 |
INTRODUCTION |
The nicotinic acetylcholine receptor
(nAChR),1 a prototypical
member of the superfamily of ligand-gated ion channels, is an integral
transmembrane protein with the subunit stoichiometry
2

(1-3). Each receptor subunit contains four
hydrophobic sequences, which are presumed to span the plasma membrane
(4, 5). The large N-terminal domain and the relatively short C-terminal part of the subunits are oriented toward the extracellular side. A
large connecting loop, which is found between transmembrane sequences
M3 and M4, is assumed to extend into the cytoplasm. The five subunits
contribute their homologous M2 sequences to the formation of the ion
channel (6), which is permeable for cations upon agonist binding. A
selectivity filter formed by the five M2 helices contributes to the
cation conductance properties of the channel. Three rings of negatively
charged amino acid residues (7, 8) located at the constriction of the
channel and on the cytoplasmic and the extracellular side of this
constriction, respectively, in particular are of functional importance.
In the absence of crystals suitable for x-ray analysis, the
three-dimensional structure of nAChR is investigated mainly by three
approaches: electron microscopy (9, 10), site-directed mutagenesis in
combination with patch clamp electrophysiology (e.g. Refs.
7, 11, and 12), and affinity labeling (e.g. Refs. 1 and
13-16). Two binding sites for agonists and competitive antagonists are
located in the extracellular region, mainly on each of the two
-subunits (1) at the
-
and
-
interfaces (14, 17, 18). A
binding site for noncompetitive inhibitors (NCIs), such as
chlorpromazine, ethidium bromide, and triphenylmethylphosphonium (TPMP+), has been found within the channel lumen (6, 19).
These NCIs are assumed to enter the ion channel from the extracellular side and to bind deep in the channel lumen, thereby inhibiting the ion
flow. Photoaffinity labels derived from well characterized NCIs have
been developed to characterize the structure of the nAChR ion channel.
[3H]chlorpromazine and
[3H]TPMP+ are preferentially
photocross-linked to amino acid residues within the M2 transmembrane
sequence of the desensitized receptor, thus demonstrating that these
compounds bind deep in the ion channel and close to the selectivity
filter (6, 19).
Philanthotoxin-433 (PhTX-433) is a neuroactive, polyamine-containing
toxin found in the venom of the digger wasp Philanthus triangulum (20). Synthetic analogues of this polyamine amide, such
as PhTX-343, have been shown to noncompetitively antagonize a range of
ionotropic receptors (21), including nAChR (22-26). These low
molecular weight compounds have a hydrophobic head group linked to a
polyamine tail. At physiological pH, they are highly positively charged
and, therefore, should bind to any surface with a corresponding
distribution of anionic functionalities (21). The binding affinities of
these compounds to nAChR are significantly influenced by modifying
their structural elements (26-28). We have synthesized a series of
polyamine-containing analogues of PhTX-343 in the search for a ligand
with high affinity and specificity for Torpedo californica
nAChR for photocross-linking studies. This approach resulted in the
discovery of a photoactivable compound, MR44, which binds to the nAChR
with high affinity (26, 29).
In the present work, we showed that two molecules of MR44 bind
with high affinity in the lumen of the nAChR ion channel. Using 125I-MR44 as a photoaffinity label, we localized the site
of interaction of the aromatic head group of MR44 in the vestibule of
the ion channel. The sequence that was labeled by 125I-MR44
was found on the
-subunit close to, but not overlapping with, the
agonist-binding site. In addition, we found that bound MR44 was
displaced by luminal NCIs and calcium, suggesting that the positively
charged polyamine moiety of MR44 binds deep in the channel lumen at the
high affinity NCI site.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Liquid nitrogen-frozen tissue from T. californica was supplied by C. Winkler (Aquatic Research
Consultants, Sa Pedro). Carbamoylcholine, calcium chloride,
ethidium bromide, and HEPES were from Sigma. Dithiothreitol,
TPMP+, and chloramine T were from Aldrich.
K125I was from Amersham Pharmacia Biotech.
125I-Labeled
-bungarotoxin (
-BTX) was purchased from
PerkinElmer Life Sciences. ArgC protease, AspN protease, LysC
protease, endoglycosidase H (Endo H), and V8 protease were obtained in
sequencing grade from Roche Molecular Biochemicals (Mannheim, Germany).
Synthesis and Purification of 125I-Labeled
MR44--
MR44 was radioactively iodinated with 125I using
the chloramine T method (31). The mono-125I derivative was
isolated by reverse-phase HPLC (Waters model 626, Eschborn, Germany) on
a Vydac C18 column applying the following linear gradient
(1 ml/min): solvent A (aqueous solution containing 0.1%
trifluoroacetic acid) and solvent B (acetonitrile containing 0.085%
trifluoroacetic acid). The UV absorption of the eluent was determined
at 305 nm, and the radioactivity of each fraction was detected using a
-counter. 125I-MR44 was characterized by matrix-assisted
laser desorption-ionization mass spectrometry.
125I-MR44 Binding Assays--
AChR-rich membranes
were prepared from frozen T. californica electric organ as
described earlier (32). Increasing concentrations of
125I-MR44 (5,000 cpm/nmol) were added to a constant amount
of nAChR-rich membranes (0.3 mg/ml protein, diluted in 100 mM NaPi, pH 7.4; total volume per sample, 200 µl) and were incubated for 45 min at room temperature. Bound ligand
was separated from the free ligand by ultracentrifugation in a Beckmann
tabletop ultracentrifuge for 10 min at 80,000 × g and
4 °C. Aliquots were withdrawn prior to centrifugation to determine
the total radioactivity, and duplicate aliquots of the supernatant were
removed after centrifugation to determine the free ligand
concentration. Nonspecific binding was determined from bound
125I-MR44 in the presence of a 100-fold molar excess of
I-MR44. 125I-Labeled
-BTX binding assays have been
performed as described previously (33). Briefly, nAChR-rich membranes
(10 µg/ml) diluted in 50 mM NaCl, 0.1% (v/v) Triton
X-100, 10 mM NaPi, pH 7.5, were incubated with
increasing concentrations of 125I-labeled
-BTX (0-1
µg/ml) for 45 min at room temperature (final volume 200 µl). 50 µl (duplicates) were adsorbed to DE81 filters. The filters were
washed three times for 10 min with 50 mM NaCl, 0.1% (v/v)
Triton X-100, 10 mM NaPi, pH 7.5. The
radioactivity of 125I-labeled
-BTX bound to the filters
was determined in a
-counter. The number of
-BTX binding sites
was calculated according to Hartig and Raftery (33).
For NCI and
-BTX competition experiments, 125I-MR44 (9 µM; 5,000 cpm/nmol) and increasing concentrations of NCI
or
-BTX were added to a constant amount of nAChR-rich membranes (0.3 mg/ml protein). In the following, the samples were treated as described above.
Calcium Competition Experiments--
125I-MR44 (9 µM; 5,000 cpm/nmol) was added to a constant amount of
nAChR-rich membranes (0.3 mg/ml of protein) diluted in 50 mM HEPES buffer, pH 7.4 (total volume per sample, 200 µl)
containing increasing concentrations of CaCl2. In the
following, the samples were treated as described above. Dissociation
constants (Kapp values) for competing calcium
were derived from analysis of its capacity to displace
125I-MR44 from its binding site at the nAChR. For
calculation of Kapp values, the binding data
were plotted according to a logarithmic formula described by Herz
et al. (34).
Cell Culture--
TE671 cells were maintained in Dulbecco's
modified Eagle's medium containing 4.5 g/liter glucose and
supplemented with 10% (v/v) fetal calf serum, 1 mM pyruvic
acid, 4 mM glutamine, 10 units/ml penicillin, and 10 µg/ml streptomycin and incubated at 37 °C in a 5% CO2
atmosphere. Cells were divided 1:10 when they were ~75% confluent.
For electrophysiology, cells were grown on glass coverslips (5 × 20 mm) in 35-mm Petri dishes and transferred to a perfusion bath
mounted on the stage of an inverted microscope.
Electrophysiology--
The whole-cell patch-clamp configuration
was used to record whole-cell currents evoked by acetylcholine (ACh).
Patch pipettes were fabricated from borosilicate glass capillaries
(GC150-10; Clarke Electromedical Instruments) using a Sutter (P-97)
programmable puller. Pipette resistances were ~5 megaohms when filled
with 140 mM CsCl, 1 mM CaCl2, 1 mM MgCl2, 11 mM EGTA, and 5 mM HEPES (pH 7.2, adjusted with CsOH). The cells were
constantly perfused with rat saline containing 135 mM NaCl,
5.4 mM KCl, 1 mM CaCl2 1 mM MgCl2, and 5 mM HEPES (pH 7.4, adjusted with NaOH). Membrane currents were monitored using a List
Electromedical L/M-EPC7 patch clamp amplifier. The patch clamp
amplifier and DAD-12 Superfusion system were controlled by pClamp 5.7.2 software (Axon Instruments), which simultaneously acquired data to the
hard disc of an IBM-compatible PC. Concentration-inhibition
relationships for MR44 were measured to determine the IC50
value for inhibition of the peak current evoked by ACh (10 µM) at holding potentials (VH) of
25,
50, and
100 mV. Experiments were performed at 18-22 °C.
All data analyses were performed on an IBM-compatible PC using pClamp
5.7.2 software (Axon Instruments). Curve fitting was performed using Graphpad Prism software. IC50 values were determined by
fitting a four-parameter logistic equation to the
concentration-inhibition/response data. p values were
determined by the unpaired Student's t test, and
differences were considered to be significant for p < 0.05.
Photocross-linking Experiments--
nAChR-rich membranes (50 µg) were diluted in 0.1 M NaPi, pH 7.4, to a
final receptor concentration of 140 nM. After the addition of carbachol (500 µM), the samples were incubated for 30 min at room temperature. Subsequently, TPMP+, ethidium,
-BTX, or unlabeled MR44 was added, and the samples were incubated
for further 30 min at room temperature. The radioactive 125I-MR44 (10 µM; 250,000 cpm/nmol) was mixed
with the sample solution and irradiated with UV light at 254 nm
(distance, 15 cm; quartz lamp; Desaga, Heidelberg, Germany) for
15 s. Longer irradiation times resulted in a significant loss of
label, presumably because the aromatic group of MR44 releases
125I, and in irreversible damage of the nAChR, resulting in
high molecular weight aggregates of the receptor (data not shown). Unbound 125I-MR44 was separated from 125I-MR44
bound to nAChR-rich membranes by centrifugation (15,000 × g, 15 min). The pellet was dissolved and separated by
SDS-PAGE using a 10% SDS-PAG (35). The stained gel was dried, and
radioactive receptor subunits were visualized by autoradiography.
Deglycosylation of nAChR Using Endo H--
180 µg of
nAChR-rich membranes were centrifuged after labeling with
125I-MR44 and resuspended in 50 µl of 100 mM
NaPi buffer, pH 6.5. 5 µl of 1% SDS was added to this
suspension, followed by 10 milliunits of Endo H in 3 µl of
NaPi buffer. As a control, buffer was added instead of Endo
H. The incubation was carried out overnight at 37 °C.
Isolation of 125I-Labeled
-Subunit--
10 mg of
nAChR (0.25 mg/ml in 0.1 M NaPi buffer, pH 7.4)
was incubated with 50 µM 125I-MR44 for 30 min
following UV irradiation at 254 nm for 15 s. Unbound toxin was
separated from 125I-MR44 bound to nAChR-rich membranes by
centrifugation (15,000 × g, 20 min, 4 °C). The
pellet was dissolved in 2 ml of gel loading buffer. For preparative gel
electrophoresis (model 491 prep cell; Bio-Rad) the sample was loaded
onto a 1.5-cm-wide cylindrical gel containing a 10-cm-long separating
gel (10% (w/v) acrylamide) and a 3-cm-long stacking gel (3%
(w/v) acrylamide). The electrophoresis was carried out overnight at 15 mA. Proteins eluting from the gel were collected in 1.5-ml fractions in
150 mM Tris/HCl, 380 mM glycine, 0.1% (w/v)
SDS, pH 8.3. Fractions were assayed by SDS-PAGE and by counting the
radioactivity of each fraction in a
-counter. Fractions containing
pure
-subunit were pooled. Typically, about 600 µg of
-subunit
was recovered containing 600,000 cpm.
Deglycosylation of Isolated 125I-Labeled
-Subunit
Using Endo H--
For deglycosylation of 20 µg of
125I-labeled
-subunit in 70 µl of 50 mM
NaPi buffer, pH 6.5, 2 µl of 10% SDS and 10 milliunits of Endo H was added. As a control, buffer was added instead of Endo H. The incubation was carried out overnight at 37 °C.
Proteolytic Mapping Using V8 Protease--
Proteolytic mapping
was performed using the method of Cleveland et al. (36) with
S. aureus V8 protease. The proteolytic cleavage takes place
during the stacking phase of electrophoresis in the Laemmli gel system.
Typically, 30 µg of pure
-subunit were lyophilized, dissolved in
25 µl of loading buffer (175 mM Tris/HCl, 0.1% (w/v)
SDS, 5% (v/v) glycerol, pH 6.8), and incubated for 10 min at 70 °C.
4 µg of V8 protease were dissolved in 6 µl of loading buffer and
added to the
-subunit. The entire sample was loaded immediately onto
the stacking gel. Stacking and proteolysis were carried out for 30 min
at 12 mA. Then the current was shut off for 30 min to allow further
digestion by the protease, after which electrophoresis was continued.
The separating gel contained 15% (w/v) acrylamide to allow adequate
separation of the low molecular weight cleavage products. After
Coomassie staining, the gel was dried, and radioactive peptides were
visualized by autoradiography.
Proteolytic Mapping Using AspN Protease--
Since the
proteolytic activity of AspN protease is drastically reduced in the
presence of 0.1% (w/v) SDS in the electrophoresis buffer (37),
proteolytic cleavage was carried out in solution prior to
electrophoretic separation of the proteolytic peptides using the
Tricine gel system described by Schägger and von Jagow (38). 25 µg of 125I-MR44-labeled
-subunit in 100 mM
NaPi, pH 7.5, were incubated with 2 µg of AspN protease
for 27 h at 37 °C. The sample was lyophilized, dissolved in 25 µl of Tricine gel loading buffer, and incubated for 10 min at
70 °C. The samples were loaded on a Tricine gel containing a 3%
spacer gel and a 16% separation gel. Electrophoresis was carried out
overnight at 30 V. After Coomassie staining, the gel was dried, and
radioactive peptides were visualized by autoradiography.
Proteolytic Mapping Using ArgC Protease--
25 µg of
125I-MR44-labeled
-subunit in 12.5 mM
CaCl2, 0.5% EDTA, 5 mM dithiothreitol, 100 mM Tris/HCl, pH 8.0, were incubated with 2 µg of ArgC
protease for 27 h at 37 °C (39). Sample preparation, Tricine
gel electrophoresis, staining of the peptides, and detection of labeled
peptides were performed as described above.
Proteolytic Mapping Using LysC Protease--
25 µg of
125I-MR44-labeled
-subunit in 50 mM
Tris/HCl, pH 8.0, 0.5% SDS were incubated with 2 µg of LysC protease
for 27 h at 37 °C (40). Sample preparation, Tricine gel
electrophoresis, staining of the peptides, and detection of labeled
peptides were performed as described above.
Peptide Sequencing by Edman Degradation--
For determination
of N-terminal amino acid sequences, the peptides obtained by V8
digestion or AspN digestion and subsequent SDS-PAGE or
Tricine-polyacrylamide gel electrophoresis, respectively, of
125I-MR44-labeled
-subunit were blotted onto
polyvinylidene difluoride membrane. After Coomassie staining, the
peptides of interest were excised and submitted to Edman degradation.
Protein sequence analysis was performed using a Type 473A protein
sequencer (Applied Biosystems).
 |
RESULTS |
High Affinity Binding of Two Molecules of 125I-MR44 per
nAChR Monomer--
Binding of the radioactively labeled
polyamine-containing toxin MR44 (Fig.
1A) to the nAChR was
investigated (Fig. 1, B and C). 125I
was introduced as radioactive label into the aromatic head group of
MR44. 125I-MR44 was purified by reverse-phase HPLC and
analyzed by matrix-assisted laser desorption-ionization mass
spectrometry (data not shown). Specific binding of
125I-MR44 to nAChR-rich membranes from T. californica was determined by subtracting the nonspecific
component from the total binding curve (Fig.
2B). Nonspecific binding was
measured in the presence of a 100-fold molar excess of nonradioactive
I-MR44. As shown in Fig. 1B, Bmax was
obtained at 2.11 ± 0.38 µM (n = 4).
The Scatchard plot shows a straight line indicating that
125I-MR44 binds to a single class of noninteracting binding
sites with a Kapp value of 0.82 ± 0.22 µM. In the presence of the agonist carbachol, which
results in nAChR desensitization, the binding affinity of
125I-MR44 was increased by a factor of 1.4, but the
Bmax value was not changed (data not shown).
Using 125I-labeled
-BTX in a binding assay to determine
the number of receptor monomers per µg of protein (33), the binding
stoichiometry of 125I-MR44 was calculated to be 2.16 ± 0.18 mol of 125I-MR44/mol of nAChR monomer,
demonstrating that two molecules of 125I-MR44 bind per
receptor monomer.

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Fig. 1.
A, chemical formula of the polymethylene
tetramine MR44. B, binding of 125I-MR44 to the
nAChR. nAChR-rich membranes (0.3 mg/ml protein) were incubated with
increasing concentrations of 125I-MR44 (1-10
µM). The specific binding of 125I-MR44 ( )
was determined by subtracting the binding in the presence of a 100-fold
molar excess of I-MR44 ( ) from the total binding ( ). Each value
is the mean ± S.D. of four separate experiments C,
Scatchard plot of 125I-MR44 binding to the nAChR. The
Kapp value was 0.82 ± 0.22 µM. Using the -BTX binding assay to determine the
number of receptor monomers per µg of protein, the binding
stoichiometry of MR44 was calculated to be 2.16 ± 0.18 mol of
MR44/mol of nAChR monomer (n = 4). D,
concentration-inhibition curves for MR44 inhibition of ACh (10 µM)-evoked whole TE671 cell current.
VH of 25 mV ( , n = 5), 50
mV ( , n = 6), and 100 mV ( , n = 7) are shown. The solid curves are the
four-parameter logistic equation fits giving IC50 values of
16.1 ± 4.6 µM, 17.5 ± 7.1 µM,
and 16.9 ± 4.0 µM, respectively.
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Fig. 2.
A, ethidium displaced bound
125I-MR44 at the nAChR. nAChR-rich membranes preincubated
with carbachol were incubated with 125I-MR44 (9 µM) in the presence of increasing concentrations of the
luminal NCI ethidium. The IC50 value was determined to be
20.4 ± 3.6 µM (n = 3).
B, calcium prevented binding of 125I-MR44 to the
nAChR. nAChR-rich membranes were incubated with 125I-MR44
(9 µM) in the presence of increasing concentrations of
calcium with ( ) and without ( ) the agonist carbachol
(n = 3). According to a logarithmic equation described
by Herz et al. (34), the Kapp values
for calcium binding were calculated to be 12.3 ± 1.8 µM, and in the presence of carbachol they were 14.6 ± 2.2 µM.
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Voltage-independent Inhibition of nAChR by MR44--
The influence
of MR44 on the agonist-mediated ion conductance of the nAChR was
investigated electrophysiologically using cells that express
muscle-type nAChR (cell line TE671). MR44 inhibited ACh-mediated
whole-cell currents of TE671 cells with IC50 values of
16.1 ± 4.6 µM (n = 5), 17.5 ± 7.1 µM (n = 6), and 16.9 ± 4.0 µM (n = 7) at VH
of
25,
50, and
100 mV, respectively (Fig. 1D).
The NCI Ethidium Displaced Reversibly Bound
125I-MR44--
The well characterized luminal NCI ethidium
interacts with a binding affinity of 1 µM (41) with the
high affinity NCI site of the nAChR. The binding of
125I-MR44 was determined in the presence of increasing
concentrations of ethidium bromide. Fig. 2A shows that bound
125I-MR44 was displaced by ethidium with an
IC50 value of 20.4 ± 3.6 µM
(n = 3). The competitive antagonist
-BTX had no
influence on the binding of 125I-MR44 (data not shown).
Calcium Displaced Reversibly Bound 125I-MR44--
It
was shown previously that the NCI ethidium could be completely
displaced by cations, indicating that NCIs and channel-permeating cations bind to the nAChR ion channel in a competitive manner (42). As
shown in Fig. 2B, the divalent cation calcium displaced 125I-MR44 from its binding site with an IC50
value of 2.4 mM. According to a logarithmic formula
described by Herz et al. (34), the Kapp value for calcium binding was calculated to
be 12.3 ± 1.8 µM (n = 3). In the
presence of carbachol, the affinity of calcium for the MR44 binding
site was not significantly changed (Kapp = 14.6 ± 2.2 µM).
125I-MR44 Photolabeled the nAChR
-Subunit--
For
photocross-linking, 125I-MR44 (10 µM) was
incubated with nAChR-rich membranes. 125I-MR44 was
covalently cross-linked to amino acid residues facing the
ligand-binding pocket by irradiation for 15 s at 254 nm. The 125I-labeled receptor subunits were separated by SDS-PAGE
and visualized by autoradiography (Fig.
3A). 125I-MR44 was
found to photolabel exclusively the nAChR
-subunit. No radioactivity
was incorporated into the other receptor subunits at detectable levels.
Since rapsyn, a 43-kDa protein associated with nAChR, migrates in the
gel close to the
-subunit, the mobility of the
-subunit was
increased by cleaving its carbohydrate moiety with Endo H. This
endoglycosidase releases high mannose and hybrid-type N-linked oligosaccharides from glycoproteins (43).
Deglycosylation of SDS-solubilized nAChR-rich membranes with Endo H
shifted the receptor subunits, but not rapsyn, to lower molecular
weights when separated by SDS-PAGE (Fig. 3A, lane
3). Incubation of the 125I-MR44-labeled nAChR
with Endo H quantitatively converted the labeled
-subunit to its
high mobility form (Fig. 3A, lane 5), indicating that 125I-MR44 was incorporated exclusively into
the
-subunit. A 100-kDa protein (Fig. 3A, lane
2; Fig. 3B, lane 2) that
most likely represents the Na+-K+-ATPase, was
faintly labeled by 125I-MR44 (Fig. 3A,
lane 4; Fig. 3B, lane
3). This protein is found in nAChR-rich membrane
preparations as a contamination. It has been shown previously that
polyamines modulate the Na+-K+-ATPase and that
other photolabile polyamine derivatives also photolabel this enzyme to
a minor extent (26).

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Fig. 3.
A, effect of Endo H on nAChR
photolabeled with 125I-MR44. nAChR-rich membranes (30 µg
each lane) were photolabeled with 10 µM
125I-MR44. Samples were incubated without Endo H
(lane 2) and with Endo H (lane
3) and separated on an 8% SDS-polyacrylamide gel. The gel
was stained with Coomassie Blue (lanes 1-3), and
radioactive protein bands present in lanes 2 and
3 were visualized by autoradiography (lanes
4 and 5). Exposure time was typically 2 days.
B, photoaffinity labeling of nAChR with
125I-MR44. nAChR-rich membranes (50 µg each lane) were
photolabeled with 10 µM 125I-MR44 in the
absence (lanes 3-5, 9, and
10) or the presence (lanes 6-8) of
500 µM carbachol and separated on an 10%
SDS-polyacrylamide gel. The gel was stained with Coomassie Blue
(lane 2 shows 50 µg of nAChR-rich membranes),
and radioactive protein bands were visualized by autoradiography
(lanes 3-10). Samples were preincubated with 560 nM -BTX (lanes 4 and 7)
or 5 mM TPMP+ (lanes 5 and 8) or 100 µM ethidium (lane
9) or 1 mM MR44 (lane 10).
The exposure time was typically 2 days. A and B,
lane 1 shows the molecular mass markers in kDa:
phosphorylase b (97 kDa), bovine serum albumin (67 kDa),
ovalbumin (43 kDa), and carbonic anhydrase (30 kDa). Exposure time was
typically 1 day.
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In the absence or presence of the agonist carbachol, photocross-linking
of 125I-MR44 with nAChR-rich membranes occurred with the
receptor being in one of the two closed conformations, i.e.
in the resting or in the desensitized state, respectively. In both
experiments, the nAChR
-subunit was labeled (Fig. 3B,
lanes 3 and 6). In the presence of
well characterized luminal NCIs, such as TPMP+ and ethidium
bromide, 125I-MR44 bound at the NCI site was completely
displaced, with the labeling intensity reduced to background levels
(Fig. 3B, lanes 5, 8, and
9). The competitive antagonist
-BTX did not significantly affect the binding of 125I-MR44 (Fig. 3B,
lanes 4 and 7). In the presence of
-BTX (lane 4) or carbachol (lane
6), the labeling intensity was reduced by 10-15%.
Cross-linking 125I-MR44 in presence of a 100-fold molar
excess of the nonradioactive analogue resulted in the complete loss of
labeling showing the specificity (saturability) of MR44 binding (Fig.
3B, lane 10). Without irradiation, no
cross-linking was observed (data not shown).
Mapping the 125I-MR44-labeled
-Subunit Using V8
Protease--
The binding site of MR44 was mapped using the method
described by Cleveland et al. (36) using S. aureus V8 protease, which specifically cleaves peptide bonds at
the carboxyl side of glutamate residues. Limited in-gel digestion of
the
-subunit with V8 protease produces preferentially four
nonoverlapping fragments (44). The largest fragment, a 20-kDa peptide
(V8-20), begins at
Ser-173 and contains the first three
membrane-spanning regions, M1, M2, and M3. The 18-kDa peptide (V8-18)
is part of the extracellular domain and carries at
Asn-141 a
carbohydrate residue of ~4 kDa. The 10-kDa peptide (V8-10) that
begins at
Asn-339 contains the fourth membrane-spanning region, M4.
The smallest fragment of 4 kDa (V8-4) represents the N-terminal part of
the
-subunit.
nAChR-rich membranes were photocross-linked with 125I-MR44,
and the labeled
-subunit was isolated by preparative tube gel
electrophoresis. The 125I-MR44 labeled
-subunit was
cleaved in the gel with V8 protease. Peptides generated were separated
using 15% SDS-PAGE, and those fragments carrying a radioactive label
were identified by autoradiography (Fig.
4A). Fig. 4A,
lanes 2-6, show Coomassie staining of the uncleaved radiolabeled
-subunit (lane 2), a V8
protease digest of 125I-MR44-labeled
-subunit
(lanes 3 and 4) and of unlabeled
-subunit (lane 5), and V8 protease alone
(lane 6). Using varying enzyme/substrate ratios,
the limited proteolysis of the radioactively labeled and the unlabeled
-subunit reproducibly yielded identical peptide patterns. V8
cleavage of the labeled and of the unlabeled
-subunit revealed two
prominent cleavage products with apparent molecular masses of about 19 kDa (V8-20) and 17 kDa (V8-18) and a smaller peptide of ~10 kDa
(V8-10) (Fig. 4A, lanes 3-5).
Additional minor poorly resolved cleavage products were found near the
dye front of the gel. V8 protease and its proteolytic fragments appear
as two major protein bands with apparent molecular masses of 29 and 27 kDa and as a 14-kDa fragment of lower intensity (Fig. 4A,
lane 6). The autoradiograph of the V8-protease
digest revealed that the V8-20 fragment carried the majority of the
radioactive label (Fig. 4A, lanes 8 and 9). No radioactivity was detected in the
V8-18 and
V8-10 peptides. As expected, in the absence of V8 protease the
uncleaved radioactive
-subunit was found in the range of 41 kDa
(Fig. 4A, lane 7).

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Fig. 4.
A, proteolytic mapping of the
125I-MR44-labeled nAChR -subunit using V8 protease.
After cross-linking of 125I-MR44 (10 µM) with
nAChR (0.2 mg/ml receptor), the 125I-MR44-labeled receptor
subunits were separated by preparative SDS-PAGE. The isolated
125I-MR44-labeled -subunit was incubated with V8
protease in the gel for 30 min, and peptide fragments generated were
separated using 15% SDS-PAGE. Lanes 2-5 show
Coomassie staining of the radioactively labeled -subunit
(lane 2, 30 µg), the radioactively labeled
-subunit incubated with 6 µg of V8 protease (lane
3, 30 µg; lane 4, 15 µg),
unlabeled -subunit incubated with 4 µg of V8 protease
(lane 5, 15 µg), and V8 protease
(lane 6, 6 µg). Lanes
7-9 show the localization of the radioactive peptides shown
in lanes 2-4 using autoradiography (exposure
time was typically 3 days). B, effect of Endo H on the
peptide pattern generated by V8 proteolytic cleavage of the
125I-MR44-labeled nAChR -subunit. The isolated
125I-MR44-labeled -subunit was incubated overnight
without (lane 2) and with (lane
3) Endo H. The two samples were incubated with V8 protease
in the gel for 30 min, and the generated peptide fragments were
separated using 15% SDS-PAGE. Lanes 2 and
3 show Coomassie staining of the peptide pattern generated
by V8 digestion, and lanes 4 and 5 show the localization of the radioactive peptides of lanes
2 and 3, respectively, using autoradiography
(exposure time was typically 3 days). A and B,
lane 1 shows the molecular mass markers in
kDa: phosphorylase b (97 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin
inhibitor (20 kDa), lysozyme (14 kDa).
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N-terminal Amino Acid Sequencing of 125I-MR44-labeled
V8-20 and of Unlabeled V8-18 and V8-10--
For the characterization
of the peptides generated by V8 digestion, their N-terminal amino acid
sequences were identified using Edman degradation. The peptides
generated by in-gel V8-protease digestion were transferred to
polyvinylidene difluoride membrane and visualized by Coomassie
staining. Single peptide bands were excised and submitted to N-terminal
amino acid sequencing. Table I shows the
results of the first five sequencing cycles for each of the peptides
examined. The most prominent N-terminal sequence found in the
125I-MR44-labeled V8-20 peptide band started from
Val-46. As a minor signal, a second peptide sequence was observed
that most likely begins with
Ser-173. The sequence was difficult to
detect, since the first two amino acids
of the N terminus of this peptide showed barely visible signals in the
chromatograph (Tables I and II). Previous studies using limited
in-gel proteolysis of the nAChR
-subunit with V8 protease (45)
clearly demonstrated that V8-20 reproducibly contained two peptides
beginning from
Val-46 and
Ser-173, respectively, confirming the
presence of the two proteolytic fragments detected in V8-20. The
N-terminal sequence of unlabeled V8-18 was found to be identical with
one of the V8-20 peptides (Table I). Microsequencing of unlabeled V8-10
revealed an N terminus starting from
Asn-339 (Table I). This peptide
starts in the middle of the putative cytosolic loop and contains the M4
transmembrane sequence.
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Table I
N-terminal amino acid sequence analysis of 125I-MR44-labeled
nAChR -subunit peptides obtained by proteolytic cleavage using V8,
AspN, ArgC, or LysC proteases. Peptides were prepared and sequenced as
described under "Experimental Procedures."
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Table II
N-terminal amino acid sequence analysis of 125I-MR44-labeled
peptides obtained by cleavage of the labeled -subunit using V8,
AspN, ArgC, or LysC proteases. The V8-20, AspN-16, ArgC-4,
and LysC-8 proteolytic peptide carried a radioactive label. Peptides
were prepared and sequenced as described under "Experimental
Procedures."
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Effect of Endo H on 125I-MR44-labeled V8-20--
To
identify the one of the two V8-20 peptides that carries the radioactive
label, the carbohydrate moiety of the peptide starting from
Val-46
was removed to increase the mobility of this fragment when separated by
SDS-PAGE. The 125I-MR44-labeled
-subunit treated with
and without Endo H was subsequently cleaved in the gel using V8
protease. Fig. 4B shows the cleavage patterns generated with
respect to Coomassie staining of the gel (Fig. 4B,
lanes 2 and 3) and photoincorporation
of 125I-MR44 as detected by autoradiography (Fig.
4B, lanes 4 and 5) after an
overnight incubation without (lanes 2 and
4) and with (lanes 3 and 5)
Endo H. Treatment of the
-subunit with Endo H prior to V8 cleavage
altered the cleavage pattern of the peptides generated. The radioactive
V8-20 band was apparently unaffected, but the intensity of the stained
band seemed to be reduced (Fig. 4B, lane
3). The V8-18 peptide disappeared, and two new bands with
apparent molecular masses of 15 kDa (V8-15) and 12 kDa (V8-12) were
generated. These results are in good agreement with earlier findings of
Pedersen et al. (45). These authors could show that V8-15
corresponds most likely to the deglycosylated form of an incompletely
cleaved form of V8-18 that comigrated with V8-20. They also
demonstrated that V8-12 was the deglycosylated from of V8-18. The
autoradiograph revealed that Endo H incubation had no influence on the
mobility of the 125I-MR44 labeled peptide in the gel (Fig.
4B, lanes 4 and 5),
demonstrating that the label and the carbohydrate moiety were
associated with different V8 proteolytic fragments. These findings
indicate that 125I-MR44 was most likely cross-linked to the
V8-20 peptide starting from
Ser-173.
Mapping the 125I-MR44-labeled
-Subunit Using AspN
Protease--
To verify the results obtained with V8 protease and to
further narrow down the region of 125I-MR44 cross-linking,
the 125I-MR44 labeled
-subunit was cleaved with AspN
protease to generate a peptide pattern different from the one obtained
with V8 protease. The 125I-MR44-labeled
-subunit was
cleaved in solution with AspN protease, and the peptides generated were
separated using Tricine gel electrophoresis (Fig.
5). Coomassie-staining of the AspN
proteolytic fragments revealed a pattern of peptides with molecular
masses of 4-16 kDa (Fig. 5, lane 2). The
largest of the peptides generated with a molecular mass of 16 kDa
(AspN-16) was found to be exclusively photolabeled by
125I-MR44 as seen in the autoradiograph (Fig. 5,
lane 3). No radioactivity was detected in the
remaining smaller peptides with apparent molecular masses of 14 kDa
(AspN-14), 9 kDa (AspN-9), 7.5 kDa (AspN-7.5), 7 kDa (AspN-7), and 4 kDa (AspN-4).

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Fig. 5.
Proteolytic mapping of the
125I-MR44 labeled nAChR -subunit
using AspN protease. After cross-linking of 125I-MR44
(10 µM) with nAChR (0.2 mg/ml receptor), the
125I-MR44-labeled receptor subunits were separated by
preparative SDS-PAGE. The isolated 125I-MR44 labeled
-subunit was incubated overnight with AspN protease, and the
generated peptide fragments were separated using Tricine-polyacrylamide
gel electrophoresis with 3% spacer and 16% separation gel.
Lane 2 shows the Coomassie-stained peptides that
were generated by incubation of the 125I-MR44-labeled
-subunit (25 µg) with AspN protease (2 µg). Lane
3 shows the corresponding autoradiograph to localize
radioactive peptides (exposure time was typically 2 days).
Lane 1 shows the molecular mass markers in
kDa on the ordinate: globine (16.9 kDa), globine I + II (14.4 kDa),
globine I + III (10.7 kDa), globine I (8.2 kDa), globine II (6.2 kDa),
glucagon (3.5 kDa).
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N-terminal Amino Acid Sequencing of 125I-MR44-labeled
AspN-16 and of Unlabeled AspN-14, AspN-9, AspN-7.5, AspN-7 and
AspN-4--
To localize these peptides in the known primary structure
of the
-subunit, the radioactively labeled fragment AspN-16 and the
other unlabeled fragments AspN-14 to AspN-4 were isolated and submitted
to Edman degradation as described above. Table II shows the results of
the sequencing cycles. The N-terminal sequence of AspN-16, which
carried the cross-linked 125I-MR44, was found to begin with
AspN-180 (Tables I and II). This finding confirms our result
obtained with V8 protease, which demonstrated that the radioactive
label is localized within the 19-kDa peptide of the
-subunit
beginning with
Ser-173. Most remarkably, the N-terminal sequence of
the unlabeled peptide migrating at 14 kDa was determined to begin with
Asp-200 (Table I). Consequently, 125I-MR44 must be
photoincorporated into the 2.5-kDa peptide
Asp-180 to
Leu-199.
Microsequencing of the unlabeled AspN-9 revealed two peptide sequences
beginning with
Ser-1 and
Asp-350, respectively. Therefore, one of
the peptides represents the N terminus presumably ending at
Ser-82,
and the other corresponds to the C terminus with part of the putative
cytosolic loop including the M4 transmembrane sequence (Table I).
AspN-7.5 (AspN-7) and AspN-4 (number 2) were identified as smaller
cleavage products of AspN-9 (number 1) and AspN-4 (number 2),
respectively. AspN-4 contained a second sequence starting from
Asp-97, which is part of the extracellular domain. All unlabeled
AspN proteolytic fragments were nonoverlapping with the 20-amino acid
sequence
Asp-180 to
Leu-199.
Mapping the 125I-MR44-labeled
-Subunit Using ArgC
Protease--
Using ArgC protease, an enzyme that cleaves peptide
bonds at the C-terminal side of arginine residues, a cleavage of the
region suggested to be labeled by the experiments described above on the
-subunit into fragments of 12 kDa (
80-182), 3.4 kDa
(
183-209), and 10 kDa (
210-301), respectively, is predicted.
When the 125I-MR44-labeled
-subunit was subjected to
cleavage using this protease and the peptides were separated by Tricine
gel electrophoresis, peptides in the molecular mass range of
4-15 kDa were found in the Coomassie-stained gel (Fig.
6A). In the autoradiograph, a single radioactively labeled peptide band of about 4 kDa (ArgC-4) was
detected. The other peptides generated carried no radioactivity (Fig.
6A, lane 3).

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Fig. 6.
Proteolytic mapping of the
125I-MR44-labeled nAChR
-subunit using ArgC protease (A)
and LysC protease (B). After cross-linking of
125I-MR44 (10 µM) with nAChR (0.2 mg/ml
receptor), the 125I-MR44-labeled receptor subunits were
separated by preparative SDS-PAGE. The isolated
125I-MR44-labeled -subunit was incubated overnight with
ArgC protease (A) or LysC protease (B), and the
generated peptide fragments were separated using Tricine-polyacrylamide
gel electrophoresis with 3% spacer and 16% separation gel.
Lane 2 (A and B) shows the
Coomassie-stained peptides that were generated by incubation of the
125I-MR44-labeled -subunit (25 µg) with ArgC protease
(A; 2 µg) or LysC protease (B; 2 µg).
Lane 3 (A and B) shows the
corresponding autoradiograph to localize radioactive peptides (exposure
time was typically 2 days). Lane 1 (A and B)
shows the molecular mass markers in kDa on the ordinate: globine (16.9 kDa), globine I + II (14.4 kDa), globine I + III (10.7 kDa), globine I
(8.2 kDa), globine II (6.2 kDa), glucagon (3.5 kDa).
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N-terminal Amino Acid Sequencing of 125I-MR44-labeled
ArgC-4 and of Unlabeled ArgC-15, ArgC-10, and ArgC-9--
N-terminal
amino acid sequencing of the 125I-MR44 labeled ArgC-4
fragment yielded two sequences starting at their N-terminal ends with
Arg-182 and
Leu-80, respectively (Tables I and II). One of the
fragments,
80-115, had previously been shown to be nonradioactive
using V8 and AspN proteases. Most importantly, the second sequence,
which therefore must carry the radioactive label, starts from
Arg-182, confirming the site of 125I-MR44
photoincorporation that was already suggested from our V8 and AspN
digest experiments. The N-terminal sequences of unlabeled ArgC-10 and
ArgC-9 were found to begin from
Ile-210 and
Ile-116, respectively
(Table I). The unlabeled 15-kDa peptide was identified as a proteolytic
fragment of the ArgC protease. The specificity of ArgC is primarily to
arginine residues, although hydrolysis proceeds to a minor degree also
after lysine residues (39) and occasionally after aromatic residues
(47). As a result, the labeled
-subunit was cleaved at the
C-terminal side of
Lys-116 and
Tyr-181 (N-terminal side of
Arg-182). Taken together with the results obtained with V8 and AspN
protease, this observation allows us to locate the site of
125I-MR44 cross-linking to the sequence
Arg-182 to
Leu-199.
Mapping the 125I-MR44-labeled
-Subunit Using LysC
Protease--
The sequence labeled by 125I-MR44 contains
at position 185 a lysine residue. LysC protease, an protease that
cleaves the peptide bond after lysine residues (40), was therefore used
to cleave the labeled
-subunit. The peptides generated were
subsequently separated using Tricine gel electrophoresis. The
Coomassie-stained gel showed peptides in the molecular mass
range of 7-14 kDa (Fig. 6B). The autoradiograph
identified a single radioactive peptide of about 8 kDa (LysC-8),
while the other peptide generated carried no radioactivity (Fig.
6B, lane 3).
N-terminal Amino Acid Sequencing of 125I-MR44-labeled
LysC-8 and of Unlabeled LysC-12, LysC-10, LysC-7, and
LysC-6--
Sequencing of the N terminus of the radioactively labeled
LysC-8 fragment yielded a single sequence starting from His-186 (Tables
I and II). The other unlabeled fragments LysC-12, LysC-10, and LysC-7
were found to start from
Ser-77,
Ile-116, and
Val-18, respectively. LysC-4 was identified as a smaller cleavage product of
LysC-12 (Table I). All identified nonradioactive peptides generated by
proteolytic cleavage with V8, AspN, ArgC, or LysC proteases represent
sequences of the
-subunits that were nonoverlapping with the
sequence
His-186 to
Leu-199. These findings allow us to further
narrow down the site of modification to the amino acid stretch
His-186 to
Leu-199.
Attempts to identify the precise site of modification within this amino
acid stretch by using the release of radioactivity during Edman
sequencing failed, since the radioactive photoaffinity label is not
stable under the chemical conditions of the Edman degradation cycle.
125I-MR44 quantitatively releases 125I from the
aromatic ring in the first sequencing step during alkaline and acidic
treatment (data not shown).
 |
DISCUSSION |
The architecture of ligand-gated ion channels can be explored
using photoaffinity derivatives of high affinity ligands. The aim of
the present studies was to identify amino acid residues facing the
ligand-binding site of T. californica nAChR and thereby to
obtain information on the structure of the luminal surface of the
channel gated by this receptor. We have used a newly developed polyamine-containing toxin carrying a hydrophobic head group in photocross-linking experiments. The novel photoaffinity label MR44 was
first characterized electrophysiologically and by binding studies and
was subsequently used to map the ligand binding site in the lumen of
the nAChR ion channel.
Using fluorescent titration, we have shown recently that PhTX analogues
including MR44 interact with the fluorescent NCI ethidium bound to the
high affinity NCI site in the desensitized state of the nAChR (26).
Like most amine NCIs containing aromatic or aliphatic rings, such as
chlorpromazine, meproadifen, and TPMP+ (41, 48, 49), MR44
was found to bind with higher affinity in the presence of the agonist
carbachol, i.e. when the nAChR is in its desensitized closed
state, as compared with the resting closed conformation. Since MR44
causes a voltage-independent block of AChR-induced ion flow, MR44
presumably acts as inhibitor of the closed channel conformation rather
than as an open channel blocker. Unlike other classical NCIs, which
show a binding stoichiometry of 1:1, we found that two molecules of
MR44 were bound per receptor monomer. In a recent study, the same
binding stoichiometry was observed for another PhTX derivative,
N3-phenyl-125I2-PhTX-343-lysine
(26). MR44 has two functional moieties; a long positively charged
polyamine tail on the one side and an aromatic head group on the other.
It is most likely that these two structural elements of the molecule,
although both contribute to the binding affinity, bind at different
sites within the receptor lumen.
For photocross-linking, 125I-MR44 was incubated with
nAChR-rich membranes and subsequently irradiated with UV light to
generate a reactive nitrene species that efficiently cross-links to any amino acid residues nearby that are facing the binding site. The finding that 125I-MR44 photolabeled the receptor
-subunit suggests that the aromatic head groups of the two MR44
bound in the channel lumen most likely interact each with one of the
-subunits, since this part of MR44 carries the photolabile group.
To localize the polyamine-binding site at the nAChR in its resting
closed state, the regions of the
-subunit that incorporated 125I-MR44 were mapped by proteolytic cleavage using V8 (36,
45), AspN (37), ArgC (39), and LysC proteases (40). The N termini of
peptides generated were microsequenced and localized in the known
primary structure of the Torpedo nAChR
-subunit (4). When
using limited V8 proteolysis, the majority of the radioactivity was
detected in a 19-kDa proteolytic fragment. Microsequencing and Endo H
treatment identified two fragments in the V8-20 peptide band beginning
with
Val-46 and
Ser-173, respectively. These data correspond to
earlier findings of Pedersen et al. (45) demonstrating that the V8-20 band contained two comigrating fragments: a carbohydrate-containing peptide starting from
Val-46 and an unglycosylated peptide beginning from
Ser-173. Removal of the oligosaccharide moiety by Endo H had no influence on the mobility of
the radioactively labeled V8-20 peptide, indicating that
125I-MR44 photolabels the fragment beginning with
Ser-173. To confirm this finding, AspN protease was used to cleave
the 125I-MR44 labeled
-subunit at sites different from
that cleaved by V8 protease. An AspN proteolytic fragment of 16 kDa was
found to carry the radioactive label. Microsequencing of this peptide revealed a single sequence beginning from
Asp-180, confirming the
results obtained with V8 protease. The N-terminal sequence of the
unlabeled proteolytic fragment migrating at 14 kDa was identified to
start from
Asp-200. Thus, the cross-linked 125I-MR44
must be located within a stretch of 20 amino acid residues from
Asp-180 to
Leu-199. Cleavage of the 125I-MR44-labeled
-subunit with ArgC protease generated a peptide pattern with only
one peptide labeled of about 4 kDa. N-terminal amino acid sequencing
identified two proteolytic fragments starting with Arg-182 and Leu-80,
respectively. The peptide beginning with
Leu-80 was shown to be
nonradioactive using V8, AspN, and LysC proteases. Consequently,
125I-MR44 must be cross-linked to the ArgC-4 fragment
starting with Arg-182, confirming the region of 125I-MR44
photoincorporation suggested from the V8 and AspN digest experiments.
The sequence
Arg-182 to
Leu-199 labeled by 125I-MR44
contains a lysine residue in position 185. Therefore, LysC protease was
used to cleave the labeled
-subunit. In mapping experiments using
LysC protease, we further narrowed down the site of modification to the
amino acid stretch
His-186 to
Leu-199.
This sequence is located in the large N-terminal domain of the
-subunit; it is found close to one of the three loops that contribute to the agonist-binding site. Photoaffinity reagents and
site-directed mutagenesis have been utilized in previous studies to
identify amino acid residues facing the agonist-binding domain. Three
discrete regions on the
-subunit primary structure have been
identified:
Trp-86 to
Tyr-93 (loop A),
Trp-147 to
Tyr-150 (loop B), and
Tyr-190 to
Tyr-198 (loop C; Refs. 50-54). Two
additional regions on each of the neighboring
- and
-subunits
were found to contribute to the binding site (loop D and E; Refs.
55-57). Based on these data, a spatial model has been developed
according to which three sequences of the
-subunit and two sequences
of the neighboring
- and
-subunits form the agonist binding
pockets of nAChR (58, 59). The amino acid residues forming loop C overlap in the C-terminal region at least partially with the sequence
His-186 to
Leu-199 labeled by 125I-MR44. Since the
agonist carbachol or the competitive antagonist
-BTX had only minor
influence on the photocross-linking yield of 125I-MR44, the
site of interaction between the aromatic ring of 125I-MR44
and the nAChR should be found outside the zone that is sterically
influenced by any bound agonist contacting
Tyr-190,
Cys-192,
Cys-193, and
Tyr-198. Therefore, the site of interaction of the
aromatic head group of 125I-MR44 lies presumably within the
hydrophobic sequence HWVY (residues
186-189) containing three
aromatic amino acid residues. Receptor desensitization induced by
carbachol might influence the accessibility of those residues that
react in the resting channel state with 125I-MR44 without
affecting the binding affinity of the whole molecule.
From various structure-activity relationship studies, it is obvious
that the aromatic moiety of PhTX derivatives has a significant influence on the binding affinity of these compounds (26, 27). Their
binding properties were considerably improved by increasing the size
and hydrophobicity of the head group. The finding that 125I-MR44 photolabeled a sequence of the
-subunit in
which aromatic amino acid residues are accumulated is in line with the
observation that PhTX derivatives that carry a large, hydrophobic head
group bind with increased affinity to the nAChR (26, 27).
Since the photolabile azido residue of MR44 is located at its aromatic
head group, the site of 125I-MR44 cross-linking identifies
the region to which this hydrophobic part of the molecule binds. In
contrast, the site of interaction of the conformationally flexible
carbon chain can be less exactly determined. Due to its positively
charged -NH2 groups interspersed with hydrophobic
-CH2 groups, the polyamine chain is expected to
preferentially interact with acidic and hydrophobic amino acid side
chains, respectively. This is similar to the active site of bacterial
polyamine binding proteins (60). In previous studies, it was suggested
that the positively charged polyamine tail binds in the lumen of the
nAChR ion channel to the negatively charged amino acids that are part
of the selectivity filter (27). To examine the site to which the
polyamine moiety of MR44 binds, the well characterized luminal NCI
ethidium was used in various displacement assays. Previous studies
located the ethidium-binding site at the high affinity NCI site deep in
the ion channel of the nAChR, presumably close to the M2 helix (34,
50). Our finding that the quaternary amine ethidium competed with
125I-MR44 supports the view that the positively charged
polyamine tail of MR44 is oriented toward the narrow part of the ion
channel lumen. Using the NCI ethidium as a fluorescent probe at the
nAChR, we determined recently the binding affinities of various PhTX derivatives including MR44. Increasing concentrations of MR44 reduced
the fluorescence of bound ethidium, indicating that MR44 displaced
ethidium from its binding site. This observation corresponds well to
the results presented here that ethidium competes with bound
125I-MR44 and that photoincorporation of
125I-MR44 was prevented in the presence of ethidium and
TPMP+. Ethidium was still displaced by MR44 even when
allosteric transitions within the nAChR had been abolished by covalent
cross-linking (26). This finding strongly suggests that MR44 interacts
with ethidium in a direct competitive manner at an overlapping luminal binding site. Channel-permeating cations, such as calcium, are known to
bind to sites within the nAChR ion channel that sterically overlap with
the high affinity NCI site (42). Using calcium in direct binding
experiments, we could show that 125I-MR44 bound to the
nAChR was completely displaced by calcium. This observation indicates a
strong influence of polar electrostatic interactions between
125I-MR44 and the NCI site of the nAChR. A site of negative
charges located deep in the lumen of the nAChR ion channel that might interact with the positively charged polyamine tail of
125I-MR44 could provide the acidic amino acid residues of
the selectivity filter as suggested in earlier studies (27). To
summarize, the polyamine moiety of MR44 interacts with the high
affinity NCI site of the nAChR, while the aromatic ring of this
compound binds to the upper part of the ion channel, i.e. in
the vestibule, and therefore to a hydrophobic region on the
-subunit
that is located in close proximity to the loop C of the agonist binding
site and is accessible from the water-filled lumen of the channel.