Changeux, Jean-Pierre

Oswald, Robert

Roques, Bernard

Sobel, André

Waksman, Gilles

Elsevier - Publisher Connector 

Selective labelling by [3H]trimethisoquin azide of polypeptide chains present in acetylcholine receptor-rich membranes from Torpedo marmorata 

Volume 111, number 1 FEBS LETTERS February 19 80 
SELECTIVE LABELLING BY [ 3H]TRIMETHISOQUIN AZIDE OF POLYPEPTIDE CHAINS 
PRESENT IN ACETYLCHOLINE RECEPTOR-RICH MEMBRANES FROM 
TORPEDO MARMORATA 
Robert OSWALD, Andr6 SOBEL, Gilles WAKSMAN+, Bernard ROQUES+ and Jean-Pierre CHANGEUX 
Neurobiologie Molkulaire et Laboratoire associi au Centre National de la Recherche Scientifique, Interactions mokktlaires 
et cellulaires, Institut Pasteur, Paris and +UER des Sciences Pharmaceutiques et Biologiques, Dipartement de Chinlie 
Organique ERA 613 (CNRS), 4, avenue de I’Observatoire, 75270 Paris Cedex 06, b’rance 
Received 17 December 1979 
1. Introduction 
Membrane fragments in which the acetylcholine 
(ACh) receptor may represent up to forty percent 
of the proteins can be purified in large quantities 
(decigram levels) from Torpedo electric organ [l-3]. 
These membrane fragments, which most likely derive 
from subsynaptic areas of the electroplaque plasma 
membrane, appear particularly convenient to study the 
structural and functional properties of the ACh 
receptor and of the associated ionophore (reviewed 
[41X 
SDS-polyacrylamide gel electrophoresis in one 
dimension of the purified membranes yields several 
polypeptide chains [3.5,6]. The predominant subunit 
has an app. mol. wt 40 000 (40 k) and is affinity 
labelled by reagents (TDF [7], MPTA [3], MBTA 
[8], BrACh [9,10], DAPA [l l] known to bind 
covalently to, or in the close vicinity of, the ACh- 
receptor site. The 40 k subunit therefore carries the 
ACh-receptor site. 
Subunits of app. mol. wt 43 k, 50 k and 66 k have 
been found in most preparations of receptor-rich 
membranes with, depending on the authors, fixed 
[12] or variable [3,13-151 stoichiometries. In addi- 
Abbreviations: ACh, acetylcholine; 5-A[)H]T, 5-azido-[3H]- 
trimethisoquin; SDS, sodium dodecyl sulfate; MWA, 
4-(N-maleimido)phenyltrimethylammonium; MBTA, 44N- 
maleimide)benzyltrimethylammonium; TDF, trimethyl- 
ammonium diazonium fluoroborate; DAPA, bis-(3-azido- 
pyridinium)-1 JOdecane perchlorate; BrACh, bromoacetyl- 
choline 
ElsevierlNorth-Holland Biomedical Press 
tion, several aboratories have reported the presence 
of a band of app. mol. wt 60 k [5,12] (but 
[3,13-151). The 43 k polypeptide can be labelled by 
iodo [14C]acetate [131 or by a covalent derivative of 
the local anesthetic procaine [16] but does not seem 
to contain the binding site for non-competitive 
blockers of the response to acetylcholine [17]. The 
suggestion has been made that the 43 k peptide repre- 
sents actin [ 181; but it does not comigrate with 
Torpedo actin (J. Hofler, unpublished) and has a dif- 
ferent amino acid composition than skeletal muscle 
actin [ 131. Photoaffinity derivatives of the snake 
a-toxins label the 66 k chain in addition to the 40 k 
one [ 12,19,34] and labelling of the 50 k and 66 k chains 
has also been reported with bis-azido-[3H]ethidium 
bromide in the presence of carbamylcholine [ 121. The 
functional significance of these transmembrane [20] 
polypeptides is still a matter of controversy. 
These receptor-rich membranes contain a category 
of sites, distinct from the ACh-receptor site, to which 
bind non-competitive blockers of the response to ACh 
such as the aminated local anesthetics and the frog 
toxin histrionicotoxin. In order to identify which of 
the polypeptide chains present in the receptor-rich 
membranes carry this site(s), we have employed a 
radiolabelled photoaffinity derivative of the non-com- 
petitive blocker trimethisoquin, 5-azido [3H] tri- 
methisoquin whose synthesis and pharmacology is 
presented in [21]. 
Specific labelling of the site for the pharmacological 
action of the non-competitive blockers should fulfil 
criteria defined by studies of the non-covalent binding 
of radioactive local anesthetics to Torpedo membranes 
29 
Volume 111, number 1 FEBS LETTERS February 1980 
[ 15,221: the labelling should be inhibited by histrio- 
nicotoxin and unlabelled local anesthetics, enhanced 
by carbamylcholine, and the effect of carbamylcholine 
blocked by ol-bungarotoxin. 
Finally, the labelling should disappear after dissolu- 
tion of the ACh-receptor protein by high concentra- 
tion of Na-cholate under conditions where the 
allosteric interaction between the ACh-receptor site 
and the site for non-competitive blockers is abolished 
[23,24]. We report here the labelling of two peptides 
of mol. wt 50 k and 66 k that fullfil the requirements 
for specific binding to the site for non-competitive 
blockers. 
2. Methods 
2.1, Preparation of nzembranes 
ACh-receptor rich membranes were purified from 
the electric organ of Tolpedo marFnorata as in [3] and 
stored in liquid nitrogen until use. Non-receptor 
peptides, including the 43 k protein [ 131, were 
removed from the membranes by pH 11 treatment as 
in [ 171 modified [14,15] yielding ‘alkaline-treated 
membranes.’ Alkaline-treated membranes were sub- 
jected to the ‘reconstitution cycle’ [ 141 which consists 
of dispersing the membranes in 3% (w/w) Na-cholate. 
dilution to 1% (w/w) Na-cholate. and centrifugation 
yielding the ‘soluble extract’, followed by reconstitu- 
tion of the extract by filtration on a Sephadex G-50 
column. The fractions excluded from the column 
which did not contain Na-cholate were pooled as 
‘reconstituted AChR’. ACh-receptor sites were 
quantitated as in [25] using 1251-labelled cw-bungaro- 
toxin. 
2.2. Affinity labelling 
Samples of ACh-receptor rich membranes were 
diluted in Torpedo physiological solution (250 mM 
NaCl, 5 mM KCl, 4 mM CaC12, 2 mM MgC12, and 
5 mM Na-phosphate (pH 7.0)), and 5-A[3H]T and 
other test compounds were added to 100 ~1 final vol. 
The final concentration of a-bungarotoxin binding 
sites was 4-5 PM, and that of 5-A[3H]T was 5 PM. 
Incubations were performed in the dark for 1 h, 
after which the tubes were cooled on ice and irradiated 
for 5 min with a Mineralight short wave ultraviolet 
lamp (Ultraviolet Products, Inc.). Following irradia- 
tion, the samples were diluted with 100 1.t1 sodium 
dodecylsulphate (SDS) sample buffer (5% SDS, 4% 
30 
&mercaptoethanol, 13% glycerol, 0.002% bromphenol 
blue, 0.2 M Tris-HCl (pH 6.8)), and 10 ~1 were 
layered directly onto SDS-polyacrylamide gels. 
Membranes were labelled with [3H]M PTA as in 
[31. 
2.3. Pol~~acn~laFFlide gel eleCtrOphOreSiS _ _ 
SDS-polyacrylamide gels (10% acrylamide) were 
prepared as in [3] with 1 .I mm thick slabs. Gels were 
stained in 0.05% Coomassie brilliant blue R-250, 
destained, and scanned with a Vernon gel scanner 
using a yellow filter. 
Fluorography was performed as in [26] using PPO 
and dimethylsulfoxide. Gels were dried and exposed 
to Kodak X-Omat R film for 48 h at --70°C. The 
developed fluorograms were scanned on a Vernon gel 
scanner. 
2.4. Chemicals 
5-Azido-trimethisoquin was prepared as in [21] 
and tritiated using [3H]methyliodide at the CEA. 
Saclay, Service des Molecules Marquees: 12sI-labelled 
a-bungarotoxin was purchased from NEN, trimethiso- 
quin was a gift from Lab. Roger Bellon; and histrio- 
nicotoxin was a gift of Dr G. Kato. Native cu-bungaro- 
toxin was a gift of Dr Cassian Bon. Live Torpedo 
marmorata were provided by the Biological Station 
of Arcachon. 
3. Results and discussion 
When native ACh-receptor-rich membranes from 
Torpedo Fxarmorata were labelled with 5-A[3H]T 
[?I] in the absence of any effector (‘resting’ condi- 
tions) following the procedure in section 2, the major 
labelled product was the 40 k polypeptide (fig.lB, 
2B). The presence of the label in this band was con- 
firmed by the comigration of the 5-A[3H]T labelled 
polypeptide with the [3H]M PTA-labelled 40 k 
protein (fig. 1 J) and by two dimensional gel electro- 
phoresis (Dr T. Saitoh, personal communication) using 
the O’Farell method [27]. In addition to the 40 k 
band, a few minor bands of app. mol. wt 50 k, 66 k and 
95 k became labelled but to a smaller extent (<lo%) 
than the 40 k polypeptide. When the labelling of the 
membranes was carried out in the presence of an 
excess of unlabelled non-azido trimethisoquin (1 Ov4 M; 
fig.lC) or of histrionicotoxin (10m4 M; fig.lD,2C) the 
incorporation of radioactivity into the 50 k and 66 k 
Volume 111, number 1 FEBS LETTERS February 1980 
40 
- --- 
ABCDEFGHIJ 
Fig.1. Effect of various competitive and non-competitive 
blocking agents on the labelling of the ACh-receptor rich 
membranes from Torpedo marmorata by 5-A [ 3H IT. The 
membranes were incubated, irradiated, solubilizcd in SDS 
sample buffer, and pcptide chains separated by electro- 
phoresis as in section 2. Illustrated are the Coomassie blue 
stain (A) of the protein components and fluorograms of 
S-A[ ‘HIT (B-I) and [3H]M PTA (J) labelling. 5-A[‘H]T- 
labelling is shown under the followmg conditions: (B) 
resting; (C) unlabelled trimethisoquin (10e4 M); (D) HTX 
(1 0e4 M);(E) ol-bungarotoxin (1 O- 5 M); (F) carbamylcholine; 
(C) unlabelled trimethisoquin (10m4 M) and carbamylcholine; 
(H) HTX (1 OT4 M) and carbamylcholine; (I) ol-bungarotoxin 
(lo-’ M) and carbamylcholine. Carbamylcholine was present 
at 10m4 M in F-I. All competitive and non-competitive 
agents were added prior to the addition of 5-A[3H]T. 
polypeptides decreased similarly, but under the 
same conditions no effect was observed on the labelling 
of the 40 k polypeptide. On the other hand, when the 
membrane fragments were pre-incubated with a-bun- 
garotoxin (fig.lE), the labelling of the 40 k chain 
decreased markedly (but not completely) indicating 
that, a significant fraction of 5-A[3H]T incorporated 
into the 40 k band results from its covalent bonding 
to the ACh-receptor site. This finding is consistent 
with the fact that the 5-AT molecule contains a 
quaternary ammonium group [21] and therefore 
might exhibit a significant affinity for the ACh- 
binding site. Moreover it is known that local anesthetics 
may bind to the ACh-receptor site in addition to their 
specific binding site [22,28,29]. 
Fig.2. Scans of the Coomassie blue stained gel (A) and 
fluorograms of S-A[ ‘HIT labelling (B-E) from tlg.1. The 
following conditions are illustrated: (B) resting; (C) HTX; 
(D) carbamylcholine; (E) HTX and carbamylcholine. Con- 
centrations are given in the legend to fig. 1. 
31 
Volume 111, number 1 F’EBS LETTERS February 1980 
equilibration of the receptor-rich membranes wrth 
carhamylcholine, under conditions where the ACh- 
receptor is stabilized into its high affinity state 129,301, 
resulted in a marked change of the labelling pattern by 
5-A[3H]T (fig.lF,2D). In the presence of carbamyl- 
choline, the incorporation of radioactivity in the 50 k 
and 66 k protein increased markedly while the labell~ng 
of the 40 k became similar to that found with the 
resting membranes in the presence of cr-bungarotoxin 
(fig.1 E). As found with the resting membranes, the 
labelling of the SO k and 66 k bands (but not that of 
40 k) was blocked by un~abeI~ed trimethiso~u~n 
(fig.IG) and by histrionicotoxin (fig.1 H,2E). Moreover, 
preincubation of the membranes with a-bungarotoxin 
abolished the effect of carbamylcholine on the labelling 
of the SO k and 66 k ~olype~tides (fig.11). These data 
A 
’ x , iii 
123 
B 
123 
are consistent with the results of binding studies carried 
out with reversible fluorescent 1311 and radioactive 
[ 15,221 local anesthetics. In the presence of 
cholinergic agonists the high affinity binding of local 
anesthetics increases and this binding is blocked by 
hjstrionicotoxin, a characteristic hgand of the binding 
site for non-competitive blockers [32,33].Occupat~on 
of the ACh-receptor site by ~-bungarotoxin both 
prevents the accessory binding of local anesthetics to 
the ACh-receptor site and the allostcric effect of car- 
bamylcholine on the high affinity binding of focal 
anesthetics to their ‘specific’ site [ 151, 
The setectivity of5.A f3H]T binding in the presence 
of carbamylcholine is evident from the rather impure 
membrane preparation used for the experiments 
illustrated in fig. I,2 (see Coomassie blue stain in 
D 
r 
123 
F~~.~.z~-A[~H]T labelling of several fractions from the reconstitution cycle. Coomassie blue staining (1) and fiuorograms of 
S-A[ ‘HIT labelling in the presence (2) and absence (3) of 10e4 M carbamylcholine are shown for native (A), alkaline-treated (B), 
Na-cholate solubilized (C) and reconstituted (D) membranes. 
32 
Volume 111, number 1 FEBS LETTERS February 1980 
fig.lA and the scan in fig.2A). In this preparation, a 
number of polypeptide chains with app. mol. wt a.5 k, 
50-66 k and <40 k were present. The only non- 
receptor peptide labelled by 5-A[3H]T was the 95 k 
band, but the labelling of this chain was unaffected 
by competitive and non-competitive blocking agents 
and therefore must be considered as non-specific. In 
addition, no small molecular weight degradation 
products were observed when the sample in fig.1 F 
was run on a 20% acrylamideeSDS gel. 
Extraction of the 43 k polypeptide by pH 11 
treatment did not change the labelling pattern of the 
receptor-rich membranes by 5-A[3H]T (fig.3B2,3B3). 
As found with the native membranes the labelling of 
the 50 k and 66 k bands was still enhanced by car- 
bamylcholine and this labelling disappeared in the 
presence of unlabelled trimethisoquin, histrionicotoxin 
and a-bungarotoxin. This confirms that the 43 k 
protein is not involved in the binding of the non-com- 
petitive blockers to their specific site and in the recip- 
rocal allosteric interaction between this site and the 
ACh-receptor site [14,17]. 
When the alkaline-treated membranes were dis- 
persed into solution by Na-cholate under conditions 
where the allosteric interaction between cholinergic 
agonists and non-competitive blockers was abolished 
[14,23], the enhanced incorporation of 5-A[3H]T 
into the 50 k and 66 k bands caused by carbamyl- 
choline was no longer observed. On the other hand, 
removal of Na-cholate from the soluble receptor 
extract under conditions which preserve the allosteric 
properties of the ACh receptor protein [ 141 yielded a 
‘reconstituted’ receptor which presented a labelling 
pattern by 5-A[3H]T similar to that found with the 
native membranes. 
Conclusively, the covalent labelling of the 50 k 
and 66 k polypeptides by 5-A[3H]T follows the same 
regulation as the reversible binding of radioactive 
local anesthetics to their high affinity sites. Most likely, 
both series of compounds label the same class of sites. 
The question remains, however, as to why 5-A[‘H]T 
labels two different polypeptide chains rather than a 
single one (e.g., [3H]M PTA and the 40 k polypeptide). 
Several explanations may account for this rather 
paradoxical observation: 
(9 The 50 k and 66 k chains would not carry the 
binding site for non-competitive blockers but lie 
in its close vicinity and possess chemical residues 
with which the azide group of 5-A[3H]T reacts 
when this compound is bound to its specific 
site (for instance on the 40 k chain). 
(ii) The 50 k and 66 k polypeptides would differ 
from each other but nevertheless possess ites for 
non-competitive blockers with similar binding 
properties. 
(iii) The 50 k chain would carry the same site as the 
66 k chain and derive from this polypeptide by 
proteolysis. 
Future studies on the relative stoichiometries of 
the 50 k and 66 k polypeptides and on their quanti- 
tative labelling by 5-A[3H]T may distinguish between 
these various alternatives. 
Acknowledgements 
The authors wish to thank J. L. Popot, A. Devillers- 
Thiery, L. P. Wennogle, T. Saitoh and T. Heidmann 
for many helpful discussions, Professor J. Staros of 
Vanderbilt University for suggestions on the use of 
nitrenes, and Mrs T. Sciuto for typing the manuscript. 
This research was supported by grants from the 
Muscular Dystrophy Association of America, the 
College de France, the Delegation GCnCrale a la 
Recherche Scientifique et Technique, the Centre 
National de la Recherche Scientifique, the Institut 
National de la Sante et de la Recherche Medicale 
(contract no. 77.4.105.6) and the Commissariat a 
1’Energie Atomique. R.O. was recipient of a fellow- 
ship from the Muscular Dystrophy Association of 
America. 
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34 


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Selective labelling by [3H]trimethisoquin azide of polypeptide chains present in acetylcholine receptor-rich membranes from Torpedo marmorata

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