Published July 1, 1969
Isologous Oxygen, Sulfur, and Selenium Compounds
as Probes of Acetylcholine Receptors
HENRY G. M A U T N E R
From the Department of Pharmacology,Yale UniversitySchool of Medicine, New Haven,
Connecticut06510
It has been postulated for a long time that acetylcholine plays a crucial
role in the conduction of the nerve impulse both at synaptic and at axonal
sites. T h e proposal has been m a d e that it acts as a trigger inducing a conformafional change in excitable membranes during electrical activity, altering the membranes' permeability to cations (Nachmansohn, 1953, 1959).
I n view of the great biological importance of acetylcholine and the simplicity
of its structure (Fig. 1), very numerous analogues of this ester have been prepared and studied. Such modifications have been centered on the onium
group, the ester group, and the alkyl chain linking these groupings (Barlow,
1964). However, while a great m a n y acetylcholine analogues have been synthesized and their pharmacological actions investigated, this approach makes
the assumption, c o m m o n among scientists working with antimetabolites, that
the modification of the molecule will be localized at the point where it is
carried out. This assumption, which has been referred to as the "stick-toy
fallacy" (Mautner, 1967), ignores the fact that molecules are not stick-toys
and that replacement of one atom by another m a y alter electron distribution
and conformation throughout the molecule being modified.
I n an attempt to minimize this problem, our laboratory has beeta investigating the physico-chemical and biological properties of analogous oxygen,
sulfur, and selenium compounds. While the atomic radii of oxygen and sulfur
are rather different, the radii of sulfur and selenium are very similar with the
result that, while the structures of oxygen and sulfur isologs tend to differ,
the structures of crystals of sulfur and selenium isologs have been found to
-~71 s
The Journal of General Physiology
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ABSTRACT Evidence is presented that while the conformations of acetylcholine
and acetylthiolcholine are different, acetylthiolcholine and acetylselenolcholine
are structurally and conformafionally very similar. Experiments with sulfur and
selenium isologs of acetylcholine, choline, and local anesthetics suggest that the
active sites of receptors of the electroplax and of electric eel acetylcholinesterase
are different, but are compatible with the postulate that acetylcholine receptors
of axonal and synapfic excitable membranes are similar.
Published July 1, 1969
272 S
EXCITABLE
MEMBRANES
CHa
CHs~OCH,CH,~ CHs
CHs
0
FIo~
+
1. Acetylcholine.
proton magnetic resonance studies in deuterium oxide that the gauche conformation prevails in solution (Culvenor and Ham, 1966), while it could be
shown recently that the trans configuration of acetylthiolcholine and acetylselenolcholine are also retained in solution (R. J. Cushley and H. G. Mautner,
unpublished data).
Acetylcholine and acetylthiolcholine exhibit considerable differences in
their activities in a variety of biological preparations (Mautner, Bartels, and
Webb, 1966; Scott and Mautner, 1967). It was difficult to determine whether
these differences were due to the fact that acetylcholine is in the gauche
and acetylthiolcholine in the trans conformation or whether they were due to
differences in electron distribution. However, when the biological actions of
acetylthiolcholine and acetylselenolcholine were compared and again found
to be different it could be assumed that, in this case, electronic rather than
configurational factors had to be responsible for the differences in activity
noted, since, as can be seen in Fig. 3, the conformation of these molecules is
almost identical.
It should be added, as a note of caution, that rotational barriers in molecules of this kind are likely to be slight (Liquori, Damiani, and De Coen,
1968), and that the possibility exists that flexible molecules of different conformation might be induced to fit a common receptor.
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be extremely similar in all cases studied. X-Ray diffraction studies have
shown the crystal structures and packing patterns of rigid sulfur and selenium
isologs (such as 2-thiouracil and 2-selenouracil (Tsernoglou, 1966) or 2,4dithiouracil (Shefter and Mautner, 1967) and 2,4-diselenouracil) to be
virtually identical. More recently, it could be shown, by means of X-ray
diffraction, that even flexible molecules of isologous sulfur and selenium
compounds have essentially identical shape, size, and conformation, different
from those of their oxygen analogs.
X-Ray diffraction studies of crystalline acetylcholine have shown that the
--+N--C--C--O-- grouping follows the gauche arrangement (Canepa,
Pauling, and S6rum, 1966; Chothia and Pauling, 1968; Pauling, 1968), while
in acetylthiolcholine (Shefter and Mautner, 1969) and acetylselenolcholine
(Shefter and Kennard, 1966), the trans arrangement is seen (Fig. 2). Studies
of Sundaralingam (1968) have shown that in 12 molecules containing the
----+C--C--O---grouping the oxygen was always gauche to the nitrogen
atom. In the case of acetylcholine, it could be demonstrated by means of
Published July 1, 1969
273 s
Invited Discussions
The fact that replacement of either of the oxygen atoms of acetylcholine or
of local anesthetics by sulfur or by selenium greatly modifies the activity of
these compounds, while the replacement of the oxygen of choline by sulfur
or by selenium converts a compound devoid of depolarizing ability into an
effective depolarizing agent, has been useful for approaching some of the
questions surrounding the roles of acetylcholine in nerve conduction.
0
S, Se
N+
Fmu~ 2.
c 3c2
c~C5
ci
0
c2
C4L~C5
0
Se~~C7
t _ ) C 7
FIOUl~ 3. Conformationsof crystallineacetylthiolcholine(left) and acetylselenol-choline
(E. Shefter and H. G. Mautner, unpublished data).
For instance, it has been suggested that acetylcholinesterase and the biopolymer carrying the acetylcholine receptor of excitable membranes might be
identical (Belleau, 1964). If these receptor polymers were identical, then
sterically closely related compounds would exhibit similar relative abilities
to interact with the active sites of acetylcholinesterase and with the active
sites of the receptor polymer of excitable membranes. No such parallelism
could be detected. It can be seen in Table I that replacement of the sidechain oxygen of acetylcholine progressively lowers depolarizing activity in
the electroplax (Scott and Mautner, 1964; Webb and Mautner, 1966). On
the other hand, while the hydrolysis product of acetylcholine, choline, is
completely inactive, its thiol analog is a potent depolarizing agent. Oxidation
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Trans
Gauche
Published July 1, 1969
274 s
EXCITABLE MEMBRANES
of cholinethiol to choline disulfide yields a compound which blocks depolarization induced by carbamylcholine. In spite of the quantitative and even qualitative differences in the depolarizing activities of these molecules, all the
above compounds are bound to acetylcholinesterase to a similar degree (G.
R. Hillman and H . G. Mautner, unpublished data). Although comparisons
of events taking place on the membranes of living cells with events involving
binding to purified enzymes are dangerous, these findings, as well as the
findings of Podleski and Nachmansohn (1966) and Changeux et al. (1967)
suggest very strongly that the active sites of the membrane receptor and of
TABLE
I
COMPARISON OF THE RELATIVE ABILITIES
OF ACETYLCHOLINE, ACETYLTHIOLCHOLINE, ACETYLSELENOLCHOLINE,
C H O L I N E , C H O L I N E T H I O L , AND C H O L I N E D I S U L F I D E T O I N D U C E
DEPOLARIZATION IN THE ELECTKOPLAX
AND T H E I R A B I L I T I E S T O BIND T O A C E T Y L C t t O L I N E S T E R A S E
Acetylcholine
Acetylthiolcholine
Acetylselenolcholine
Choline
Cholinethiol
Choline disulfide
3 X
5 X
1 X
1X
3 X
1X
10-6
10-~
10-~ (to 65 my)
10-1 (inactive)
10-5
10-4 (blocks depolarization)
Acetylcholinestera~
1.0
0.6
0.3
4.0
5.0
3.0
X
X
X
X
X
X
10-4
10-4
10-~
10-4
10-4
10-*
K~
K,~
K,~
K¢
Ki
K¢
CHs
C6Hr-C--B--CH~CH2N/~CH3
A = O, S, Se
B = O, S, Se
F1ouIu~ 4.
Isologs related to local anesthetics.
acetylcholinesterase are different, although it is likely that these receptors
are related both functionally and structurally.
Studies of isologs of this type have also proved to be useful for throwing
light on the drug-receptor complex formed when compounds related to
acetylcholine interact with the active sites of membrane receptor or acetylcholinesterase. Thus, the observation that cholinethiol (pEa = 7.7) exhibits
higher depolarizing activity at pH's below the p K , than at pH's above the
pK~ of this molecule (Mautner, Bartels, and Webb, 1966), coupled with the
observation that methylation of its sulfur atom increases depolarizing activity
even further, emphasizes the importance of hydrophobic bonding and the
lack of importance of hydrogen-bonding in the depolarizing action of these
compounds.
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(Averagemolar concentrationto
depolarizeF_Aectroplaxto 45 mv)
Compound
Published July 1, 1969
Invited Discussions
275 s
Systematic variations carried out on simple local anesthetics have provided a useful approach to the question whether synaptic and axonal acetylcholine receptors are similar. The compounds synthesized (Chu and Mauther, 1968) are summarized in Fig. 4: Unfortunately, the selenocarbonylesters
did not prove stable enough for biological work.
When activities of these compounds were tested in axonal (giant axon of
the squid, Loligo pealei) and synaptic (electroplax, Electrophorus electricus) preparations, a striking parallelism in the relative activities of the various compounds could be seen (Rosenberg and Mautner, 1967; and G. D. Webb and
H. G. Mautner, unpublished data) (Table II). In both preparations replaceT A B L E II
A
B
Bloc.Hngaction in electroplax
Blocking
action, giant a x o n
of
squid
Esters
% inhibition (10-~ u solution)
0
0
0
0
s
Se
7
% InAibition (3XIO-' u solution)
0
40
99
40
100
Thionoesters
S
S
S
0
S
Se
% inhibition (10TM u solution)
°7oinhibition (1X 10TM u solution)
57
63
99
50
55
95
ment of the side-chain oxygen of the substituted benzoate by sulfur and
selenium resulted in a progressive increase in blocking action. On the other
hand, in both preparations, replacement of the side-chain oxygen of the
thionobenzoate by sulfur had only a negligible effect, while replacement of
this oxygen by selenium increased blocking activity significantly. Parallelism
of this kind does not prove the identity of synaptic and axonal receptors, it is,
however, compatible with the postulate that these receptors are similar.
T h e observation that reduction of disulfide groups to thiol groups inactivates electrical activity in the electroplax as well as in axonal preparations,
a process that can be reversed by reoxidation of the thiol groups, has been
used by Karlin (1968) as a guide for the successful design of active-sitedirected compounds for inactivating thiol groups near the "anionic sites" of
the electroplax. The finding that some selenolesters such as succinoylselenol-
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B L O C K I N G A C T I V I T Y O F 2 - D I M E T H Y L A M I N O E T H Y L BENZOATE,
2 - D I M E T H Y L A 1 V I I N O E T H Y L - T H I O N O B E N Z O A T E AND OF T H E I R
T H I O L E S T E R AND S E L E N O L E S T E R I S O L O G S IN T H E E L E C T R O P L A N P R E P A R A T I O N AND T H E G I A N T A N O N OF T H E S Q U I D
Published July 1, 1969
276 s
EXCITABLE
MEMBRANES
choline or benzoylselenolcholine block b o t h synaptic a n d a x o n a l r e c e p t o r s
irreversibly ( G o o d y e r a n d M a u t n e r , 1967; R o s e n b e r g a n d M a u t n e r , 1967),
c o u p l e d w i t h the o b s e r v a t i o n t h a t selenolesters are c a p a b l e of acylating
b o t h thiol a n d disulfide groups ( M a u t n e r a n d G/inther, 1961; G f i n t h e r
a n d M a u t n e r , 1965) suggests t h a t these c o m p o u n d s m a y also act as actives i t e - d i r e c t e d inactivators of m e m b r a n e receptors.
I t is interesting to n o t e t h a t in a x o n a l p r e p a r a t i o n s , the action of sulfh y d r y l reagents m a y be p o t e n t i a t e d b y electrical stimulation ( H i l l m a n a n d
M a u t n e r , 1968). I t m a y b e postulated t h a t electrical stimulation m i g h t
increase the nerve's p e r m e a b i l i t y to sulfhydryl reagents, or it m i g h t result
in the u n c o v e r i n g of " b u r i e d " thiol groups or in cleavage of disulfide groups.
I t is h o p e d t h a t c o n t i n u a t i o n of these studies m a y p r o v e to be useful for
a p p r o a c h i n g the p r o b l e m of a possible link b e t w e e n electrical activity a n d
c o n f i g u r a t i o n a l changes of excitable m e m b r a n e s d u r i n g n e r v e c o n d u c t i o n .
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It is a pleasure to acknowledge the interest and the frequent, generous hospitality of ProfessorDavid
Nachmansohn and his collaborators at Columbia University.
This work was supported by grants from the National Science Foundation (GB-6835) and the
National Institute of Neurological Diseases and Blindness (NB-07853).
Published July 1, 1969
Invited Discussions
~77 s
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