Transcript [ 3 H]DAE specifically incorporates within a fragment containing M1

Abstract
[3H]3,8-Diazidoethidium
Figure 5. Within  subunit, there is specific incorporation
of [3H]DAE within a 24 amino acid fragment containing
only M2.
([3H]DAE),
a photoactivatible analog of the noncompetitive antagonist (NCA)
ethidium has been used to map the ethidium binding site in the Torpedo nicotinic acetylcholine receptor in the
presence of the agonist carbamylcholine. Within the  subunit, [3H]DAE incorporation inhibitable by the
NCA phencyclidine (PCP) was in a 20kDa fragment containing the first three transmembrane segments (M1,
M2, and M3). Digestion of this fragment with EndoLysC (EKC) created two peptides with PCP-inhibitable
labeling: one from His-186 through Lys-242, containing M1, and one beginning at Met-243, the
beginning of M2. The observed photoincorporation of [3H]DAE into Leu-251 and Ser-252, residues in
M2 which line the pore of the channel, was inhibited by PCP. In , the PCP-inhibitable incorporation was in
a 14kDa fragment which included M1 and M2. EKC digestion of this fragment created two peptides with
[3H]DAE incorporation: Phe-206 through Lys-256, containing the M1 segment, and Met-257 through
Glu-280, the M2 segment. These results provide evidence that [ 3H]DAE (and ethidium) binds within the
channel lumen and interacts simultaneously with M2 and M1.
Figure 3. Within -subunit, [3H]DAE specifically
incorporates within a fragment containing M1 and
another containing M2 and M3.
Figure 1. In desensitized nAChR,
photoincorporation of [3H]DAE into the 
and  subunits is inhibited by PCP.
E
mep
L
V
L
dae
L
S
V8-14
A
Ile-192
M1
KF
T
KM M2
E
Figure 7. Incorporation of NCAs
into M2
V
L
cpz
S
T
In the desensitized state within M2, meproadifen
mustard (mep) reacts with Glu262; chlorpromazine
(cpz) with Ser248, and diazidoethidium with
Leu251, Ser252, and Leu258.
EndoLysC digest to SDS-PAGE
A
V8-20
173-S
M1
KH
KM M2
M3
Ile-192
M1
206-F
257-M
~3 kD
EndoLysC digest to HPLC
173-S
M1
186-H
M2
243-M
M2
M3
Molecular Weight Markers
3
|
5000
5
14000
cpm
25
30
35
40
B
M243 126 pmol
18
28
43
||
|
||
||
|
-PCP
+PCP
3000
1000
8000
H186 128 pmol
6000
0
C
2000
1500
0
0.72
1000
Fluorescence
Absorbance
0.48
Digested V8-14
M257
2000
-PCP
+PCP
500
0
0.00
0
10
15
2. [3H]Diazidoethidium specifically incorporates into the  subunit in a
fragment containing the M1 segment, as well as into residues in M2
which line the pore of the channel.
3. [3H]Diazidoethidium labels the  subunit specifically in the M2
segment and incorporates into a fragment containing the M1 segment
without M2.
0.24
5
Conclusions
1. [3H]Diazidoethidium photoincorporates into nAChR at the high affinity
NCA site of ethidium.
2000
4000
nAChR-rich membranes were equilibrated with 10 um
[3H]ethidium diazide in the presence and absence of 2mM
carbamylcholine (carb) and 100 mM PCP and then irradiated.
Polypeptides were resolved by SDS-PAGE. Panel A: Coomassie
Blue stained polypeptides with subunits running as indicated.
Panel B: Fluorogram: Lane 1: -carb, -PCP; Lane 2: +carb, -PCP;
Lane 3: +carb, +PCP.
14
Undigested V8-14
4000
-PCP
+ PCP
10000
Absorbance
Results of affinity labeling and mutational analysis provide abundant evidence that
the channel pore of the nicotinic acetylcholine receptor (nAChR) is composed primarily
of the M2 segments from all of the five subunits of the pentamer (for review see Arias,
1996). Mutations in M2 affect the potency of QX-222 as an open channel blocker, and
the binding sites for aromatic amine noncompetitive antagonists (NCA) are localized to
M2 in the desensitized receptor. In addition, part of the M1 segment also contributes to a
portion of the channel pore. Cysteine scanning mutagenesis has identified N-terminal
residues of the M1 segment that are solvent-accessible (Akabas and Karlin, 1995), and
quinacrine azide, a photoactivatible analog of quinacrine, photoincorporates into residues
in the amino terminus of the M1 segment. Mutations in the N-terminal portion of the M1
segment affect the potency of block by quinacrine; however, they have no effect on the
block by QX-222 (Tamamizu et al, 1995).
Ethidium is both a NCA as well as a competitive antagonist. While ethidium binds
competitively with PCP and other NCAs expected to bind in the channel pore,
fluorescence energy transfer studies indicate that ethidium binds 52 Å from the
membrane surface, presumably at the most extracellular portion of the receptor (Johnson
and Nuss, 1994). To map the site of binding of ethidium in the desensitized nAChR, we
used [3H]3,8-diazidoethidium ([3H]DAE) as a photoactivatible analog of ethidium. DAE
binds the agonist site with two affinities, KI1=10 mM, KI2=160 mM, similar to the binding
of ethidium. Additionally, DAE inhibits the binding of PCP in the desensitized state with
a KI of 1 mM, near the value for ethidium (Pedersen, 1995). Here we show that there is
PCP-inhibitable incorporation of [3H]DAE into nAChR in the desensitized state, and that
this incorporation is reduced in the presence of PCP. Specific incorporation into the 
and  subunits is present in the M2 segments. Incorporation is also present in peptide
fragments of  and  containing M1, but not M2 or M3.
20
DigestedV8-20
12000
Introduction
15
cpm
B
10
|
6
20
25
30
35
10
20
30
40
40
50
60
70
80
90
mm from bottom of gel
Fraction #
A 14kD band, V8-14 was isolated from a V8 digestion of  as described in the methods.
A portion of V8-14 was cleaved with EndoLysC, and the cleavage products, as well as
undigested V8-14, were separated by SDS-PAGE. Panel A: expected sites of cleavage of
V8-14 by EndoLysC. 3H eluted from gel slices of undigested V8-14 (Panel B) or from
gel slices of V8-14 digested with EndoLysC (Panel C). The radioactivity near 3 kD is
expected to be the fragment beginning at Met257. The 3H between 10 and 14kD is
expected to include undigested V8-14 as well as the fragment beginning at Phe206.
Panel A: Expected sites of cleavage of V8-20 by EndoLysC. Panel B: HPLC separation of
EndoLysC digest of V8-20 labeled in the absence () and presence () of PCP. Sequence
analysis of HPLC fraction 30 showed the presence of only one fragment, that beginning at
His186, for both conditions (-PCP: 5290 cpm loaded, 128 pmol; +PCP: 3330 cpm loaded,
208 pmol). Sequence analysis of fraction 34 showed the presence of only the fragment
beginning at the N-terminus of M2, Met243, for both conditions (-PCP: 11600 cpm
loaded, 123 pmol; +PCP: 7900 cpm loaded, 172 pmol).
4. Based on these photolabeling results, the high affinity ethidium site in
the desensitized nAChR is within the pore of the ion channel domain and
is made up of amino acids from M1 and M2 hydrophobic segments. These
results provide the first evidence of a NCA which interacts with both the
M2 and M1 hydrophobic segments.
Figure 6. Within  subunit, there is also incorporation of
[3H]DAE in a fragment containing M1 without M2.
Figure 2. In the  subunit, [3H]DAE photoincorporation
inhibitable by PCP is localized to the V8-20 fragment.
Reference List
M1
M2
M3
M4
NH2
COOH
V8-4 V8-18
V8-20
V8-10
Figure 4. Sequence analysis shows PCP-inhibitable
incorporation in M2 at Leu251, Ser252, and Leu258.
A
V8-20
Ile-192
M1
KF
KM M2
Akabas, M.H. and Karlin, A. Identification of acetylcholine receptor
channel-lining residues in the M1 segment of the -subunit. Biochemistry
34:12496-12500, 1995.
M3
EndoLysC digest to HPLC
Ile-192
M1
206-F
257-M M2
M3
243- M T L S I S V L L S L T V F L L V I V E L I P S T S S A V P
HPLC: Subunit fragments were purified by reversed-phase HPLC using a Brownlee Aquapore C4 column (100 x 2.1
mm). Solvent A was 0.08% trifluoroacetic acid in water; solvent B was 0.05% trifluoroacetic acid in 60%
acetonitrile, 40% 2-propanol. The elution gradient was from 25% to 100% solvent B in 80 minutes with a flow rate of
0.2 ml/minute. The elution of peptides was monitored by absorbance at 210 nm, and the elution of azidopyrenelabeled fragments was monitored by fluorescence emission. The elution of 3H was monitored by scintillation
counting of 50 ml out of each 500 ml fraction.
Sequencing: N-terminal sequence analysis was performed on an Applied Biosystems model 477A protein sequencer
using gas phase cycles. HPLC fractions were loaded directly onto glass fiber disks. Approximately 30% of the
released PTH-derivatives from each cycle were separated on a Model 120A PTH-derivate analyzer, while the
remaining derivates were analyzed by scintillation counting. Initial yields and repetitive yield were calculated by
nonlinear least squares regression of the observed release for each cycle.
10
60
4000
1
3000
2000
4
6
8
10 12 14 16 18 20 22 24 26 28 30
cycle
AChR-rich membranes were labeled with [3H]ethidium diazide in the presence of
2 mM carbamylcholine and the presence or absence of 100 mM PCP. The samples
were photolyzed and then digested with V8 protease as described in the methods.
The resulting gel was stained by Coomassie Blue (Panel A) and processed for
fluorography (Panel B). Samples in lanes 2 and 4 were treated with
endoglycosidase H prior to SDS-PAGE. Endoglycosidase H deglycosylates the
fragment which runs at 18kD and shifts its mobility to 12 kD.
Lanes 1 and 2: -PCP; lanes 3 and 4: +PCP.
Fraction 34 from the HPLC purification of V8-20 digested with EndoLysC
was subjected to Edman degradation as described in methods. ,: cpm
released per cycle (for absence and presence of PCP, respectively); , :
pmols of PTH-derivatives released per cycle (for absence and presence of
PCP, respectively). The solid and dotted lines indicate the exponential decay
fit to the detected PTH-amino acid derivatives.
Absorbance
0
15
20
20
25
30
25
35
30
35
40
F206 27 pmol
M257 19 pmol
40
Undigested V8-20
10
15 20 25
fraction #
30
35
F206 53 pmol
M257 4 pmol
40
Blanton, M.P. and Cohen, J.B. Identifying the lipid-protein interface of the
Torpedo nicotinic acetylcholine receptor: secondary structure implications.
Biochemistry 33:2859-2872, 1994.
Johnson, D.A. and Nuss, J.M. The histrionicotoxin-sensitive ethidium
binding site is located outside of the transmembrane domain of the nicotinic
acetylcholine receptor: A fluorescence study. Biochemistry 33:9070-9077,
1994.
Pedersen, S.E. Site-selective photoaffinity labeling of the Torpedo
californica nicotinic acetylcholine receptor by azide derivatives of ethidium
bromide. Mol.Pharmacol. 47:1-9, 1995.
Digested V8-20
-PCP
+PCP
1000
cpmPMOL79
released
-PCP
+PCP
2
1500
1250
1000
750
500
250
0
15
10
5
40
20
10
5
cpm
cpm
80
5000
B
5
cpm
-PCP
+PCP
100
Photoincorporation: nAChR-rich membranes isolated from Torpedo californica (2 mg/ml) were incubated in
Torpedo physiological saline (TPS: 250 mM NaCl, 5 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 5 mM sodium phosphate,
pH 7.0) with 10 mM [3H]diazidoethidium and other drugs as indicated. After a one hour incubation, oxidized
glutathione was added to a final concentration of 10 mM to act as a scavenger. Suspensions were irradiated for 30
seconds with a 254 nm lamp (EN-Spectroline). For analytical experiments, following irradiation, sample loading
buffer was added to each, and samples were submitted to SDS-PAGE. In some cases, after photolysis, the samples
were divided, and half were incubated with endoglycosidase H overnight prior to SDS-PAGE. For preparative
labelings, samples were pelleted and resuspended in TPS after photolysis. Membranes were incubated with 1azidopyrene for 90 minutes and then photolyzed for 15 minutes with a 365 nm lamp. Samples were then pelleted,
resuspended in sample loading buffer, and submitted to SDS-PAGE (Blanton and Cohen, 1994).
100
pmol
Methods
SDS-PAGE and proteolytic fragmentation: Polypeptides were separated by SDS-PAGE on an 8% acrylamide gel
(Blanton and Cohen, 1994). For preparative labeling, subunits were visualized using 1-azidopyrene fluorescence. For
S aureus V8 protease digestion, subunits were excised from an SDS-PAGE gel and placed in the well of a 15%
mapping gel. The subunit was overlayed with V8 protease, and the subunit was cleaved during electrophoresis. The
azidopyrene-labeled bands were excised, and the fragments eluted, concentrated, and acetone precipitated. For
EndoLysC digestion, fragments were resuspended in 15 mM Tris pH 8.1, 0.1%SDS, and 1.5 U EndoLysC
(Boeringher Mannheim) were added. Digestion proceeded for 7 days before purification of fragments.
pmol released
120
Arias, H.R. Luminal and non-luminal non-competitive inhibitor binding
sites on the nicotinic acetylcholine receptor (review). Mol Membrane Biol
13:1-17, 1996.
Tamamizu, S., Todd, A.P., and McNamee, M.G. Mutations in the M1 region
of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by
quinacrine. Cell.Mol.Neurobiol. 15:427-438, 1995.
0.72
Fluorescence
Absorbance
0.48
0.24
0.00
5
10
15
20
25
30
35
40
Fraction #
A 20 kD band (V8-20) was isolated from a V8 digest of  as described in the methods. A portion
of V8-20 was cleaved with EndoLysC, and the cleavage products were separated by HPLC.
Panel A: sites of cleavage of V8-20 by EndoLysC. Panel B: HPLC separation of EndoLysC
digest of V8-20 labeled in the absence () and presence () of PCP. Sequence analysis of
HPLC fraction 31 showed the presence of two fragments: -PCP: 3800 cpm loaded, 27 pmol
beginning at F206, 19 pmol beginning at M257; +PCP: 2200 cpm loaded, 12 pmol beginning
at F206, 5 pmol beginning at M257. The fragment beginning at F206 present in fraction 31 is
expected to contain the M257 fragment because, as is seen in the inset, undigested V8-20 also
runs in fraction 31. Sequence analysis of fraction 26/27 also showed the presence of two
fragments. -PCP: 2500 cpm loaded, 53 pmol F206, 4 pmol M257; +PCP: 1020 cpm loaded,
15 pmol F206, 6 pmol M257. Data from other experiments showed that the fragment in
fraction 27 beginning at Met257 is expected to end between the M2 and M3 segments.