscience
(R.M.H., O.R.P., A.D.R., P.A.F., R.L., V.A.E.),
Universidad 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-
-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.
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Introduction |
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 (Abramson
et al., 1991), a member of the ligand-gated ion channel superfamily, which also includes neuronal AChRs and the
-aminobutyric acid type A, glycine and serotonin type 3 receptors
(Karlin, 1993). These cembranoids led to identification of tyrosine 190 on the 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
(Eterovi et al., 1993a, b). These cembranoids inhibit
binding of the noncompetitive inhibitor, [3H]-PCP, to
its high-affinity site (Eterovi 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.
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Fig. 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.
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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). Female
Xenopus 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 Rodríguez, 1991), pseudoplexaurol [4], 14-deoxycrassin [16] and crassin acetate [17] from
Pseudoplexaura porosa (Rodríguez and
Martínez, 1993), methylpseudoplexaurate [5], euniolide
chlorohydrin [8], eupalmerin [11], eupalmerin acetate [12] and
eupalmerone [15] from Eunicea mammosa (Rodríguez
et al., 1993; Fontán and Rodríguez, 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; Rodríguez and
Dhasmana, 1993; Rodríguez 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) (Rodríguez 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 (Eterovi 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:
![[<UP>RL</UP>]=<UP>B</UP>−<UP>N</UP>](/content/vol287/issue1/fulltext/253/img001.gif)
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(1)
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where 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:
![[<UP>RL</UP>]=[<UP>R</UP>]<SUB><UP>t</UP></SUB>/(1+(K<SUB>d</SUB>/[<UP>L</UP>]<SUB><UP>f</UP></SUB>)<SUP><UP>n</UP></SUP>)](/content/vol287/issue1/fulltext/253/img002.gif)
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(2A)
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where [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:
![[<UP>RL</UP>]/[<UP>L</UP>]<SUB><UP>f</UP></SUB>=(<UP>−</UP>1/K<SUB>d</SUB>)[<UP>RL</UP>]+([<UP>R</UP>]<SUB><UP>t</UP></SUB>/K<SUB>d</SUB>)](/content/vol287/issue1/fulltext/253/img003.gif)
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(2B)
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For 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:
![[<UP>RL</UP>]/[<UP>RL</UP>]<SUB><UP>o</UP></SUB>=1/(1+([<UP>I</UP>]/<UP>IC</UP><SUB>50</SUB>)<SUP><UP>n</UP></SUP>)](/content/vol287/issue1/fulltext/253/img004.gif)
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(3A)
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to 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):
![[<UP>RL</UP>]/[<UP>RL</UP>]<SUB><UP>o</UP></SUB>={[<UP>L</UP>]×([<UP>L</UP>]<SUB><UP>o</UP></SUB>+K<SUB>d</SUB>)}/{[<UP>L</UP>]<SUB><UP>o</UP></SUB>](/content/vol287/issue1/fulltext/253/img005.gif)
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(3B)
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where [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) and
Kd 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:
![1/[<UP>RL</UP>]={K<SUB>d</SUB>×(1+[<UP>I</UP>]/K<SUB>i</SUB>)/[<UP>R</UP>]<SUB><UP>t</UP></SUB>}(1/[<UP>L</UP>]<SUB><UP>f</UP></SUB>)+1/[<UP>R</UP>]<SUB><UP>t</UP></SUB>](/content/vol287/issue1/fulltext/253/img007.gif)
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(4)
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Data 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 (Eterovi 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 M
, 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 CaCl2
0.2 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:
![<UP>current </UP>(% <UP>of control</UP>)=100/(1+([<UP>I</UP>]/<UP>IC</UP><SUB>50</SUB>)<SUP><UP>n</UP></SUP>)](/content/vol287/issue1/fulltext/253/img008.gif)
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(5)
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where [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 a
Kd 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.
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Fig. 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 equation
2A 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 equation
2B.
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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).
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Fig. 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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[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.
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Fig. 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.
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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 ± 0.12 vs. 1.02 ± 0.32).
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Fig. 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 equation
3A.
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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).
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Fig. 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.
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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 ± 0.04 vs. 0.83 ± 0.06). Alone, n-octanol inhibited [3H]-PCP binding to electric organ AChR with an
IC50 of 1.1 ± 0.1 mM.
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Fig. 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.
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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).
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Fig. 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.
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The potency of eupalmerin acetate decreased with time after injection
of the RNA transcripts into the oocyte (fig. 8B). The IC50
was 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 (table
2). As with cembranoid [12], the
concentration-inhibition curves were shallow and reached only about
60% inhibition at the highest concentration of cembranoid.
| |
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 (equation
4) and agrees with previously reported findings on cembranoids [9],
[12] and [13] at single cembranoid concentrations (Eterovi
et 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 (Heidmann et 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; table
1), 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 figure
6 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--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 (Forman et 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]
(Eterovi 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.
| |
Acknowledgments |
The authors thank Derick Vergne, Dinely Pérez and Myriam
Rosado for their excellent technical assistance.
| |
Footnotes |
Accepted for publication June 3, 1998.
Received for publication March 17, 1998.
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.
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.
| |
Abbreviations |
AChR, nicotinic acetylcholine receptor;
[3H]-PCP, [piperidyl-3,4-3H]-phencyclidine;
[125I]-Bgt, [125I]Tyr54--bungarotoxin;
ACh, acetylcholine;
CCh, carbamoylcholine;
DMSO, dimethylsulfoxide;
HPTLC, high-performance thin-layer chromatography;
Rf, ratio of
sample front to solvent front on HPTLC.
| |
References |
-
Abramson SN,
Li Y,
Culver P and
Taylor P
(1989)
An analog of lophotoxin reacts covalently with tyr190 in the -subunit of the nicotinic acetylcholine receptor.
J Biol Chem
264:
12666-12672[Abstract/Free Full Text].
-
Abramson SN,
Trischman JA,
Tapiolas DM,
Harold EE,
Fenical W and
Taylor P
(1991)
Structure/activity and molecular modeling studies of the lophotoxin family of irreversible nicotinic receptor antagonists.
J Med Chem
34:
1798-1804[Medline].
-
Akaike H
(1976)
An information criterion (AIC).
Math Sci
1:
5-9.
-
Amitai G,
Herz JM,
Bruckstein R and
Luz-Chapman S
(1987)
The muscarinic antagonists aprophen and benactyzine are noncompetitive inhibitors of the nicotinic acetylcholine receptor.
Mol Pharm
32:
678-685[Abstract].
-
Aronstam RS,
King CT,
Albuquerque EX,
Daly JW and
Feigl DM
(1985)
Binding of [3H]-perhydrohistrionicotoxin and [3H]-phencyclidine to the nicotinic receptor-ion channel complex of Torpedo electroplax.
Biochem Pharm
34:
3037-3047[Medline].
-
Eldefrawi ME,
Eldefrawi AT,
Aronstam RS,
Maleque MA,
Warnick JE and
Albuquerque EX
(1980)
[3H]-Phencyclidine: A probe for the ionic channel of the nicotinic receptor.
Proc Natl Acad Sci USA
77:
7458-7462[Abstract/Free Full Text].
-
Eterovi VA,
Li L,
Palma A and
McNamee MG
(1990)
Regulation of nicotinic acetylcholine receptor function by adenine nucleotides.
Cell Mol Neurobiol
10:
423-433[Medline].
-
Eterovi VA,
Hann RM,
Ferchmin PA,
Rodríguez AD,
Li L,
Lee YH and
McNamee MG
(1993a)
Diterpenoids from caribbean gorgonians act as noncompetitive inhibitors of the nicotinic acetylcholine receptor.
Cell Mol Neurobiol
13:
99-110[Medline].
-
Eterovi VA,
Li L,
Ferchmin PA,
Lee YH,
Hann RM,
Rodríguez A and
McNamee MG
(1993b)
The ion channel of muscle and electric organ acetylcholine receptors: differing affinities for noncompetitive inhibitors.
Cell Mol Neurobiol
13:
111-121[Medline].
-
Fontán LA,
Yoshida WY and
Rodríguez AD
(1990)
Application of two-dimensional NMR spectroscopy in the structural determination of marine natural products. Total structural assignment of the cembranoid diterpene eupalmerin acetate through the use of two-dimensional 1H-1H, 1H-13C and 13C-13C chemical shift correlation spectroscopy.
J Org Chem
55:
4956-4960.
-
Fontán LA and
Rodríguez AD
(1991)
Isolation of eupalmerin, a minor cembranoid diterpene from the Caribbean gorgonian Eunicea mammosa. J Natl Prod 54:1101-1113.
-
Forman SA,
Miller KW and
Yellen G
(1995)
A discrete site for general anesthetics on a postsynaptic receptor.
Mol Pharm
48:
574-581[Abstract].
-
Heidmann T,
Oswald RE and
Changeux J-P
(1983)
Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from Torpedo marmorata.
Biochemistry
22:
3112-3127[Medline].
-
Higgins LS and
Berg DK
(1988)
Cyclic AMP-dependent mechanism regulates acetylcholine receptor function on bovine adrenal chromaffin cells and discriminates between old and new receptors.
J Cell Biol
107:
1157-1165[Abstract/Free Full Text].
-
Karlin A
(1993)
Structure of nicotinic acetylcholine receptors.
Curr Opin Neurobiol
3:
299-309[Medline].
-
Li L,
Schuchard M,
Palma A,
Pradier L and
McNamee M
(1990)
Functional role of the cysteine 451 thiol group in the M4 helix of the subunit of Torpedo californica acetylcholine receptor.
Biochemistry
29:
5428-5436[Medline].
-
Lowry OH,
Roseborough NJ,
Farr AL and
Randall JR
(1951)
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275[Free Full Text].
-
Morales JJ,
Espina JR and
Rodríguez AD
(1990)
The structure of euniolide, a new cembranoid diterpene from the Caribbean gorgonians Eunicea succinea and Eunicea mammosa.
Tetrahedron
46:
5889-5894.
-
Peniston M and
Rodríguez AD
(1991)
The isolation of ()-sarcophytol and (+)-marasol from the Caribbean gorgonian Plexaura flexuosa.
J Natl Prod
54:
1009-1016.
-
Rodríguez AD and
Dhasmana H
(1993)
Further bioactive cembranolide diterpenes from the gorgonian Eunicea succinea.
J Natl Prod
56:
565-570.
-
Rodríguez AD and
Martínez N
(1993)
Marine antitumor agents: 14-Deoxycrassin and pseudoplexaurol, new cembranoid diterpenes from the Caribbean gorgonian Pseudoplexaura porosa.
Experientia
49:
179-181[Medline].
-
Rodríguez AD,
Li Y,
Dhasmana H and
Barnes CL
(1993)
New marine cembrane diterpenoids isolated from the Caribbean gorgonian Eunicea mammosa.
J Natl Prod
56:
1101-1113.
-
Rodríguez AD
(1995)
The natural products chemistry of West Indian Gorgonian octocorals.
Tetrahedron
51:
4571-4618.
-
Rodríguez AD,
Piña IC and
Barnes CL
(1995)
Synthesis and biological evaluation of cembranolide analogs containing cyclic ethers.
J Org Chem
60:
8096-8100.
-
Rodríguez AD and
Acosta AL
(1997)
New cembranoid diterpenes and a geranylgeraniol derivative from the common Caribbean sea whip Eunicea succinea.
J Natl Prod
60:
1134-1138.
-
Schmidt J and
Raftery MA
(1973)
A simple assay for the study of solubilized acetylcholine receptors.
Anal Biochem
52:
349-354[Medline].
-
Szczawinska K,
Ferchmin PA,
Hann RM and
Eterovi VA
(1992)
Electric organ polyamines and their effects on the acetylcholine receptor.
Cell Mol Neurobiol
12:
95-106[Medline].
-
Wahlberg I and
Eklund AM
(1992)
Cembranoids, pseudopteranoids and cubitanoids of natural occurrence, In: Progress in the Chemistry of Organic Natural Products, Vol 59 (Herz W, Kirby GW, Moore RE, Steglich W and Tamm C eds) pp 141-294, Springer Verlag, New York.
-
White BH,
Howard S,
Cohen SG and
Cohen JB
(1991)
The hydrophobic photoreagent 125I-TID is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor.
J Biol Chem
266:
21595-21607[Abstract/Free Full Text].
-
White MM,
Mixter-Mayne K,
Lester HA and
Davidson N
(1985)
Mouse-Torpedo hybrid acetylcholine receptors: Functional homology does not equal sequence homology.
Proc Natl Acad Sci USA
82:
4852-4856[Abstract/Free Full Text].
-
Wood SC,
Forman SA and
Miller KW
(1991)
Short chain and long chain alkanols have different sites of action on nicotinic acetylcholine receptor channels from Torpedo.
Mol Pharm
39:
332-338[Abstract].
-
Wood SC,
Hill WA and
Miller KW
(1993)
Cycloalkanemethanols discriminate between volume- and length-dependent loss of activity of alkanols at the Torpedo nicotinic acetylcholine receptor.
Mol Pharm
44:
1219-1226[Abstract].
-
Wood SC,
Tonner PH,
Armendi AJ,
Bugge B and
Miller KW
(1995)
Channel inhibition by alkanols occurs at a binding site on the nicotinic acetylcholine receptor.
Mol Pharm
47:
121-130[Abstract].
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Abstract
Introduction
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