From the Department of Biochemistry, Microbiology,
and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
and the § Departments of Anesthesiology and Pharmacology,
Texas Tech University Health Sciences Center,
Lubbock, Texas 79430
Received for publication, August 4, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The structural changes induced in the nicotinic
acetylcholine receptor by two noncompetitive channel blockers,
proadifen and phencyclidine, have been studied by infrared difference
spectroscopy and using the conformationally sensitive photoreactive
noncompetitive antagonist
3-(trifluoromethyl)-3-m-([125I]iodophenyl)diazirine.
Simultaneous binding of proadifen to both the ion channel pore and
neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm The magnitude of the cation flux response elicited by
acetylcholine at the postsynaptic membrane is dependent upon a variety of factors. These factors include both the number of nicotinic acetylcholine receptors
(nAChRs)1 present in the
postsynaptic membrane and the proportion of these receptors that exist
in active versus inactive conformations. For example, the
nAChR in native Torpedo membranes exists in at least two
distinct conformations: a low affinity closed (resting) and a high
affinity inactive (desensitized) state. In the absence of
acetylcholine, the equilibrium between these two strongly favors the
resting state with only ~20% adopting the desensitized conformation (1-3). Prolonged exposure to acetylcholine, however, shifts the equilibrium in favor of the desensitized conformation, thus diminishing the postsynaptic response. The magnitude of the cation flux can be
modulated further by endogenous factors including receptor phosphorylation (4, 5) and membrane lipid composition (6), which
influence the proportion of nAChRs in the resting versus desensitized state and/or the kinetics of the resting-to-desensitized conformational transition.
The flux response elicited by acetylcholine is also affected by a class
of structurally diverse exogenous compounds collectively referred to as
noncompetitive blockers (NCBs). NCBs of the nAChR include both general
and local anesthetics, the hallucinogenic drug phencyclidine
hydrochloride (PCP), and the frog toxin histrionicotoxin (7, 8). These
compounds sterically inhibit cation flux through the nAChR. In some
cases, they also modulate the affinity of the nAChR for acetylcholine.
PCP and the local anesthetics dibucaine, prilocaine, lidocaine, and
proadifen increase the affinity of the nAChR for acetylcholine and are
thought to stabilize the desensitized state (9, 10). Other local
anesthetics such as tetracaine and adiphenine decrease the affinity of
the nAChR for acetylcholine and are thought to shift the equilibrium in
favor of the resting state (11). Most NCBs also bind with comparable
affinity to the neurotransmitter site as well as to numerous low
affinity sites on the periphery of the nAChR (9). The conformational effects, if any, that result from binding to the latter are not well characterized.
We previously examined the structural consequences of local anesthetic
binding to the nAChR using FTIR difference spectroscopy (12). The
difference between spectra of the nAChR recorded in the presence and
absence of the agonist carbamylcholine (referred to as a Carb
difference spectrum) exhibits a complex pattern of positive and
negative bands that provides a spectral map of the structural changes
that occur upon Carb binding and desensitization (12-14, 25). Carb
difference spectra recorded in the presence of the desensitizing local
anesthetics dibucaine, prilocaine, and lidocaine all exhibit a pattern
of band intensity changes that is consistent with the formation of a
desensitized nAChR. The spectral changes, however, indicate that local
anesthetic-induced desensitization occurs upon binding to the
neurotransmitter as opposed to the NCB site. The data also suggest that
NCB-site binding may lead to the formation of a conformation that is a
structural intermediate between the resting and desensitized states, as
opposed to the normally assumed desensitized state. Unfortunately, the overlapping binding affinities of the studied local anesthetics for the
NCB and neurotransmitter binding sites prevented an unequivocal assessment of the structural changes elicited upon binding specifically to the ion channel pore.
The potential existence of a conformation that is a structural
intermediate between the resting and desensitized states has important
implications for our understanding of both nAChR conformational equilibria and the mechanisms of agonist-induced conformational change.
An intermediate conformation in vivo could also play a role
in the modulation of a postsynaptic response. To determine unequivocally whether or not such a conformational intermediate does
exist, we probe here using both FTIR difference spectroscopy and the
conformationally sensitive chemical probe, [125I]TID, the
structural changes induced in the nAChR by two NCBs, proadifen and PCP,
that have distinct affinities for the NCB and neurotransmitter binding
sites. Our data show conclusively that the binding of either proadifen
or PCP to the NCB site leads to the formation of a conformation that is
distinct from either the resting or the desensitized states of the
nAChR. In contrast, binding to the neurotransmitter site leads to full
desensitization. A revised model of nAChR conformational equilibria is presented.
Sample Preparation--
NAChR-rich membranes were prepared by
sucrose density centrifugation as described by Chiara and Cohen (15).
Affinity purification and reconstitution of the nAChR into membranes
composed of 3:1:1 egg phosphatidylcholine:dioleoylphosphatidic
acid:cholesterol was as described by McCarthy and Moore (16). The
Torpedo electric organ was purchased from either Aquatic
Research Consultants (San Pedro, CA) or Marinus, Inc. (Long Beach, CA).
Egg phosphatidylcholine and dioleoylphosphatidic acid were both from
Avanti Polar Lipids (Alabaster, AL). Cholesterol, Carb, and dibucaine
were from Sigma. PCP was either a gift from Health Canada or was
purchased from Sigma/RBI
FTIR Difference Spectroscopy--
FTIR spectra were recorded
using the ATR technique on an FTS-40 spectrometer equipped with a DTGS
detector. Each spectrum was recorded at 8 cm
Carb difference spectra were recorded as described in detail elsewhere
(12). Briefly, two consecutive resting state spectra of an nAChR film
deposited on the surface of a germanium internal reflection element
were recorded in the absence of Carb with buffer (250 mM
NaCl, 5 mM KCl, 2 mM MgCl2, 3 mM CaCl2, and 10 mM Tris, pH 7.0)
flowing continuously through the sample compartment of the ATR cell at
a rate of ~1.5 ml/min (see schematic in Ref. 12). The flowing
solution was switched to an identical one containing 50 µM Carb and after 1 min a spectrum recorded of the
desensitized state. The differences between both the two resting state
spectra (absence of Carb; control spectra) and the consecutive resting and desensitized (presence of Carb) state spectra were calculated, stored, and the flowing buffer switched back to buffer without Carb.
After a 20-min washing period to remove Carb from the film and convert
the nAChR back into the resting conformation, the process was repeated
many times.
Both proadifen- and PCP-induced structural changes in the nAChR were
monitored by recording Carb difference spectra while maintaining the
nAChR in continuous contact with either proadifen or PCP (Fig. 1,
bottom schematic). The structural changes that result from PCP binding to the NCB site were also monitored by recording spectra of the nAChR in the presence and absence of PCP,
referred to as PCP difference spectra. The PCP difference spectra were
recorded as described above for the Carb difference spectra, except
that PCP was used instead of Carb to induce the conformational change
(Fig. 1, middle schematic).
The measurement of difference band intensity changes for plotting
dose-response curves is difficult because of base-line distortions that
can occur in the 1500-1800 cm [125I]TID Labeling of the nAChR--
For
photolabeling experiments, Torpedo nAChR-rich membranes (1 mg/ml in Vesicle Dialysis Buffer (10 mM MOPS, 100 mM NaCl, 0.1 mM EDTA, and 0.02%
NaN3, pH 7.5) were incubated for 2 h at room
temperature with [125I]TID (0.4 µM) in the
absence or presence of 400 µM Carb, or PCP at various
concentrations. In some experiments nAChR-rich membranes were
pre-equilibrated with 10 µM FTIR Difference Spectra--
The difference between infrared
spectra of the nAChR recorded in the presence and absence of the
agonist Carb exhibits a complex pattern of positive and negative bands
that provides a vibrational map of the Carb-induced structural change
(Fig. 1, top trace) (13, 25). These difference bands reflect specifically the vibrations of
nAChR-bound Carb, as well as vibrational changes associated with both
the formation of physical interactions between Carb and the nAChR and
the Carb-induced resting-to-desensitized conformational transition
(Fig. 1, top schematic) (6, 12, 24, 25).
A vibrational map of NCB-induced structural change could similarly be
obtained by calculating the difference between spectra of the nAChR
recorded in the presence and absence of a NCB (referred to as a NCB
difference spectrum; Fig. 1, middle schematic).
Unfortunately, the bands of interest in NCB difference spectra are
usually masked by overlapping bands due to the partitioning of the NCB
into the lipid bilayer and a consequent expansion of the nAChR film on the germanium optical element, as shown for the NCB dibucaine in Fig. 1
(middle trace; note that PCP is an exception, see
below). An alternative approach is to record Carb difference spectra
while maintaining the nAChR in continuous contact with the NCB of
interest, as shown for dibucaine (Fig. 1, lowest
schematic and trace). Although the resulting
difference spectra do not provide a direct map of the NCB-induced
structural change, they are informative because NCBs influence both the
equilibrium between the resting and desensitized states and in turn the
affinity of the nAChR for Carb. As Carb difference spectra exhibit
bands that reflect both the resting-to-desensitized conformational
change and the formation of physical interactions between Carb and the
nAChR, NCB-induced variations in Carb difference band intensity provide
insight into the structural basis of NCB action at the nAChR.
Proadifen--
Carb difference spectra recorded in the presence of
proadifen exhibit changes in the intensities of a number of difference bands that reflect structural consequences of proadifen binding to the
nAChR. In particular, proadifen leads to dose-dependent decreases in the intensities of five bands, centered near 1663, 1655, 1547, 1430, and 1059 cm
The binding of proadifen to the NCB site leads to a loss of intensity
in three of the five conformationally sensitive bands centered near
1663, 1547, and 1059 cm
In contrast, positive band intensity centered near 1655 and 1430 cm
Higher concentrations of proadifen lead to additional changes in the
Carb difference spectra, although these are slightly distorted by a
broad positive artifact between 1700 and 1500 cm
The decrease in the 1655/1637 cm
Note that the proadifen-sensitive vibration near 1547 cm PCP--
Additional evidence for the existence of a conformational
intermediate between the resting and desensitized states of the nAChR
was obtained from both Carb difference spectra recorded in the presence
of PCP and direct PCP difference spectra. PCP binds to the pore of the
ion channel with dissociation constants of 1-6 µM,
whereas binding to the neurotransmitter site is relatively weak
(Kd ~ 250 µM) (9, 18).
Carb difference spectra recorded while maintaining the nAChR in
continuous contact with PCP at concentrations consistent with NCB site
binding exhibit the same spectral changes observed upon proadifen
binding to the NCB site, although the changes are less pronounced (Fig.
4). The presence of PCP reduces both the
1668/1680 cm
The difference between spectra of the nAChR recorded in the presence
and absence of PCP, referred to as a PCP difference spectrum, probes
directly the vibrational/structural changes that occur upon PCP binding
to the nAChR (Fig. 1, middle schematic). PCP difference spectra exhibit three relatively intense bands located near
1663, 1643, and 1547 cm
The appearance of a strong positive difference band near 1663 cm Effect of PCP on [125I]TID Labeling of the Resting
nAChR Channel--
The conformational changes in the nAChR elicited by
NCB binding to the ion channel pore were investigated further using the uncharged photoreactive compound [125I]TID.
[125I]TID is a potent NCB of the nAChR, binding with
micromolar affinity to both the resting and desensitized state of the
receptor (18, 19). In the resting state, [125I]TID
specifically photolabels homologous aliphatic residues at positions 9 and 13 in each channel-lining M2 segment (e.g.
To assay the effect of both proadifen and PCP on
[125I]TID incorporation into the resting nAChR,
nAChR-rich membranes were equilibrated in the absence of agonist, with
[125I]TID and increasing concentrations of both NCBs.
Following irradiation and SDS-PAGE, all four nAChR subunits were
efficiently labeled in the absence of NCB with 4-fold greater labeling
of the Numerous studies have shown that a variety of NCBs modulate nAChR
conformational equilibria by binding to sites within the ion channel
pore. Proadifen and PCP, etc., bind to histrionicotoxin-sensitive sites
in the ion channel and stabilize a high affinity agonist binding
conformation. Others, such as chlorpromazine and dimethisoquin, bind to
histrionicotoxin-insensitive sites in the ion pore with similar effects
on agonist binding affinity. In contrast, binding of the NCB tetracaine
to the ion channel leads to the formation of a low affinity agonist
binding state. Most blockers also bind to the neurotransmitter sites as
well as to numerous low affinity sites on the nAChR with unclear
effects on agonist binding affinity.
The changes in agonist binding affinity elicited upon NCB binding to
the ion channel pore are usually interpreted in terms of a two-state
conformational model. This model is based on the fundamental assertion
that in the absence of bound ligands the nAChR exists in equilibrium
between two conformations, a low affinity agonist binding resting and a
high affinity agonist binding desensitized state. The equilibrium
between these two strongly favors the resting state with only ~20%
of the nAChRs adopting a desensitized conformation (1-3). Blockers
such as proadifen, PCP, chlorpromazine, and dimethisoquin, etc., which
stabilize a high affinity agonist binding conformation, are thought to
shift the equilibrium in favor of the desensitized conformation (9,
10). Conversely, those such as tetracaine, which stabilize a low
affinity agonist binding conformation, are thought to shift the
equilibrium in favor of the resting state (11).
The data presented here indicate a more complex model of NCB action and
illustrate a rich conformational diversity of the nAChR (Fig.
8). As a starting point for the
interpretation of our data, we have also made the assumption that in
the absence of bound ligand the nAChR exists in an equilibrium between
two conformations, the resting and desensitized states. Our data
indicate that "desensitizing" NCBs bind to the ion channel pore and
stabilize a conformation intermediate between the resting and
desensitized states that must, based on previous studies (9, 10), bind acetylcholine with a high affinity. In contrast, sensitizing NCBs bind
to the ion channel pore and stabilize a complimentary intermediate between the resting and desensitized states that binds acetylcholine with low affinity. In addition, the binding of NCBs to the
neurotransmitter site leads to the formation of a fully desensitized
state, regardless of whether or not NCB-site binding favors low or high
affinity agonist binding conformations.
1 in
carbamylcholine difference spectra, suggesting the stabilization of a
desensitized conformation. In contrast, only the positive markers near
1663 and 1059 cm
1 are maximally affected by
the binding of either blocker to the ion channel pore suggesting that
the conformationally sensitive residues vibrating at these two
frequencies are stabilized in a desensitized-like conformation, whereas
those vibrating near 1655 and 1430 cm
1 remain
in a resting-like state. The vibrations at 1547 cm
1 are coupled to those at both 1663 and
1655 cm
1 and thus exhibit an intermediate
pattern of band intensity change. The formation of a structural
intermediate between the resting and desensitized states in the
presence of phencyclidine is further supported by the pattern of
3-(trifluoromethyl)-3-m-([125I]iodophenyl)diazirine
photoincorporation. In the presence of phencyclidine, the subunit
labeling pattern is distinct from that observed in either the resting
or desensitized conformations; specifically, there is a
concentration-dependent increase in the extent of
photoincorporation into the
-subunit. Our data show that domains of
the nicotinic acetylcholine receptor interconvert between the resting
and desensitized states independently of each other and suggest a
revised model of channel blocker action that involves both low and high
affinity agonist binding conformational intermediates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
resolution using 512 scans, which took roughly 7 min per spectrum. All
difference spectra were base-line-corrected between 1800 and 1000 cm
1 and were interpolated to an effective
resolution of 4 cm
1. The presented spectra
are averages of between 30 and 70 difference spectra recorded, for each
experiment, from at least two new films prepared from each affinity
purification/reconstitution.
1 region as a
result of nAChR film instability and temperature fluctuations.
Distortions are particularly evident in difference spectra recorded at
high concentrations of proadifen, because partitioning of large amounts
of this NCB into the bilayer leads to nAChR film instability. Extensive
studies have shown that the difference intensity near 1700, 1680, 1637, and 1530 cm
1 is at or near base line, as is
evident in the difference spectra recorded at low concentrations of
proadifen (Fig. 2, arrows in the second
trace from bottom). In contrast, higher
concentrations of proadifen lead to base-line distortions and an
apparent increase in intensity at each of these frequencies (Fig. 3,
arrows in the second trace from
bottom). Peak heights were measured relative to the
intensity at an adjacent frequency that is close to the base line and
that, based on experience, is not affected by NCB binding to the nAChR.
The intensity centered at 1655 cm
1 was
measured relative to 1637 cm
1 and is referred
to as the 1655/1637 cm
1 ratio. The changes in
positive intensity at 1663 cm
1 were best
quantified by measuring the changes in negative intensity near 1668 cm
1 relative to 1680 cm
1 and is referred to as the 1668/1680
cm
1 ratio. The binding of proadifen to the
neurotransmitter sites was quantified by integrating the intensity of
the negative proadifen band centered near 1740 cm
1.
-bungarotoxin for 20 min
prior to the addition of PCP, and in some experiments affinity-purified nAChRs reconstituted into asolectin-lipid vesicles were substituted for
nAChR-rich membranes. Incubations were performed in 10 × 75-mm glass culture tubes in the dark. The samples were then irradiated with
a 365-nm UV lamp (Spectroline EN-280L) for 7 min at a distance of <1
cm, the membrane suspensions transferred to 1.5-ml microcentrifuge tubes and centrifuged at 39,000 × g for 1 h.
Pellets were solubilized in electrophoresis sample buffer and nAChR
subunits resolved by SDS-PAGE (26) using 1.0-mm-thick separating gels
comprising 8% polyacrylamide, 0.33% bisacrylamide. Following
electrophoresis, gels were stained with Coomassie Blue R-250, dried,
and autoradiographs prepared using Kodak X-Omat LS film at
80 °C
in the presence of an intensifying screen (15-h exposure).
[125I]TID incorporation into AChR subunits was quantified
by excising the bands from the dried gel and determining the amount of
125I by counting in a Packard Cobra II Gamma counter. The
concentration-response data were curve-fitted by nonlinear
least-squares analysis using the graphical curve-fitting program Prism
(Graphpad Software, Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conformational changes probed by difference
spectroscopy. The difference between FTIR spectra of the nAChR
recorded in the presence and absence of Carb (top
panel on left and top
spectrum on right) exhibits features due to
nAChR-bound Carb (short dashed lines),
the formation of physical interactions between Carb and nAChR binding
site residues, and the Carb-induced resting-to-desensitized
conformational change (asterisks). A signature spectrum of
Carb is found in Baenziger et al. (13). The difference
between spectra of the nAChR recorded in the presence and absence of
200 µM dibucaine (middle panel on
left and middle spectrum on
right) exhibit similar features indicative of
dibucaine-induced structural change in the nAChR, but these are masked
by the strong absorption bands of dibucaine partitioned into the lipid
bilayer (long dashed lines) as well as
the intense negative protein and lipid bands that reflect expansion of
the nAChR film beyond the penetration depth of the infrared light from
the germanium internal reflection element. At 200 µM
concentrations, dibucaine binds to both the two neurotransmitter and
NCB sites in the ion channel pore. A signature spectrum of dibucaine is
found in Ryan and Baenziger (12). To avoid the spectral changes
associated with partitioning into the lipid bilayer, the difference
between spectra of the nAChR are recorded in the presence and absence
of Carb, but while continuously maintaining the nAChR in contact with
200 µM dibucaine (bottom panel on
left and bottom spectrum on right).
The resulting difference spectrum exhibits positive and negative
features due to the binding of Carb to and consequent displacement of
dibucaine from the neurotransmitter site, respectively
(short and long dashed
lines, respectively). Bands indicative of the
resting-to-desensitized conformational change noted with
asterisks in the top Carb
difference spectrum are absent because dibucaine
stabilizes the nAChR in a desensitized state prior to the addition of
Carb. Note that the Carb vibration near 1720 cm 1 (dashed line on
far left) in both the top and
bottom spectrum has an intensity of roughly
7.5 × 10
5 absorbance units while the
negative lipid vibration near 1740 cm
1 in the
dibucaine difference spectrum has a negative intensity of roughly
2.5 × 10
4 absorbance units.
1, which serve as
markers of the resting-to-desensitized conformational transition (12,
14). Bands centered near 1663 and 1655 cm
1
are in the amide I region (predominantly peptide C=O stretch) and are
coupled to the 1547 cm
1 amide II vibration
(predominantly peptide N-H bend). All three likely reflect a
conformational change in the polypeptide backbone upon desensitization.
The bands centered near 1430 (not shown) and 1059 cm
1 remain to be assigned, but likely reflect
a structural change in individual side chains. Spectral changes that
occur at proadifen concentrations up to 50 µM, where
binding is restricted to the NCB site (Kd ~ 3 µM), are presented in Fig.
2. Those that occur at proadifen
concentrations between 100 and 400 µM Carb, where
additional binding occurs at the neurotransmitter site
(Kd ~ 400 µM), are presented in Fig.
3 (17). The dose-dependent
spectral changes are summarized in Fig. 6.
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Fig. 2.
Selected regions of Carb difference spectra
recorded in the presence of the noted concentrations of proadifen.
At these concentrations proadifen binding is restricted to the NCB site
in the ion channel pore. The short dashed
lines denote positive intensity in the difference spectra
that reflects specifically the resting-to-desensitized conformational
change (see text). Long dashed lines denote the
frequencies at which proadifen itself absorbs infrared light. The
lowest trace is an absorbance spectrum of
proadifen recorded using the ATR technique in aqueous solution. The
absorbance due to water has been subtracted. The bar at
top right denotes the absorbance scale for the
Carb difference spectra. Arrows on the second
trace from bottom designate frequencies that are
typically at or close to the base line.
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Fig. 3.
Selected regions of Carb difference spectra
recorded in the presence of the noted concentrations of proadifen
consistent with proadifen binding to both the NCB and neurotransmitter
sites. The short dashed lines denote
positive intensity in the difference spectra that reflects specifically
the resting-to-desensitized conformational change (see text).
Long dashed lines denote the frequencies at which proadifen
itself absorbs infrared light. The negative features at these
frequencies in the difference spectra corresponds to the binding of
proadifen to the neurotransmitter site (see text and bottom
schematic in Fig. 1). The lowest trace
is an absorbance spectrum of proadifen as in Fig. 2. Arrows
on the second trace from bottom
designate frequencies which are typically at or close to the base line,
but are distorted to follow a rainbow-like pattern between 1700 and
1500 cm 1 because of nAChR film instability at
high concentrations of proadifen (see "Experimental Procedures").
The Carb difference spectrum recorded at 100 µM proadifen
(dashed line) is superimposed with a slight
y axis offset on the Carb difference spectrum recorded at
400 µM proadifen (solid line,
second trace from bottom).
1 (Fig. 2). The loss
of intensity near 1663 is best monitored as a change in the 1668/1680
cm
1 intensity ratio (see "Experimental
Procedures"). Both the decrease in the 1668/1680
cm
1 ratio and loss of intensity centered near
1059 cm
1 are maximal at 50 µM
proadifen, whereas additional changes in intensity near 1547 cm
1 are observed at higher proadifen
concentrations (see below). The decrease in the 1668/1680
cm
1 ratio and loss of intensity centered near
1059 cm
1 indicate that the conformationally
sensitive residues vibrating at 1663 and 1059 cm
1 are not able to undergo the
resting-to-desensitized conformational change upon the binding of Carb.
The simplest interpretation is that proadifen binding to the NCB site
stabilizes the conformationally sensitive residues vibrating at these
two frequencies in a desensitized conformation prior to the addition of
Carb (see below).
1 (latter not shown) is essentially
unaffected by concentrations of proadifen up to 50 µM. A
change in intensity at 1655 cm
1 is best
monitored as a change in the 1655/1637 cm
1
ratio. At 50 µM proadifen, the conformationally active
residues that vibrate near 1655 and 1430 cm
1
retain the ability to undergo a Carb-induced resting-to-desensitized conformational change and must therefore remain in a resting-like conformation despite the presence of bound proadifen. The contrasting effects of proadifen on the intensities of the conformationally sensitive bands near 1663/1059 cm
1 and
1655/1430 cm
1 suggest that the binding of
proadifen to the NCB site shifts some nAChR residues into a
desensitized-like conformation while others remain in a resting-like
state. Proadifen binding to the NCB site thus leads to the formation of
a conformation of the nAChR that is an intermediate between the resting
and desensitized states and that shares structural features in common
with both conformations.
1 due to increasing instability of the
nAChR film at higher proadifen concentrations (Fig. 3). Regardless of
these distortions, it is clear that concentrations of proadifen between
100 and 400 µM lead to a substantial decrease in the
1655/1637 cm
1 ratio and a loss of intensity
near 1547 and 1430 cm
1. These intensity
changes are most easily visualized upon superimposition of the
difference spectra recorded at 100 and 400 µM proadifen (Fig. 3, see dashed line spectrum). No
additional intensity changes were consistently detected near either
1663 or 1059 cm
1.
1 ratio and
loss of intensity near 1547 and 1430 cm
1 can
be attributed to changes in nAChR structure resulting from proadifen
binding to the neurotransmitter site. This interpretation is based on
the known Kd of proadifen for the neurotransmitter site (17) as well as the appearance of negative band intensity in the
difference spectra (Fig. 3, long dashed
lines) at frequencies that match the molecular vibrations of
proadifen itself. These negative bands reflect the competitive
displacement of proadifen from the neurotransmitter site upon the
addition of Carb (Fig. 1, see bottom schematic).
In addition, there appear to be subtle changes in the intensities of
two bands near 1620 and 1516 cm
1. The latter
spectral changes likely result from an ability of proadifen to mimic
some of the physical interactions that normally occur between Carb and
neurotransmitter binding site residues (12). The absence of positive
intensity at the five noted conformationally sensitive frequencies
centered near 1663, 1655, 1547, 1430, and 1059 cm
1 in Carb difference spectra recorded at
elevated concentrations of proadifen indicates that the additional
binding of proadifen to the neurotransmitter site completely abolishes
the ability of the nAChR to undergo the Carb-induced
resting-to-desensitized conformational transition. In agreement with
data obtained for several other NCBs (12), the binding of proadifen to
the neurotransmitter site thus stabilizes the nAChR in a fully
desensitized state.
1 is a multicomponent band that is coupled
to the two vibrations near 1663 and 1655 cm
1.
The coupling of these vibrations explains why changes in nAChR structure reflected by a loss of intensity near 1547 cm
1 occur as a consequence of proadifen
binding to both the NCB and neurotransmitter binding sites.
1 ratio and the positive band
intensity near 1547 and 1059 cm
1, but has no
effect on the 1655/1637 cm
1 ratio or the
positive band intensity near 1430 cm
1 (Fig.
6). As with proadifen, PCP binding to the NCB site stabilizes the
conformationally sensitive residues that vibrate near 1663 and 1059 cm
1 in a desensitized-like state, whereas
those that vibrate near 1655 and 1430 cm
1
remain in a resting-like conformation. Note that the PCP-induced changes in intensity near 1663 and 1059 cm
1
are, as expected, small compared with those induced by proadifen (Fig.
2). In contrast to proadifen, saturation of PCP binding to the NCB site
leads to only a modest shift of the nAChR into a high affinity binding
conformation. The relatively minor effects of PCP on both the Carb
difference spectra and nAChR agonist binding affinity is likely a
consequence of the only slight preference of PCP for the desensitized
(Kd ~ 1 µM) versus
resting (Kd ~ 6 µM) states (9,
18).
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Fig. 4.
Selected regions of Carb difference spectra
recorded in the presence of the noted concentrations of PCP consistent
with PCP-binding to the NCB site in the ion channel pore. The
short dashed lines denote positive
intensity in the difference spectra that reflects specifically the
resting-to-desensitized conformational change (see text).
1, as well as several
less intense bands in the 1500-1000 cm
1
region (Fig. 5, second
trace from bottom). The latter include vibrations
due to nAChR-bound PCP and are difficult to accurately assign due to
limited signal-to-noise. The negative and a positive amide I difference
bands centered near 1643 cm
1 and 1663 cm
1, respectively, likely result from a shift
in an amide I vibration of one or more residues from 1643 in the
absence of PCP to 1663 cm
1 in the PCP-bound
state. Both vibrations are likely coupled to the amide II difference
band near 1547 cm
1 and likely reflect a
PCP-induced change in the conformation of the polypeptide backbone.
View larger version (30K):
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Fig. 5.
Comparison of Carb difference spectra
recorded in the presence and absence of 50 µM PCP with a PCP difference
spectrum. The top trace is a Carb difference spectrum
recorded in the absence of NCB (Fig. 1, top
schematic). The second trace from top
is a Carb difference spectrum recorded in the continuous presence of 50 µM PCP (Fig. 1, bottom schematic).
The second trace from bottom is a PCP difference
spectrum (Fig. 1, middle schematic). The
bottom trace is an absorbance spectrum of powdered PCP
recorded using the ATR technique. Hydration likely leads to a
broadening of the absorbance bands in the PCP absorbance spectrum. The
short dashed lines denote the bands that reflect
the resting-to-desensitized conformational change.
1 in the PCP difference spectrum is
consistent with the loss of band intensity near 1663 cm
1 in Carb difference spectra recorded in
the continuous presence of PCP (Fig. 5). The lack of an
intense band near 1655 cm
1 in the PCP
difference spectrum is also consistent with the lack of a substantial
intensity change in this region of the Carb difference spectra (Fig.
6). These data conclusively show that PCP
binding to the NCB site leads to a subtle conformational change from
the resting state. It can thus be concluded that the conformationally sensitive residues that vibrate at 1663 cm
1
in the Carb difference spectrum are stabilized in a desensitized-like conformation upon PCP binding to the NCB site.
View larger version (23K):
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Fig. 6.
Dose-dependent effects of
proadifen and PCP on the intensities of selected bands in the Carb
difference spectra. A, dose-dependent
effects of proadifen on the 1668/1680 cm 1
intensity ratio (
), 1655/1637 cm
1
intensity ratio (
) and the intensity centered near 1740 cm
1 (
). The appearance of negative
intensity at 1740 cm
1 reflects the binding of
proadifen to the neurotransmitter site and its subsequent displacement
upon the binding of Carb. The proadifen-induced
concentration-dependent changes in intensity were
curve-fitted using nonlinear least-squares resulting in
EC50 values for the 1668/1680 cm
1
ratio and 1655/1637 cm
1 ratio of 3.2 and 140 µM, respectively. These values correspond to binding to
the NCB and neurotransmitter sites, respectively (EC50 for
1740 cm
1 is 210 µM).
B, dose-dependent effects of PCP on the
1668/1680 cm
1 ratio (
) and 1655/1637
cm
1 ratio (
). Vibrations due to PCP itself
cannot be detected in the Carb difference spectra. The calculated
EC50 value for the 1663 cm
1 band
intensity change in the presence of PCP is 12 µM.
Leu-265 and
Val-269; Ref. 20). While TID binds with equal affinity to both
the resting and desensitized channel, [125I]TID
photoincorporates into the resting channel ~10-fold more efficiently
than into the desensitized channel. In the absence of agonist the vast
majority of [125I]TID incorporation into individual
receptor subunits reflects labeling of the resting channel. The
addition of nAChR agonist or NCBs such as tetracaine reduces by greater
than 75% the incorporation of [125I]TID into receptor
subunits (20, 21).
-subunit compared with that of
-,
-, or
-subunits
(Fig. 7A, lane
1). Addition of agonist alone (400 µM
carbamylcholine) decreased the extent of incorporation of
[125I]TID into each nAChR subunit by 75% or greater
(93% for
-subunit; Fig. 7A, lane
8). Equilibration of the resting state nAChR with proadifen
led to a complete loss of [125I]TID labeling (data not
shown) likely due to competitive displacement of
[125I]TID from the ion channel pore, as has been observed
for other NCBs (21). In contrast, PCP had no detectable,
concentration-dependent effect on the extent of
[125I]TID incorporation into either the
-,
-, or
-subunits (Fig. 7A), but led to a
concentration-dependent increase in the extent of
[125I]TID incorporation into the
-subunit. The
concentration dependence of the increase in [125I]TID
labeling by PCP was quantified by excising the
-subunit bands from
the dried polyacrylamide gel and determining the amount of
125I present by
-counting. The amount of cpm
([125I]TID) associated with the
-subunit band for each
concentration of PCP was plotted as a percentage of the incorporation
detected in the
-subunit in the absence of any ligand. For the data
presented in Fig. 7B, an EC50 value of 3.9 µM was calculated for the increase in
[125I]TID incorporation into the
-subunit by PCP with
a maximal increase of 146%. Preincubation of the nAChR with
-bungarotoxin, which should prevent PCP binding to the agonist
recognition site, had no detectable effect on the ability of PCP to
increase the extent of [125I]TID incorporation into the
-subunit. Further, substitution of nAChR-rich membranes with
affinity-purified nAChRs resulted in qualitatively similar results for
the PCP-induced increase in [125I]TID incorporation into
the
-subunit.
View larger version (52K):
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Fig. 7.
Effects of PCP on the photoincorporation of
[125I]TID into the nAChR channel in the resting
state. nAChR-rich membranes were equilibrated (2 h) with
[125I]TID (0.4 µM) in the absence
(lanes 1-7) and in the presence (lane
8) of 400 µM Carb or in the presence of
increasing concentrations of PCP (lanes 2-7).
nAChR-rich membranes were then irradiated at 365 nm for 7 min, and
polypeptides resolved by SDS-PAGE. A, shown is the
corresponding autoradiograph of the gel containing the
concentration-response labeling experiments for [125I]TID
versus PCP. The positions of the nAChR subunits are
indicated on the left. B, for each concentration
of PCP, individual nAChR subunit bands were excised from the dried gel
and the amount of [125I]TID photoincorporated into each
subunit determined by counting. The amount of
[125I]TID subunit incorporation (
,
) determined for
each concentration of PCP is expressed as a percentage of the
[125I]TID subunit incorporation detected in the absence
of any ligand. No significant (>10%)
concentration-dependent difference in the extent of
[125I]TID incorporation was detected in the
-,
-,
or
-subunit (
-subunit (
)). The
concentration-dependent increase in [125I]TID
incorporation into the
-subunit (
) was curve-fitted using
nonlinear least-squares resulting in a calculated EC50
value of 3.9 µM and a maximal increase of 146%. The
dashed line indicates the amount of
[125I]TID incorporation into the
-subunit in the
presence of agonist and defines the level of nonspecific
labeling.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 8.
Schematic model of the conformational effects
of NCB binding to the nAChR. In the absence of bound ligand, nAChR
exists in an equilibrium between the low affinity resting and high
affinity desensitized states. "Desensitizing" NCBs bind to the ion
channel pore and stabilize a high affinity conformation intermediate
between the resting and desensitized states. "Sensitizing" NCBs
bind to the ion channel pore and stabilize a low affinity intermediate
between the resting and desensitized states. The binding of NCBs to the
neurotransmitter site leads to the formation of a fully desensitized
state.
Our modified model of NCB action is based on the following
observations. First, we have identified five positive bands near 1663, 1655, 1547, 1430, and 1059 cm1 in Carb
difference spectra that reflect the vibrational changes in the nAChR
that are associated specifically with the resting-to-desensitized conformational transition (6, 12, 14). Carb difference spectra recorded
in the presence of proadifen lose intensity at all five frequencies, an
indicator that the nAChR has adopted a desensitized conformation, only
at concentrations consistent with binding to the neurotransmitter sites
and where spectral features indicative of neurotransmitter site binding
are observed (see "Results"). Similarly, spectral changes
indicative of full desensitization are only observed upon the binding
of the NCBs dibucaine, prilocaine, and lidocaine to the
neurotransmitter binding sites (12). In addition, the binding of
tetracaine to the neurotransmitter site leads to spectral changes
indicative of desensitization, despite the fact that tetracaine binding
to the ion channel pore stabilizes a low as opposed to a high affinity
agonist binding conformation (12). These results illustrate that
neurotransmitter site binding is not only required but is alone
sufficient for NCBs to stabilize a fully desensitized nAChR.
Second, Carb difference spectra recorded in the presence of
concentrations of proadifen, and PCP consistent with binding
exclusively to the NCB site exhibit a loss in the intensities of only
two of the noted conformationally sensitive bands centered near 1663 and 1059 cm1, while the two conformationally
sensitive vibrations centered near 1655 and 1430 cm
1 are essentially unaffected. The loss of
intensity at both 1663 and 1430 cm
1 indicates
that the residues giving rise to these bands do not undergo the
resting-to-desensitized conformational change upon Carb binding and are
likely stabilized in a desensitized conformation prior to the addition
of Carb. In contrast, the lack of a change in intensity near 1655 and
1430 cm
1 indicates that the residues giving
rise to the latter bands remain in a resting-like conformation that can
still respond to Carb binding. PCP difference spectra, which probe
directly the PCP-induced conformational change, exhibit positive
intensity near 1663 cm
1 and possibly 1059 cm
1 with no change in intensity near 1655 cm
1, confirming that NCB site binding leads
to a structural change in only those residues that vibrate at the
former two frequencies from the resting to a desensitized-like
conformation. In addition, similar patterns of band intensity changes
are observed in Carb difference spectra recorded at concentrations of
prilocaine and lidocaine consistent with NCB site binding, although the
overlapping affinities of these two blockers for the NCB and
neurotransmitter sites make the data difficult to interpret (12).
Collectively, these results conclusively show that NCB site binding
leads to the formation of a conformation that is a structural
intermediate between the resting and desensitized states. In this
structural intermediate, some residues remain in a resting-like while
others adopt a desensitized-like conformation. This intermediate is
referred to as the high affinity intermediate.
Third, Carb difference spectra recorded in the presence of the
sensitizing NCB tetracaine at concentrations consistent with binding to
the NCB site exhibit an increase in intensity centered near 1663 and
1059 cm1 with little or no effect on band
intensity near 1655 and 1430 cm
1 (12).
Tetracaine binding to the NCB site thus influences the same residues
that are affected by proadifen and PCP binding to the NCB site binding,
but in contrast the data suggest a shift in these residues from a
desensitized-like to a resting-like conformation. This interpretation
is consistent with the decrease, as opposed to an increase in agonist
binding affinity observed upon tetracaine binding to the NCB site. It
can thus be concluded that tetracaine stabilizes an intermediate
between the resting and desensitized states that is complimentary to
that stabilized by either proadifen or PCP and that binds acetylcholine
with a low affinity. This intermediate is referred to as the low
affinity intermediate.
Finally, the [125I]TID labeling pattern of the nAChR in the presence of PCP is distinct from the [125I]TID labeling pattern observed in either the presence (desensitized conformation) or absence (predominantly resting conformation) of Carb. Although inconclusive with respect to the formation of a conformational intermediate in response to PCP binding, the [125I]TID labeling suggest the formation of a conformation that is distinct from both the resting and desensitized states and is thus consistent with the FTIR data. Note also that a similar labeling pattern has been detected in the presence of ketamine, memantine, and amantadine.2
The observation that both proadifen and PCP stabilize a conformation intermediate between the resting and desensitized states has important implications. Numerous studies have shown that the binding of either NCB to the ion channel leads to the formation of a conformation that has a high affinity for acetylcholine. In fact, increased acetylcholine binding affinity is typically used as an indicator of desensitization. Our data show that the high affinity conformation stabilized by proadifen and PCP is an intermediate between the resting and desensitized states. It can thus be concluded that an increase in acetylcholine binding affinity is not sufficient to define the desensitized conformation. More comprehensive methods are required to define the conformation status of the nAChR.
The Carb difference spectra recorded in the presence of PCP and proadifen also show that regions of the nAChR inter-convert between the resting and desensitized conformations independently of each other. Although it is tempting to suggest that this independent conformational change of domains leads to an uncoupling of the transmembrane domain from the extramembranous neurotransmitter binding site, the fact that proadifen and PCP binding to the to the ion channel alters both agonist binding affinity indicates that this is not the case (see also Ref. 22). In this light it is interesting to note that a recent linear free energy analysis of nAChR channel gating suggested that the binding affinity of the nAChR for acetylcholine increases along the pathway between agonist binding and the open state (23). The conformational intermediate detected here upon NCB binding to the ion channel pore may share structural similarities to this transient conformational intermediate.
Finally, it should be noted that the high affinity structural
intermediate between the resting and desensitized states detected in
this study is also detected in Carb difference spectra recorded from
the nAChR reconstituted into egg phosphatidylcholine membranes containing small amounts of either phosphatidic acid or cholesterol. A
structural intermediate between the resting and desensitized states may
therefore exist under a variety of physiological conditions, in both
the absence and presence of bound ligands. Conformational diversity may
play a role in the modulation of the post-synaptic response by both
endogenous and exogenous factors. Further studies are required to
define the biophysical properties of this intermediate to understand
its possible biological significance.
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FOOTNOTES |
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* This work was supported in part by grants from the Canadian Institutes of Health Research (to J. E. B.) and by National Institutes of Health NINDS Grant NS35786 (to M. P. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd., Ottawa, ON K1H 8M5, Canada. Tel.: 613-562-5800 (ext. 8222); Fax: 613-562-5440; E-mail: jebaenz@uottawa.ca.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M007063200
2 M. P. Blanton and E. A. McCardy, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: nAChR, nicotinic acetylcholine receptor; Carb, carbamylcholine; FTIR, Fourier transform infrared; PCP, phencyclidine hydrochloride; [125I]TID, 3-(trifluoromethyl)-3-m-([125I]iodophenyl)diazirine; NCB, noncompetitive blocker; MOPS, 4-morpholinepropanesulfonic acid; ATR, attenuated total reflection.
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