From the Receptor Biology Laboratory, Department of Physiology and
Biophysics, Mayo Foundation, Rochester, Minnesota 55905
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INTRODUCTION |
Epibatidine recently emerged as one of the most potent nicotinic
receptor ligands thus far characterized. In addition to its femtomolar
to picomolar affinity for certain neuronal acetylcholine receptor
(AChR)1 subtypes, epibatidine
also induces analgesia and is approximately 200 times more potent than
morphine as an analgesic (1-3). Despite the intense interest in the
neuronal actions of epibatidine, its interactions with muscle-type AChR
are not well characterized.
Muscle-type AChRs are heteropentamers of homologous but functionally
distinct subunits with compositions
2

in fetal
and
2

in adult muscle (4). Within the pentamer
are two binding sites for acetylcholine (ACh); one is formed at the

subunit interface, whereas the other is formed at the 
interface in the fetal receptor and at the 
interface in the
adult. Each type of binding site displays distinct selectivities for
agonists and competitive antagonists. For example, carbamylcholine
binds 30-fold less tightly to the 
binding site than to the

and 
binding sites owing to different contributions of the
,
, and
subunits (5, 6). Previous studies used
-
and
-
subunit chimeras to identify residues in these subunits which confer site selectivity for curare, conotoxin M1, and carbamylcholine (6-9). The overall findings show that four loops, well separated in
the primary sequence of the non-
subunits, contribute to the binding
site interface (10).
To investigate further the structure of the ligand binding site, the
present work examines binding of epibatidine to sites of fetal and
adult muscle AChRs. We reasoned that a structurally constrained ligand
such as epibatidine would be less able to accommodate differences in
binding site structure and thus might show greater or different
selectivity compared with flexible agonists such as carbamylcholine and
ACh. We show that epibatidine binds with novel site selectivity to
muscle AChRs, selecting strongly for the 
and 
binding
sites over the 
site. Further, unlike carbamylcholine,
epibatidine maintains strong site selectivity when the receptor is
converted to the high affinity desensitized state. Owing to its unique
site and state selectivity, epibatidine is a potentially valuable probe
of binding site structure and of elements that confer
state-dependent selectivity.
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EXPERIMENTAL PROCEDURES |
Materials--
125I-Labeled
-bungarotoxin
(
-BTX) was obtained from NEN Life Science Products. Proadifen and
(
)-, (+)-, and (±)-epibatidine were purchased from Research
Biochemicals Inc. The 293 human embryonic kidney-293 (HEK-293) cell
line was obtained from the American Type Culture Collection. Sources of
the mouse AChR subunits were as described previously (7, 11).
Expression of Receptor Complexes--
HEK-293 cells were
transfected at about 50% confluence using calcium phosphate
precipitation as described previously (7). Cells expressing
intracellular 
, 
, or 
complexes or cell surface
pentamers (
2

or
2

) were
maintained at 37 °C for 48 h after transfection. Cells
expressing triplet pentamers (
2
2,
2
2, or
2
2) were maintained at 37 °C for
24 h after transfection and then at 31 °C for 48 h.
Ligand Binding Measurements--
Cells expressing surface
pentamers were harvested by gentle agitation in phosphate-buffered
saline containing 5 mM EDTA, centrifuged at 1000 × g for 1 min, and resuspended in potassium Ringer's solution (140 mM KCl, 5.4 mM NaCl, 1.8 mM
CaCl2, 1.7 mM MgCl2, 25 mM HEPES, 30 mg/l bovine serum albumin, adjusted to pH 7.4 with 10-11 mM NaOH). Cells expressing intracellular
complexes were permeabilized with saponin before harvesting (6).
Agonist binding was determined by competition against the initial rate
of 125I-labeled
-BTX binding as described previously
(6). In brief, the receptor complexes were equilibrated with agonist
for 40 min before addition of 5 nM 125I-labeled
-BTX. The cells were then incubated for a further 20-40 min to
allow occupancy of at most 50% of the binding sites by 125I-labeled
-BTX. The total number of sites was
determined by incubating with 25 nM
125I-labeled
-BTX for 20-40 min and subtracting a blank
determined in the presence of 10 mM carbamylcholine. The
cells were harvested using a Brandell cell harvester and counted in a
counter.
Data Analysis--
We fit the following two equations to our
data using Prism 2.0 (GraphPad Software):
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(Eq. 1)
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(Eq. 2)
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where [L] is the concentration of competing ligand,
K is an apparent dissociation constant, n is the
Hill coefficient, K1 and
K2 are intrinsic dissociation constants, and
P is the fraction of sites with dissociation constant
K1.
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RESULTS |
Binding to Intracellular Complexes--
Transfection of HEK-293
cells with
and
,
, or
subunit results in robust
expression of agonist displaceable intracellular
-BTX binding sites.
To determine selectivity of epibatidine for these intracellular
complexes, we measured binding by competition against
125I-labeled
-BTX binding. Both enantiomers of
epibatidine bind with highest affinity to 
complexes,
intermediate affinity to 
, and lowest affinity to 
(Fig.
1A and Table
I). Although both isomers of epibatidine
bind with the same affinity to 
complexes, the (
)-isomer binds
with significantly higher affinity to 
and 
complexes
compared with the (+)-isomer. By contrast, carbamylcholine shows
opposite site selectivity to epibatidine, binding with high affinity to

and 
and low affinity to 
complexes (Fig.
1B and Table I). In this and previous studies (6) we found
that agonists bind to intracellular complexes with Hill coefficients
significantly less than 1. Because complexes of an
and a non-
subunit should form only one type of binding site, the shallow binding
curves we observe indicate some type of site heterogeneity. Despite
this heterogeneity, selectivities of carbamylcholine and acetylcholine
for intracellular complexes (5, 6) coincide with selectivities of
receptors in the closed activable state determined by single channel
kinetic analysis (12, 13). Further, intracellular complexes show the
same rank order of affinity for the competitive antagonist
d-tubocurarine to that observed in native receptors, 

> 
(14). Intracellular complexes, unlike fully
assembled native receptors, do not enter a high affinity desensitized
state and are presumably incapable of channel gating (6). Thus the
binding site selectivity of intracellular complexes may most closely
resemble that of closed activable receptors.

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Fig. 1.
Epibatidine and carbamylcholine binding to
intracellular complexes. A, epibatidine binds with high
affinity to  complexes, with intermediate affinity to 
complexes, and with low affinity to  complexes. B,
carbamylcholine binds with high affinity to  and  complexes
and with low affinity to  complexes. Binding of agonists to
intracellular complexes was determined as described under
"Experimental Procedures." In both panels the data are means of
three to five experiments with the error bars representing
the standard error. The curves are fits of Equation 1 (see
under "Experimental Procedures") to the data with parameters given
in Table I.
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Table I
Binding of (+)- and ( )-epibatidine and carbamylcholine to
intracellular complexes
The parameters (Kd, dissociation constant;
n, Hill coefficient) are derived from fits of Equation 1
(see under "Experimental Procedures") to the data in Fig. 1 and are
expressed ± S.E.
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Binding to Cell Surface Pentamers--
To assess site selectivity
of epibatidine for receptors containing the full complement of
subunits, we expressed adult or fetal receptors in HEK-293
cells and measured binding to intact cells under control and desensitizing conditions (Fig. 2 and Table II). Under control conditions, both
enantiomers of epibatidine displace 125I-labeled
-BTX
with much higher affinity than carbamylcholine. Although fetal and
adult receptors bind epibatidine with similar affinities, fetal
receptors distinguish between (+)- and (
)-epibatidine, whereas adult
receptors do not. Hill coefficients for epibatidine are only ~0.8
compared with values for carbamylcholine of ~1.3. Although a Hill
coefficient less than 1 implies multiple binding sites, for agonists
equilibrium binding cannot be interpreted using a simple two site
model. At equilibrium, agonist binding is determined by affinities of
the sites in the resting, open, and desensitized states as well as the
allosteric constants governing channel opening and desensitization (see
under "Discussion").

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Fig. 2.
Binding of carbamylcholine and epibatidine to
cell surface pentamers. Fetal ( 2  )
(A) and adult ( 2  ) (C)
receptors bind (+)- and ( )-epibatidine in a monophasic manner under
equilibrium conditions (no additions). In contrast, the
desensitized states of these receptors have clearly biphasic binding
curves for both isomers of epibatidine (+ proadifen). In all
cases dissociation constants of the two desensitized epibatidine
binding sites differ by more than 170-fold. B (fetal) and
D (adult), under both equilibrium (no additions)
and desensitizing (+ proadifen) conditions carbamylcholine
binds in a monophasic manner. Receptors were transiently expressed in
HEK-293 cells and agonist binding determined as described under
"Experimental Procedures." For fetal receptors, desensitization was
induced by 200 µM proadifen and for adult receptors by 60 µM proadifen. The curves are fits of Equation 1 or 2 to the data (see under "Experimental Procedures") with
parameters given in Table II. In all cases the data are means of three
to five experiments with standard errors of less than 5% of
total.
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Table II
Binding of (+)- and ( )-epibatidine and carbamylcholine to adult and
fetal receptors
The parameters (K1, K2,
dissociation constants; P, proportion of binding sites with
dissociation constant K1, n, Hill
coefficient) are derived from fits of either Equation 1 or 2 (see under
"Experimental Procedures") to the data in Fig. 2 and are
expressed ± S.E. Maximally desensitizing concentrations of
proadifen were used, 60 µM for adult receptors and 200 µM for fetal receptors.
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To measure binding of agonist to a single functional state of the
receptor, we used the local anesthetic proadifen to promote entry into
the high affinity desensitized state (15, 16). When fully desensitized
with proadifen, adult and fetal receptors bind carbamylcholine with
high affinity and Hill coefficients close to 1, indicating binding to a
single class of high affinity sites (Fig. 2, B and
D, and Table II; however, see below). Epibatidine, on the
other hand, binds in a distinctly biphasic manner in the presence of
proadifen; fits to a two-site equation (Equation 2) yield intrinsic
dissociation constants different by more than 170-fold (Fig. 2,
A and C and Table II). Thus, whereas
carbamylcholine does not distinguish between the 
and 
binding sites in the desensitized fetal receptor nor between the 
and 
sites in the desensitized adult receptor, epibatidine
selects strongly between the two binding sites of each receptor.
To determine which subunits form the high affinity sites in fetal and
adult receptors, we expressed triplet
2
2,
2
2, or
2
2 receptors and measured agonist
binding under control and desensitizing conditions (Fig.
3 and Table
III). Unlike native receptors, the two
binding sites in triplet receptors are formed by
and identical
non-
subunits (17). Under control conditions both enantiomers of
epibatidine bind more tightly to
2
2
and
2
2 than to
2
2 pentamers. Carbamylcholine, by
contrast, binds more tightly to
2
2
than to
2
2 or
2
2 pentamers. In the presence of
proadifen, the affinities of epibatidine and carbamylcholine for
2
2 pentamers increase markedly, as
with native pentamers, whereas affinities for
2
2 pentamers are only slightly
affected; similar results were obtained for carbamylcholine by Sine and Claudio (17).

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Fig. 3.
Agonist binding to
2 2 and
2 2 triplet receptors.
A, binding of (+)- and ( )-epibatidine; B,
binding of carbamylcholine. A concentration of 100 µM
proadifen (+ PRO) was used to promote desensitization. The
data are means of at least three experiments with the error
bars representing the standard error. The curves are
fits of Equation 1 (see under "Experimental Procedures") to the
data with parameters given in Table III.
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Table III
Agonist binding to 2 2,
2 2, and 2 2 triplet
receptors in the absence or presence of proadifen
The parameters (Kd, apparent dissociation constant;
n, Hill coefficient) are derived from fits of Equation 1
(see under "Experimental Procedures") to the data in Fig. 3 and are
expressed ± S.E. A concentration of 100 µM
proadifen was used to desensitize 2 2 and
2 2 receptors. Low expression of
2 2 receptors prevented measurement of a
desensitized Kd.
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Comparison of epibatidine binding to desensitized fetal and the
corresponding
2
2 and
2
2 triplet receptors shows that the
high affinity component corresponds to the 
site, whereas the low
affinity component corresponds to the 
site. Although expression
of
2
2 pentamers was too low to
accurately measure under desensitizing conditions, at equilibrium
epibatidine binds to these receptors 30-fold more tightly than to the
low affinity binding site of desensitized adult receptors. Thus in the
desensitized adult receptor, the high affinity component corresponds to
the 
site, whereas the low affinity component corresponds to the 
site.
Effects of Proadifen: Concentration Dependence--
Our
preliminary experiments with (±)-epibatidine tested a range of
proadifen concentrations to determine an optimum concentration for promoting desensitization. As observed previously, increasing proadifen concentrations progressively increase carbamylcholine affinity to approach a limiting value corresponding to the agonist affinity for the desensitized state (15). For the fetal receptor, the
limiting carbamylcholine affinity is reached at 200 µM
proadifen; at all concentrations of proadifen carbamylcholine binding
is well described by Hill coefficients of 1 or greater, implying binding to a single class of sites (Fig.
4 and Table
IV). For the adult receptor the limiting
affinity is achieved by only 30-60 µM proadifen; at
proadifen concentrations up to 60 µM, carbamylcholine binding is described by Hill coefficients of 1 or greater, again indicating binding to a single class of high affinity sites (Fig. 4 and
Table IV). However, in the presence of 100-200 µM
proadifen, carbamylcholine binds to adult receptors with Hill
coefficients of 0.7-0.8. As carbamylcholine affinity reaches a limit
at lower proadifen concentrations, the observed broadening of the
binding curve at high proadifen concentrations likely results from an action distinct from enhancement of desensitization.

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Fig. 4.
Concentration dependence of proadifen-induced
desensitization. A, binding of (±)-epibatidine to the fetal
receptor. B, binding of carbamylcholine to the fetal
receptor. C, binding of (±)-epibatidine to the adult
receptor. D, binding of carbamylcholine to the adult
receptor. The curves are fits of Equation 1 or 2 (see under
"Experimental Procedures") to the data (means of three to five
experiments). Curve fit parameters are given in Table IV.
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Table IV
Dependence of desensitized state agonist binding on proadifen
concentration
Adult or fetal receptors were incubated with the indicated
concentration of proadifen, and agonist affinity was determined. The
parameters (K1, K2, dissociation
constants; n, Hill coefficient; P, fraction of
sites with dissociation constant K1) are derived
from fits of Equation 1 or 2 (see under "Experimental Procedures")
to the data in Fig. 4 and are expressed ± S.E. For the adult
receptor, carbamylcholine binding curves at 100 and 200 µM proadifen were fit using both the Hill equation and
the sum of two equally weighted sites.
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At both fetal and adult receptors, epibatidine binds in a distinctly
biphasic manner in the presence of proadifen; biphasic binding is
observed at all proadifen concentrations greater than 10 µM. Furthermore, for both sites of the adult receptor,
epibatidine affinity increases to reach a limiting value with proadifen
concentrations of 30 µM or greater, as observed for
carbamylcholine (Table III). For the fetal receptor, the maximum
increase in affinity is reached at 60-100 µM proadifen,
similar to results with carbamylcholine (Table IV). Thus for both fetal
and adult receptors, a given concentration of proadifen increases
carbamylcholine and epibatidine affinities to similar extents.
We noticed for epibatidine, however, that the relative weights of the
high and low affinity components depend on proadifen concentration
(Fig. 4 and Table IV). For fetal receptors in the presence of 30 µM proadifen, the high affinity component dominates, whereas for adult receptors the two components are approximately equally weighted. When proadifen concentration is increased to 200 µM, fetal receptors show equally weighted low and high
affinity components, whereas adult receptors show a dominant low
affinity component. These results indicate that in addition to
promoting desensitization, proadifen noncompetitively inhibits the
binding of 125I-labeled
-BTX binding. Furthermore, the
extent of inhibition differs for each site in the two types of
receptors.
To examine this additional action of proadifen, we measured the initial
rate and total number of 125I-labeled BTX binding sites at
varying concentrations of proadifen. The results show that increasing
proadifen concentrations reduce the rate of toxin binding and the total
number of sites approximately in parallel (Fig.
5). As these determinations reflect
contributions of both sites in each receptor type, they do not reveal
which of the 
, 
, or 
sites are preferentially
affected. However, our results on site selectivity of epibatidine show
that proadifen decreases the rate and number of BTX sites in a
site-selective manner (Fig. 4); site selectivity for proadifen follows
the rank order, 
> 
> 
.

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Fig. 5.
Proadifen inhibits binding of
125I-labeled -bungarotoxin. A, proadifen
reduces the total number of binding sites labeled by
125I-labeled -BTX in a
concentration-dependent manner. Cells expressing either
fetal or adult AChR were preincubated for 40 min with the indicated
concentration of proadifen. 125I-Labeled -BTX was then
added to a final concentration of 25 nM and the incubation
continued for a further 30 min. Nonspecific binding was determined in
the presence of 300 µM d-tubocurarine. For
both adult and fetal receptors, maximal binding in the absence of
proadifen was typically 200-600 fmol/10-cm plate of cells.
B, proadifen slows the initial rate of
125I-labeled -BTX binding. Cells were preincubated with
proadifen for 40 min, after which 125I-labeled -BTX was
added to a final concentration of 5 nM. The binding
reaction was allowed to continue for 30 min. In both panels the data
are the means of three experiments. The error bars represent
the standard error.
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DISCUSSION |
The present study examines various combinations of AChR subunits
to characterize the interaction of (+)- and (
)-epibatidine with the
three types of binding sites of muscle AChRs. Our results reveal that
epibatidine is unique among AChR agonists thus far characterized in
that it binds with high affinity to 
and 
sites and low
affinity to 
sites. Further, unlike classical agonists,
epibatidine selects between these binding sites in the desensitized
state of the receptor.
Binding of agonists to the AChR can be described by the following
conventional scheme:
where R is the closed, activable state of the receptor, O is the
open state, D is the desensitized state, and A is agonist. K1 and K2 are the
dissociation constants for the activable state binding sites, and
Kd1 and Kd2 are the dissociation constants for the desensitized state binding sites, M is an
allosteric constant governing the transition between the activable and
desensitized states, and
is the channel opening equilibrium
constant (18, 19). Because apparent affinity measured in equilibrium
binding assays depends on the four intrinsic dissociation constants as well as the state equilibrium constants, such measurements do not
directly reflect intrinsic agonist affinities of a particular binding
site.
Nevertheless, estimates of parameters in the upper limb of Scheme 1 can
be derived from the kinetics of single channel currents. Single channel
kinetic analysis established that K1 and
K2 for ACh differ by 30-100-fold in fetal mouse
and Torpedo receptors (12, 20) and by 5-fold in adult human
receptors (21) but are indistinguishable in adult mouse receptors (13).
Here, we observe a similar selectivity pattern for carbamylcholine
binding to mouse intracellular complexes: 
and 
complexes
bind agonist with high affinity but 
complexes bind agonist
20-30-fold less tightly. As in previous studies (6) we find that
intracellular complexes bind agonists with Hill coefficients less than
1. Because complexes of an
and a single type of non-
subunit
should form only one class of binding site, a Hill coefficient less
than 1 suggests heterogeneous binding sites. Heterogeneity of
intracellular complexes could arise from 1) formation of tetrameric
complexes of the form
x
x, where
x is
,
, or
(22); 2) access of agonist to immature
or partially degraded complexes due to permeabilization by saponin; 3)
a fixed proportion of the receptors arrested in the low affinity
activable and high affinity open channel and desensitized states.
Despite this apparent heterogeneity, carbamylcholine selectivity
determined in our binding assay parallels selectivity of the activable
state of the native receptor determined from single channel kinetic
analysis (12, 13). The overall results suggest that the ligand
selectivities of 
, 
, and 
complexes most closely
resemble those of the corresponding closed activable binding sites in
native pentamers. Thus in activating the receptor, epibatidine likely
binds with high affinity to the 
and 
sites but with low
affinity to the 
site.
Unlike the closed activable state, the desensitized state can be
studied in isolation by measuring agonist binding in the presence of a
desensitizing agent such as proadifen (15, 16). Here we observe that
carbamylcholine does not distinguish between the desensitized 
,

, and 
binding interfaces. This is evidenced by the
monophasic binding curves of desensitized fetal and adult receptors and
the similar affinities of desensitized
2
2 and
2
2 triplet receptors. Similar results
were obtained previously for carbamylcholine and ACh (17, 23).
Epibatidine, by contrast, distinguishes between the two sites in
desensitized fetal and adult receptors.
Previous studies showed that
2
2 and
2
2 pentamers bind a range of ligands
with affinities similar to those of the two sites in native fetal
receptors (7, 17), suggesting that binding sites of subunit-omitted
receptors are close in structure to binding sites in the corresponding
native receptor. For epibatidine, we observe similar dissociation
constants for desensitized
2
2 receptors and the high affinity component of desensitized fetal receptors and similar dissociation constants for desensitized
2
2 receptors and the low affinity
components of desensitized fetal and adult receptors. These results
strongly suggest that the biphasic binding of epibatidine under
desensitizing conditions is due to intrinsic differences in the 
and 
binding sites, i.e. the different contributions
of the
and
subunits to the binding sites confer different
epibatidine affinities. A second possibility is that biphasic binding
might represent uncoupled binding sites of a single receptor type where
one component corresponds to the high affinity desensitized state and
the other corresponds to the low affinity activable state,
i.e. proadifen might eliminate the cooperativity of the
binding sites predicted by the allosteric model of Monod et
al. (24). However, this hypothesis seems unlikely because the low
affinity component in the presence of proadifen is shifted to the left
of the overall curve in its absence. In addition, uncoupling of the
binding sites by proadifen should result in a biphasic binding curve
for carbamylcholine, whereas we observe essentially monophasic binding
of carbamylcholine to both adult and fetal receptors. Thus the weight
of evidence suggests that unlike acetylcholine and carbamylcholine,
epibatidine selects between the desensitized 
and 
binding
sites in the fetal receptor and between the 
and 
binding
sites in the adult receptor. Whereas previous studies with the
competitive antagonist d-tubocurarine showed site
selectivity for desensitized Torpedo receptors (25), to our
knowledge epibatidine is the first agonist that selects between the
binding sites of the desensitized receptor.
Our preliminary experiments sought to determine the optimal
concentration of proadifen for desensitizing each receptor type. These
studies revealed additional complexities in the interaction of
proadifen with the AChR. In the absence of agonist, high concentrations of proadifen inhibit the binding of 125I-labeled
-BTX;
this inhibition manifests as decreases in both the initial rate and
maximal number of toxin binding sites. A decrease in the maximal number
of toxin binding sites suggests that proadifen shifts the receptor into
a conformation that no longer binds
-BTX. On the other hand, slowing
of the rate of
-BTX association could be due to either
noncompetitive or competitive inhibition by proadifen. If proadifen
competitively inhibits
-BTX association, one might expect a decrease
in agonist affinity as the concentration of proadifen increases.
However, we observed no decrease in agonist affinity even at the
highest concentration of proadifen. Thus if the interaction between
-BTX and proadifen is competitive, the site of interaction does not
overlap with the agonist binding site.
We observed an additional action of proadifen when we measured
epibatidine binding in the presence of varying concentrations of
proadifen. Our results establish that epibatidine binds in a biphasic
manner in the presence of proadifen. However, the apparent ratio of
high to low affinity sites varies with proadifen concentration. Whereas
for both adult and fetal receptors the fraction of high affinity sites
decreases as the proadifen concentration increases, the effects of
proadifen differ quantitatively for the two types of receptors. At 60 µM proadifen, adult receptors show approximately equally
weighted high and low affinity components, whereas fetal receptors show
about 3-fold more high than low affinity sites. In contrast, at 200 µM proadifen, fetal receptors show equally weighted high
and low affinity components, whereas adult receptors show a dominant
low affinity component. The changes in the apparent ratio of sites
indicate different proadifen sensitivities of the 
, 
, and

interfaces to the inhibitory effects of proadifen. In the fetal
receptor, the 
site is more sensitive to proadifen than the

site. Thus at low concentrations of proadifen,
-BTX binding
to the 
site is inhibited more, and the apparent weight of the

site is greater. As the proadifen concentration is increased,
-BTX binding to the 
also becomes inhibited while effects on the 
site reach a maximum. This yields equally weighted high and
low affinity components at 200 µM proadifen. Conversely,
in the adult receptor the 
site is more sensitive to proadifen than the 
site. At low concentrations of proadifen, the adult receptor shows two equally weighted components, but as the proadifen concentration is increased the weight of the high affinity 
component decreases. Further experiments are required to determine whether changes in the ratio of high and low affinity binding sites owe
to changes in the rates at which the 
, 
, and 
interfaces bind
-BTX or whether the binding sites differ in their propensity to enter a conformational state which does not bind
-BTX.
Our overall results demonstrate that epibatidine binds with unique site
and state selectivity to fetal and adult muscle AChRs. In contrast to
carbamylcholine, epibatidine selects strongly for binding sites formed
by the 
and 
subunit interfaces compared with the 
interfaces. Further, unlike carbamylcholine, selectivity of epibatidine
is maintained in the desensitized state. Structure-function studies
using epibatidine should allow identification of new binding site
determinants in the AChR thus refining our understanding of this
receptor.