From the Department of Pharmacology 0636, University of California,
San Diego, La Jolla, California 92093
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INTRODUCTION |
The nicotinic acetylcholine receptor
(nAChR)1 in muscle is a
pentamer composed of four homologous subunits present in a
stoichiometry
2

and arranged to surround a
central channel (1-3). Simultaneous occupation of agonist at the two
binding sites that reside at the interfaces of the 
and 
subunits activates the nAChR by increasing the probability of channel
opening. Continued exposure to agonist shifts the receptor into the
desensitized state characterized by a greater than two orders of
magnitude increase in agonist affinity and by a closed channel.
The extracellular domain in each subunit is formed principally from the
amino-terminal 210 amino acids; this domain is followed by four
transmembrane spanning regions. Since only a small segment between
membrane spans 2 and 3, and the very carboxyl terminus of ~8 residues
reside at the extracellular face, residues within the amino-terminal
210 amino acids should largely contribute to the agonist binding site.
Three segments of the
-subunit, residues surrounding Tyr-93, the
regions between residues 149 and 153 and between residues 180 to 200, have been shown by chemical labeling and site-directed mutagenesis to
contribute to agonist binding (2, 4). Four segments on the opposing
subunit interface of the
and
subunits (identified by residues
34, 55-59, 111-119, and 172-174 on the
subunit and corresponding
residues on the
subunit) present surfaces which also appear to
contribute to ligand binding (5-12). A model of the extracellular
portion of the nAChR based on sequence identity with proteins of known
structure and on identification of interacting residues details the
known information on the extracellular domain of the receptor (13).
Long range electrostatic attractive forces and stabilization of paired
charges through Coulombic interactions have long been known to control
binding of charged ligands to proteins. More recently, the importance
of interaction between quaternary ammonium moieties and aromatic side
chains became evident through studies of model binding sites (14) and
crystallographic studies of proteins that associate with quaternary
ammonium ligands (15, 16).
Charged residues involved in ligand binding on the
and
subunits
of the nAChR have been identified by chemical cross linking (17, 18)
and site-directed mutagenesis (7, 19). Residue proximity revealed from
cross-linking and the reduction in agonist affinity associated with
charge removal point to
Asp-174 and
Asp-180 as critical residues.
Our search for anionic residues in
-subunit identified
Asp-152 to
be at the 
and 
subunit interfaces, where it affects the
binding of agonists and competitive alkaloid antagonists as well as
subunit assembly (20). Since
Asp-152 and
Asp-174/
Asp-180 are
critical to ligand binding, yet differentially affect agonist and
antagonist binding, herein we examine the role of these residues in
conferring specificity and affecting state changes in the receptor.
These residues are compared with other anionic residues critically
positioned in the extracellular region of the receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
Carbamylcholine, suberyldicholine, decamethonium,
and d-tubocurarine were purchased from Sigma.
Dimethyl-d-tubocurarine was obtained from Lilly.
125I-
-Bungarotoxin (specific activity ~16 µCi/µg)
was a product of NEN Life Science Products.
Site-directed Mutagenesis--
cDNAs encoding mouse nAChR
subunits were subcloned into a cytomegalovirus-based expression vector,
pRBG4. Single-stranded cDNAs were prepared as described previously
(20). All mutations were introduced using single-stranded templates.
After the mutagenesis reaction, restriction fragments containing the
mutated site were subcloned into the original pBRG4 plasmid.
Confirmation of the mutations and the absence of spontaneous mutations
in the polymerase generated segment were established by dideoxy
sequencing.
Cell Transfection--
cDNAs encoding the wild type and
mutant subunits were transfected into human embryonic kidney (HEK-293)
cells using Ca3(PO4)2 in the ratios
of
(15 µg)/
(7.5 µg)/
(7.5 µg)/
(7.5 µg) and
(15 µg)/
(7.5 µg)/
or
(15 µg).
Ligand Binding Measurements--
Cells were harvested in
phosphate-buffered saline, pH 7.4, containing 5 mM EDTA,
2-3 days after transfection. They were briefly centrifuged,
resuspended in potassium-Ringer's buffer, and divided into aliquots
for binding assays. Specified concentrations of agonists, antagonists,
and proadifen were added to the samples 20 min prior to initiating the
rate of association assay with 125I-
-bungarotoxin.
Dissociation constants of the ligands were determined from their
competition with the initial rate of 125I-
-bungarotoxin
association (21, 22).
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RESULTS |
Influence of nAChR
Subunit Mutations at Positions 99, 152, 180, and 200 on Ligand Binding--
Candidate anionic residues were
selected in three segments of the extracellular domain of the
subunit known to affect ligand binding (Table
IA). In segment A (amino acids
88-99), aspartate 99 was mutated to asparagine. In segment B (amino
acids 144-154), aspartate 152 was mutated to glutamine. Glutamine
substitution precludes insertion of a glycosylation signal. In segment
C (amino acids 180-200), two anionic residues glutamate 180 and
aspartate 200 were mutated into asparagine and glutamine,
respectively.
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Table I
A, Aligned amino acid sequences of three main ligand binding segments
in the subunit of the nicotinic acetylcholine receptor and B, four
segments conferring ligand specificity found in the / subunits
Mutations studied are denoted by arrows. Conserved residues are marked
with asterisks. Residues detected by affinity labeling are underlined.
Residues replaced by mutagenesis in referenced studies are shown in
bold type.
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Fig. 1A shows the effect of
charge neutralization of these anionic side chains. Both the
D152Q
and
D200Q mutations shift the binding curves to more than 10-fold
higher agonist concentrations, whereas
D99N and
E180N mutations
produce only slight shifts in concentration dependence and a loss of
positive cooperativity for the agonists (Table
II). For the antagonist
d-tubocurarine, the
D152Q mutation produces a reduction
of affinity similar to that seen for the agonist (Fig. 1B)
(20). In contrast, the
D200Q mutation showed only a small influence
on d-tubocurarine binding (Fig. 1B, Table II).
This was also true for the
D99N and
E180N mutations.

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Fig. 1.
Agonist and antagonist inhibition of the
initial rate of 125I- -bungarotoxin binding to cell
surface nAChRs after transfection of HEK cells. Panels A and
B, transfection with cDNAs encoding wild type ( ) and
mutant subunits, D99N ( ), D152Q ( ), E180N ( ),
D200Q ( ), along with , , and subunit cDNAs.
Panels C and D, transfection with cDNAs
encoding wild type ( ), D152Q ( ), D174N/ D180N ( ),
D152Q and D174N/ D180N ( ) along with the complementary ,
, , and cDNAs to achieve a pentameric receptor of
stoichiometry 2  .
kobs/kmax is the ratio of
initial rates for 125I- -bungarotoxin binding in the
presence and absence of the respective ligands (19). The
lines represent least squares fits of the Hill equation to
the data with the fitted dissociation constants and Hill coefficients
given in Table I (20).
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Table II
The influence of anionic residue mutations in the , , and subunits on carbamylcholine and d-tubocurarine dissociation constants
Dissociation constants were calculated from competition with the
initial rate of the 125I- -bungarotoxin binding. Receptor was
expressed as 2  ,
2 2, or
2 2 by transfection of the 4 or 3 respective sets of subunits. Kequil is an overall
dissociation constant for carbamylcholine at equilibrium.
Kd1 and Kd2 are
dissociation constants for the  and  sites for
d-tubocurarine. The ratios of dissociation constants of
mutant (mt) to wild type (wt) were calculated using an average or mean
value of at least two measurements involving separate transfections.
 G is a change of free energy of binding associated
with the mutations.
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Influence of
D174N/
D180N Mutations on Ligand
Binding--
Asp-174 and
Asp-180 are situated in the most
carboxyl-terminal position of the four segments in the
and
subunits known to contribute to ligand binding (Table IB). After
cotransfection of
,
,
, and
subunits carrying the
D174N/
D180N mutations, the binding curve for the agonist
carbamylcholine shifts over two orders of magnitude to higher
concentration (Fig. 1C), whereas less than a 10-fold shift
is seen for the antagonist d-tubocurarine (Fig.
1D). To distinguish between the two binding sites, subunits containing
D174N or
D180N were expressed as pentamers of either
2
2 or
2
2 subunit composition on the cell
surface by transfection of the requisite three cDNAs; these subunit
arrangements possess apparently equivalent subunit interfaces for
binding (
2
2 with two
-
sites,
2
2 with two
-
sites). A
reduction in agonist affinity that is greater than those for
antagonists is again evident with these unnatural subunit compositions
(Fig. 2). The loss of cooperativity on
agonist binding for
2
2 pentameric
receptor was also evident in previous studies (23), and likely reflects the diminished capacity of this subunit combination to elicit state
transitions in response to agonists. Binding of
d-tubocurarine is largely unaltered by these mutations with
the
2
2 and
2
2 subunit combinations. As expected
for a pentamer containing two equivalent sites, the respective Hill
coefficients also approach 1.0.

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Fig. 2.
Agonist and antagonist inhibition of binding
of 125I- -bungarotoxin to cell surface nAChRs expressed
as 2 2 (panels A and
B) or 2 2 (panels
C and D). Open circles represent wild type
and filled circles represent mutant receptor; D174N in
panels A and B, and D180N in panels
C and D. The analysis of data is similar to Fig.
1.
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Ligand Specificity of
D152Q and
D174N/
D180N Receptor
Mutations--
The bis-quaternary agonist, suberyldicholine, shows a
reduction in affinity similar to carbamylcholine for the charge
neutralization mutations in the
and the
/
subunits, 10-fold
for the
D152Q mutation and about 100-fold for the
D174N
or the
D180N mutation (Table III).
Thus, mono- and bis-quaternary agonists are not differentially affected
by these mutations. For bis-quaternary antagonist, dimethyl d-tubocurarine, the
D152Q mutation showed over a 10-fold
reduction in affinity, whereas the
D174N or the
D180N mutation
showed only a 3-4-fold reduction; values similar to
d-tubocurarine, which contains single cationic tertiary and
quaternary moieties. For the bis-quaternary partial agonist,
decamethonium, the loss of affinity with the mutations falls between
d-tubocurarine and suberyldicholine, but somewhat closer to
the antagonist (Table III).
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Table III
The effect of mutations, D152Q, D174N/ D180N, and
D174Y/ D180Y on the dissociation constants of a bisquaternary
agonist, partial agonist, and antagonist
Dissociation constants were calculated as Table I. Receptors were
expressed as 2  on the cell surface.
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Combining the Mutations of
D152Q and
D174N/
D180N--
Upon expression of receptor containing charge
neutralization mutations on both the
and the
/
subunits, the
change of free energy (
G), between the double subunit
mutant combining
D152Q with
D174N/
D180N and wild-type receptor
for carbamylcholine is in close accord with the sum of

G calculated from separate mutations on the individual
subunits contained in the pentameric receptor (Fig. 1C;
Table II). For d-tubocurarine, 
G between the wild-type receptor and the receptor with combined
and
/
substitutions is also in close accord with the sum of

G values calculated for two respective receptors with
individual substitutions in the two subunits (Fig. 1D; Table
II). These results suggest that the charged residues,
D152Q and
D174N/
D180N, confer independent, noninteracting electrostatic
contributions to the binding site.
Influence of the
D152Q and
D174N/
D180N on Receptor
Desensitization--
The selectivity of the
D174N/
D180N
mutations for agonist, but not antagonist, affinity might be a
consequence of these residue positions affecting the characteristic
state transitions associated with the binding of agonists, but not
antagonists. When agonists are allowed to equilibrate the receptor, the
overall equilibrium constant (Kequil) reflects
binding to at least three discrete receptor states: activable, active
(open channel), and desensitized. The last state exhibits the highest
affinity for agonists, and allosteric ligands such as local anesthetics
promote a shift in the equilibria between states so that the receptor
resides primarily in the desensitized state (24-30). At saturation
with local anesthetics, such as proadifen (27), the apparent
dissociation constant for carbamylcholine is equivalent to that for the
desensitized state (KR').
We examined the possibility that a diminished capacity for
desensitization was responsible for the apparent reduction of agonist affinity associated with the charge neutralization on the receptor. Fig. 3 shows binding profiles for the
agonist carbamylcholine at various concentrations of a local
anesthetic, proadifen. Upon increasing the concentration of proadifen,
the binding curves shift to toward lower concentrations of
carbamylcholine. The degree of enhancement of affinity by proadifen can
be monitored from the shift in the family of carbamylcholine binding
curves. In both wild type and two mutant receptors (
D152Q and
D174N/
D180N), 10 µM proadifen is nearly sufficient
for saturation (Fig. 3, panels A-C).

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Fig. 3.
Carbamylcholine inhibition of
125I- -bungarotoxin binding to surface membrane nAChRs in
the presence of various concentrations of proadifen. Panel
A, wild type receptor 2  ; panel
B, ( D152Q)2  ; panel C,
2 D174N/ D180N; panel D,
( D200Q)2  ; and panel E,
2 D174Y/ D180Y. Proadifen concentrations are 1 µM ( ), 10 µM ( ), 30 µM
( ), 90 µM ( ), and 200 µM ( ), no
proadifen ( ). The analysis of data is similar to Fig. 1.
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Similar degrees of shift of carbamylcholine affinity in the presence
and absence of proadifen are observed for the wild type (Kequil/KR' = 120),
D152Q
(Kequil/KR' = 88) and
D174N/
D180N (Kequil/KR' = 130) mutant receptors (Fig. 4).
Therefore, the
D152Q and the
D174N/
D180N mutants retain their
capacity for desensitization or conversion to a high affinity state,
and the large influence of the charge neutralization mutation on
agonist binding does not arise from a compromised capacity of the
mutant receptor to desensitize. Furthermore, the concentration
dependence for proadifen in effecting the increase in carbamylcholine
affinity for the
D152Q and the
D174N/
D180N mutant receptors
are superimposable with that found for wild-type receptor (Fig. 4,
panels A-C).

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Fig. 4.
Ratios of carbamylcholine dissociation
constants in the absence and presence of proadifen. Panel A,
wild type receptor 2  ; panel B,
( D152Q)2  ; panel C,
2 D174N/ D180N; panel D,
( D200Q)2  ; and panel E,
2 D174Y/ D180Y. Data are derived from the family
of curves similar to those shown in Fig. 3.
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Influence of
D200Q Mutation on Receptor Desensitization--
A
previous study of the permeability response of the mouse nAChR
expressed from mRNAs encoding the individual subunits when injected
into Xenopus oocytes showed a only minor influence of
D200N on the concentration dependence for activation and functional antagonism (8). Our study shows a larger influence of this mutation on
carbamylcholine binding at equilibrium, over one order of magnitude
reduction in affinity (Fig. 1). d-Tubocurarine affinity is
less affected by this mutation (Fig. 1). Exposure of the ligand to the
receptor prior to assay allows all of the receptor states to
equilibrate in the binding measurement, whereas the concentration dependence of the permeability response does not reflect the fraction of receptor in the desensitized state. Therefore, transitions to the
desensitized states may explain the difference between the two
studies.
For the
D200Q mutant, the ratio of dissociation constants
(Kequil/KR' = 22) (Figs. 3
and Fig. 4, panel D) is significantly reduced compared with
wild-type and the other receptor mutations with charge neutralization
in the
or
/
subunits. The proadifen concentration dependence
also increases where half-maximal conversion occurs at 61 µM, a value more than 10-fold higher than for wild type
(Figs. 3D and 4D). These data indicate that the
capacity for desensitization and for conversion to the high affinity
state contributes to the overall dissociation constant measured at
equilibrium and may well account for the interesting differences seen
for this mutant between the previous measurements of permeability (8)
and our binding studies.
Substitutions of Tyrosine for the Anionic Residues in the
and
/
Subunits--
Since both anionic and aromatic residues are
known to stabilize complexes of quaternary ligands and their binding
sites, we determined whether substitutions of tyrosine for the charged
residues would still result in a binding site that accommodates the
bound agonist and antagonist. Although capacity for surface expression of receptor with the
D152Y mutation is about 30% of that of its
D152Q mutation, ligand binding affinities for carbamylcholine and
d-tubocurarine are similar to those seen with
D152Q (Fig. 5). The
D174Y/
D180Y mutation also
results in a loss of affinity for carbamylcholine, but leads to an
enhanced affinity for d-tubocurarine (Fig. 5; Table II).
This increase in affinity is also seen for structurally related
bisquaternary antagonist, dimethyl d-tubocurarine (Table
III).

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Fig. 5.
Ligand inhibition of the initial rate of
binding of 125I- -bungarotoxin to cell surface
nAChRs. Wild type ( ), D152Q ( ), D152Y ( ),
D174N/ D180N ( ), and D174Y/ D180Y ( ) mutant receptors.
All nAChRs are expressed as 2  and carry wild
type sequences at the nonmutated positions. The analysis of data is
similar to Fig. 1.
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The Influence of Proadifen on the
D174Y/
D180Y Mutant
Receptor--
The profile of binding curves for carbamylcholine in the
presence of proadifen differs between the
D174Y/
D180Y and
D174N/
D180N (Fig. 3, panels E and C).
Proadifen at 1 µM does not affect carbamylcholine binding
and only at 10 µM gives a shift in the binding profile comparable to that found for wild type and the
/
D174/D180N mutant receptors at 1 µM proadifen. The proadifen concentration
which gives a half-maximal shift in carbamylcholine affinities is
nearly an order of magnitude greater than the wild-type receptor (Fig. 3, A and E). Moreover, the magnitude of proadifen
elicited shift in carbamylcholine binding to higher affinity for the
D174Y/
D180Y mutant is approximately 4 times smaller than that
found for wild-type receptor.
 |
DISCUSSION |
Hydrophobic and Electrostatic Forces Affecting Ligand-nAChR
Complexes--
The forces involved in stabilization of quaternary
ammonium complexes with macromolecules have been studied for nearly a
half-century and reveal both hydrophobic and electrostatic
contributions to their stabilization (31). Electrostatic forces have
been quantified by ionic strength masking of rates of association
enhanced by electrostatic attraction or slowed by repulsion. Apart from
long range forces generated from an electrostatic dipole in the binding site, charge may serve to stabilize particular orientations of the
bound ligand or to entrap the ligand in the target site (16, 32).
Recent studies with model compounds and crystallographic studies of
complexes with quaternary ligands also show the importance of aromatic
residues in interacting with cations through an ion quadripole
interaction (14-16). Perspectives on the importance of charged
versus aromatic amino acid side chains depend in large part
on the measurement technique. Photolytic labeling of the nAChR reveals
the involvement of proximal aromatic residues in binding since those
very residues are susceptible to photolysis (33). The crystal
structures of the phosphorylcholine antibody (15) and
acetylcholinesterase (16) show their binding sites for quaternary
ligands to be heavily surrounded with aromatic residues. Nevertheless
strategically placed charges, where longer range electrostatic forces
(1/r in coulombic interactions versus 1/r6 and 1/r3 for
hydrophobic dispersion forces and ion-quadripole interactions), appear
important for stabilization of these complexes.
Formation of site-specific mutants of the cholinesterase have clearly
delineated the importance of both aromatic and charged residues on the
enzyme in stabilizing quaternary ions (34, 35). With the nAChR,
chemical cross-linking (17, 18) and subsequently mutagenesis studies
have pointed to the cationic residue, on the
subunit Asp-174 (7),
and the corresponding residue Asp-180 (19), on the
subunit, to lie
within 10 Å of cysteine 192 or 193 of the
subunit and to be
critical for the binding of agonists.
Martin and Karlin (23) have introduced several mutations at the
Asp-174 and
Asp-180 positions and have analyzed the electrostatic and steric influences of these residues on ligand binding and activation. Their studies, conducted by co-injection of mouse subunit
receptors into Xenopus oocytes, also revealed that these mutations primarily decreased agonist, rather than antagonist affinity
(19, 23), and that the loss of affinity was observed equivalently in
receptor states involving activation and desensitization (23). Other
studies analyzing the electrostatic potential by introduction of
charged methanethiosulfonates at cysteines are consistent with an
agonist binding site containing 2-3 negative charges (36). Elimination
of the charge on
Asp-152 substantially reduces both agonist and
antagonist affinities (20). Thus, distinctions between the charged
residues at
152,
/
-174/180, and
200 are evident, for the
charge at
152 influences the binding of both agonists and
antagonists, whereas the charges on
174,
180, and
200
selectively influence agonist binding.
Distinguishing the Role of Anionic Charges in Ligand Binding to the
nAChR--
Charge neutralization in the vicinity of a ligand
recognition site and subunit interface can be expected to exert an
influence on ligand selectivity and subunit assembly by multiple
mechanisms. Indeed the mRNA encoding the
D174N mutation, when
co-injected with mRNAs encoding the
and
subunits, did not
result in expressed receptor on the Xenopus oocyte surface
(19); yet we find upon cDNA transfection into mammalian cells, the
combination of
2
(
D174N)2 yields an
expression level similar to the wild-type
2
2. The
D152N mutation inserts an
additional glycosylation signal in the sequence and increased
glycosylation of the
-subunit receptor expressed in mammalian cells
(20). This mutation yields diminished receptor expression which appears
to be a consequence of compromising the initial dimer formation between
the
and
subunits, and particularly the
and
subunits.
Since
D152Q and especially
D152Y also yield diminished receptor
expression when cotransfected with the complement of other subunits,
this perturbation of subunit assembly can not be attributed solely to
steric hindrance by added oligosaccharide. Assembly of the pentamer can
be monitored by sedimentation analysis, but even when assembly occurs,
slight alterations in residue apposition at the subunit interface may influence the capacity of the receptor to undergo state changes. Diminished cooperativity seen with receptors of
2
2 and
2
2 compositions with Hill slopes less
than unity (28) are indicators of either a compromised state transition
or a lack of identity of the two binding sites in these receptors
assembled from three distinct subunits.
To underscore the differences in agonist and antagonist selectivity
seen between charge neutralization at
152 and at
/
174/180, we
examined these mutations in combination and by substitution of tyrosine
for the charges. The influence of the two charges on distinct subunits
for both agonist and antagonist binding shows a summation of free
energy contributions, presumably reflecting the independent influence
of the charges of the receptor on the ligand and, for agonists, the
preservation of cooperative state transitions in these particular
mutants.
Agonist binding is compromised by substitution of tyrosine for the
diacidic amino acid at
152 and
174/
180, but antagonists show
an enhanced affinity with the substitutions of tyrosine at
174/
180. This is of particular interest since
d-tubocurarine is a rigid molecule with a fixed distance of
10.5 Å between the two cationic nitrogen centers and distinct
hydrophilic and hydrophobic surfaces (37). Hence, the
d-tubocurarine, with its multiple aromatic moieties, may
actually bind in a different orientation on the receptor where tyrosine
has replaced aspartate in the
and
subunits. For agonists, it
appears on basis of proadifen sensitivity that at least part of the
affinity reduction seen for carbamylcholine when tyrosine is
substituted at
174 and
180 arises from the compromised state
transition elicited by the agonist.
Mutations in Relation to Multiple States of the Receptor--
Our
studies also reveal that elimination of charge of the various anionic
residues can affect agonist binding by influencing the degree of
desensitization. A multistate scheme where two agonists, L,
and one heterotropic ligand, H, bind to the receptor is
shown in Scheme 1.
In equilibrium binding for agonists, the observed dissociation constant
at equilibrium (Kequil), not only reflects the
intrinsic dissociation constants for the activable and desensitized
states, KR and KR', respectively,
but is influenced by the allosteric constant, M
(=R'R'/RR), for the state transitions between the low
affinity, activable state and desensitized state (24-30). For
simplicity, the channel opening constant,
/
, for the doubly
liganded species, LRLR, has been omitted. A heterotropic ligand, such as proadifen, will bind to a distinct site. Its relative affinities for the R'R', and RR receptor states
will influence the ratio of high affinity, desensitized to low
affinity, activable states of the receptor.
Fractional occupation of the agonist (L), carbamylcholine in
our case, can be described in the absence of H by
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(Eq. 1)
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A heterotropic ligand H, such as proadifen, may be
described in terms of altering the allosteric constant M, to
yield an apparent constant, M'
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(Eq. 2)
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KR, H and KR', H represent
dissociation constants of the heterotropic ligand for the R
and R' state of the receptor. Previous studies have shown
that proadifen and related ligands preferentially associate with the R' receptor state at a site distinct from the agonist site
(25). This shifts the equilibrium toward the high affinity state. Since KR', H
KR, H, in the
presence of saturating concentrations of heterotropic ligand such as
proadifen, Kequil should approach
KR'. Table IV shows a
tabulation of constants employing Scheme 1. The data reveal differences
between the various mutations where an anionic charge is eliminated.
The
D152Q and
D174N/
D180N mutations yield receptors which have similar shifts to the high affinity state with proadifen and similar concentration dependencies. By contrast,
D174Y/
D180Y and
D200Q show smaller shifts induced by proadifen and require larger
concentrations of proadifen to affect the shifts.
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Table IV
Influence of proadifen on carbamylcholine binding to wild-type and
mutant acetylcholine receptors
The data are fit to Equations 1 and 2 in the text. M is the
allosteric constant.
[C]H=0/[C]H
is the ratio of carbamylcholine concentrations giving equivalent
occupancies in the absence and presence of saturating concentration of
proadifen. [H0.5] is the concentration of
proadifen producing a half maximal shift in the carbamylcholine
concentration dependence (Fig.
4).
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When the ligand concentration, [L] is equal to
Kequil,
is 0.5. Under
this condition, Equation 1 can be reduced to
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(Eq. 3)
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A previous study estimated KR for
carbamylcholine and the wild type receptor to be 6 × 10
5 (21). Using this value, M for wild type
receptor is calculated to be 6.6 × 10
5, a value
similar to 1.0 × 10
4 derived from mouse receptors
in BC3H-1 cells (25). Although we have no data on KR
for
D174N/
D180N mutant, a dose-response analysis for
acetylcholine with this mutant receptor showed activation to occur at a
140-fold higher concentration (23). Since the difference in
concentrations for activation and binding is similar for acetylcholine
and carbamylcholine (38), KR for
D174N/
D180N
can be approximated to 8.2 × 10
3 M.
Then M may be calculated to be 5.6 × 10
5, a value also very close to the constant for wild
type receptor. Furthermore, the proadifen concentration dependence of
the
D174N/
D180N mutant receptor is superimposable with that of
wild type (Fig. 4, panels A and C), suggesting
the allosteric constant M in Scheme 1 is similar for this
mutant and wild type receptor.
The
D200Q mutation shifts the proadifen concentration dependence to
higher concentration as well as diminishes the total capacity for
desensitization. Hence,
Asp-200 appears crucial for not only for
receptor channel opening (39), but also for achieving full
desensitization. The lack of influence of
D174N/
D180N mutations
on the capacity to desensitize shown previously (23) and in these
studies suggests that these charges are not required for the state
transition associated with desensitization. Hence, the overall
dissociation constant measured for agonists at equilibrium is a
composite of various contributory equilibria between receptor states.
For agonists, at least three states (activable, open channel, and
desensitized) are involved and the charge mutations differentially affect the various equilibria.