ARCHIVES
OF
BIOCHEMISTRY
Cholinergic
T. LEE,
AND
BIOPHYSICS
183,
57-63
(19771
Ligand-Induced Affinity Acetylcholine V. WITZEMANN,
Changes Receptor’
M. SCHIMERLIK,
in Torpedo
califomica
M. A. RAFTERY
AND
Church Laboratory of Chenzical Biology, Diuisior1 0fChfvnistry rind Chcmiccd Engirwcring.” cazifwnia Institute
of Twhnologv. Received
Pasadena, September
CaIifornicl
9112.5
13, 1976
The binding of cholinergic ligands to Torpedo californica acetylcholine receptor has a-bungarotoxin been studied in vitro by inhibition of the time course of ‘“‘I-labeled receptor complex formation. The extent of inhibition was dependent on the duration of exposure to the ligand, the apparent affinity for ligand increasing with time, and was reversible upon removal of ligand. Ligand concentration, temperature. and Ca’+ ions influenced this effect which is reminiscent of receptor desensitization in uivo. Such effects were observed both for a cholinergic agonist. carbamylcholine, and for an antagonist, bis(3-aminopyridiniuml-1.10.decane diiodide. A minimal model is discussed which can account for these effects and for receptor ligand association leading to postsynaptic depolarization.
The association of AcCh3 with the nicotinic AcChR constitutes the first step in postsynaptic depolarization at the neuromuscular junction. Due to recent advances in AcChR identification and purification, this event can now be studied in vitro by direct binding methods using radiolabeled or fluorescent ligands, or indirectly by ligand-induced inhibition of the kinetics of AcChR-““I-labeled cu-Bgt complex formation. This latter method is relatively uncomplicated due to the irreversible nature of the receptor-toxin complex and is convenient using a simple direct assay procedure (1). In addition, the availability of membrane preparations highly enriched in AcChR from sources such as Torpedo
californica facilitates studies of the binding properties of the AcChR in its native membrane environment. Such studies provide an opportunity to determine various molecular parameters for the binding process, such as the number of ligandbinding sites on a receptor molecule, the possible existence of cooperative site-site interactions, and possible ligand-induced conformational changes related to receptor mechanism. In this communication we report studies of cholinergic ligand binding to T. californica AcChR-enriched membrane preparations and describe the characteristics of time-dependent effects which such ligands have on receptor properties. A minimal model for AcChR-ligand interaction leading to physiological effects is discussed.
’ This research was supported by U.S. Public Health Service Grant NS-10294 and by a grant from the Sloan Foundation. The authors are grateful for an NIH postdoctoral fellowship (to M. S.1, a fellowship from the Deutsche Forschungsgemeinschaft (to V. W.), and for an NIH Research Career Development Award (to M. R.). ” Contribution No. 5336. ‘I Abbreviations used: AcCh. acetylcholinc; AcChR, acetylcholine receptor; cu-Bgt. a-bungarntoxin; Dap, bis(3-aminopyridinium).1, 10.decane diiodide; Carb, carbamylcholinc; DEAE. diethylaminoethyl.
EXPERIMENTAL
PROCEDURES
Materials. Il’or&o californica was obtained live from local sources and was kept in a tank at 16”C, or electric organs were excised and frozen at -90°C. ““I-Labeled u-Bgt was prepared from purified Bgt (2) obtained from Bungurus multicinctus venom (Sigma Chemical Co.). Carbamylcholine tCarb) was obtained from Aldrich Chemical Co. and Dap was synthesized according to published procedures (3). AcChR-rich membrane fragments were prepared by 57
Copyright All rights
8 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
0003~9R61
58
LEE
ET
AL
preincubation a concentration in excess of this (lOPfi M, see Fig. 1) did not cause significant complexation. The half-time for conversion of the AcChR preparation to a high-affinity form for Carb (Figs. 1 and 2) was found to be dependent on ligand concentration (Table I). In all these cases the final (equilibrium) values for the 12”1-labeled cw-Bgt-AcChR complex formed were identical whether in the absence or presence of ligand, in accord with the apparent irreversible nature of this process. A further interesting property was that the conversion of the recepRESULTS tor to a high-affinity form was reversible The effects of preicubation with Car-b on (Fig. 3). Following pretreatment for 20 the kinetics of Y-labeled cY-Bgt-receptor min with 1.8 x lo-” M Carb, a preparation complex formation are shown in Figs. 1 was diluted (40 X) and a solution of Carb and 2. Pretreatment with the cholinergic (1 PM) and 12”1-labeled (Y-Bgt was added at agonist resulted in a dramatic reduction of varying time intervals followed by deterthe time course of receptor-toxin complex mination of the kinetics of receptor-12”1formation in the presence of the agonist. It labeled ~Bgt complex formation. As was demonstrated that the toxin bound shown in Fig. 3, the receptor reverted to a with similar observed rates to ligand-unlow-affinity form for the ligand as a functreated preparations and to ones which tion of time with a half-time of recovery of were treated with ligand for 30 min fol140-160 sec. lowed by dilution (X 40) just prior to lz51Such interconvertible forms of differing labeled (u-Bgt addition (Fig. 1, right). It ligand affinity were not restricted to Carb was therefore evident that (a) ligand such but were also observed with AcCh and as Carb dissociate rapidly from their rewith a variety of other cholinergic comceptor complexes and (b) pretreatment pounds. The most illustrative of these is with ligand does not alter the time course the compound Dap, an antagonist. The of toxin association with ligand-uncomdata in Fig. 4 demonstrate the affinity plexed AcChR. Obviously, pretreatment change observed using this ligand to inwith ligand depleted the amount of uncomhibit ‘““I-labeled a-Bgt-receptor complex plexed AcChR available for reaction with formation. This Dap-induced affinity 12”1-labeled a-Bgt (Scheme I). change was also reversible (Fig. 4) as in T the case of Carb and with a similar rate k, RL RT-k,R + L km, constant for recovery of low ligand afinSCHEME I ity. Thus the change from low- to high(R = AcChR, T = ‘2”I-labeled a-Bgt, L = ligand (Garb); affinity form induced by an antagonist as RT complex formation is irreversible; RL complex formawell as by an agonist and recovery of lowtion is reversible.) affinity binding, upon removal of ligand, are independent of the nature of the liSince we could independently show that gand. RL complex formation was rapid compared It is possible that the time-dependent with RT complex formation (to be pubincrease in affinity for agonist (Car-b) and lished), a reasonable explanation of the (Dap) is due to conversion of results in Figs. 1 and 2 is that a change of antagonist the AcChR from its ground state to the ligand affinity (to higher affinity) occurred species, since Carb upon pretreatment with a ligand such as same high-affinity shows an increased affinity for DapCar-b. The apparent dissociation constants treated AcChR-enriched membrane (Fig. for Car-b from all its binding sites in the high-affinity form range from 1 x lop8 to 5 4) and vice versa (data not shown). Alternatively, different but related high-affinx 10eR M (13). It was evident that without homogenization of electroplax in Ringer’s solution and fractionated by procedures previously detailed (4, 5) using Ringer’s-containing solutions throughout. Methods. ‘Y-Labeled a-Bgt-AcChR complex formation was determined using a DEAE-cellulose tilter-disc assay procedure (11. AcChR-rich membrane preparations (lo-‘-3 x IO-” M cY-Bgt binding sites) were treated with excess 12”I-labeled u-Bgt (8- to 12fold excess) with cholinergic ligand added simultaneously, or were pretreated with the ligand for periods of time varying from 1 to 30 min prior to izaIlabeled ~Bgt addition. The precise conditions for each experiment are given in the figure legends.
ACETYLCHOLINE
RECEPTOR
T,ne :m “1
AFFINITY
CHANGES
59
T,rrc ‘mrl
FIG. 1. Left: Time-dependent desensitization of AcChR-rich membrane fragments with Carb. AcChR (lo-’ M ol-Bgt-binding sites) was incubated in the presence of Carb (lo-” M) in Ringer’s solution at 25°C. At times indicated, n-Bgt (5 x lo-’ M) was added and the time course of toxin binding was determined. (A) Time course of toxin binding in absence of Carb. Final values for all curves were within 3% of 3 x lo4 cpm. Insert: Half-time of “desensitization” process: Toxin binding was plotted as In 2 (C, - C,/C,) (where C, is the toxin binding as measured at given time periods and C, is the toxin binding at equilibrium1 versus time, as described in Ref. (12). To determine half-time of “desensitization,” initial rates were extrapolated (m = In [2 (C, - C,lC,ll/min) and plotted versus incubation times of AcChR with Carb. Right: AcChR stock solution = 3.1 x lo-” M in ol-Bgt-binding sites. Concentration of ““I-labeled cy-Bgt in all assays = 0.3 x lo-” M. (--a--, AcChR diluted 40 x and time course of reaction with ““I-labeled a-Bgt measured. (-0-J AcChR stock solution made 0.9 x lo--” M in Carb, incubated for 30 min, diluted 40 x , and ‘?“I-labeled u-Bgt added. (-0-l AcChR diluted 40 x into Ringer’s solution containing lo-” M Carb and ““I-labeled n-Bgt. (-W--j AcChR stock pretreated for 30 min with 0.9 x lo-” M Carb and diluted 40 x into Ringer’s solution containing lo-” M Carb and ““I-labeled ol-Bgt. Infinity values were 18,000 ? 500 cpm for all time courses (measured after 6 hr of reaction).
ity forms could be involved. These results, coupled with the lack of dependence of recovery of low affinity on the nature of the ligand, are highly indicative of a process involving a change in receptor conformation. The conversion of AcChR to a highaffinity conformation(s) by cholinergic ligands was found to be dependent on additional factors (Table 1) such as ligand (Garb) concentration, temperature, and the presence of Ca’+ ions, all of which affected the rate of conversion to high-afflnity form(s). DISCUSSION
Several features of the in vitro conversion of the AcChR to a higher affinity form(s) in the presence of agonists are suggestive of this phenomenon being an in
vitro parallel of in vivo desensitization. Specifically, the dependence of the rate of onset on the parameters listed in Table 1 and the half-times observed are consistent with this notion. Katz and Thesleff (6) proposed a simple kinetic scheme to account for the phenomenon. In uivo experiments of Rang and Ritter (7) demonstrated that agonist-treated AcChR appeared to have a greater affmity for some antagonists, a phenomenon they termed a metaphilic effect. Recently Weber et al. (8) reported on studies of Torpedo marmorata electroplax membrane-bound AcChR being converted to a high-affinity form by several agonists but not by antagonists such as Dtubocurarine or gallamine. They also showed that hexamethonium behaved in an anomalous manner and suggested that
60
LEE
I ‘Ir -
1 a I ,I,lJii’
:
ET
1 I
FIG. 2. Effect of preincubation of AcChR-rich membrane fragments with Carb on the kinetics of ‘“:I-labeled cu-Bgt-receptor complex formation. Conditions of excess toxin-binding sites over toxin ([Carbl = 10 PM; [toxin-binding sites] = 8 x 10 i M; I toxin] = 5 x IO-’ M). (-0-l Membrane fragments preincubated with 10 pM Carb for 20 min followed by addition of “‘I-labeled cu-Bgt. The reaction followed simple first-order kinetics as expected. The slope, which is the pseudo first-order rate constant. reflects theK,, of the ligand; i.e., the greater the slope, the larger the K,, (C,: cpm of “‘I-labeled cu.Bgtt AcChR complex formed at time t). (-(J-j Reaction started by adding Carb and “‘I-labeled cu-Bgt simultaneously to the membrane fragments (final Carb concentration, 10 @MI. The slope was greater than that given above (-O--i at t = 0 and decreased gradually with time to a final value equal to that of (-0-j yielding evidence for a slow conversion of AcChR caused by Carb from a low-affinity to a highaffinity form. This process was estimated to have a half-time of 47 SW under the conditions used. See Table 1 for additional experiments. TABLE PARAMETERS
AFFECTING
AFFINITY
Solvent Ringer’s Ringer’s Ringer’s Ringer’s Ringer’s (Ca’*-free)
I HALF-TIME
Method” A B B B B
OF LICAND
FOR AcChR
CHANGE
T (“C)
[CarbJ
fl (set)
(/AM)
25 25 25 0 25
1.0 10 50 50 50
AL
80 47 17 37 39
k k -t+ k
8 5 3 3 3
” A, See legend to Fig. 1. B, Receptor concentrations ranged from 8 x 10 i to 2.8 x lo-” M oc-Bgtbinding sites. Initial I25I-labeled cu-Bgt concentrations correspondingly changed from 0.5 x 10 -’ to 1.7 X lom7 M.
its behavior could be explained by the metaphilic effect (7). As shown here, antagonists can also cause a change, for themselves or for agonists, to a conformation of higher affinity with the greatest effect thus far being observed with the antagonist Dap. The simplest model to account for desensitization first proposed by Katz and Thesleff (61, and subsequently discussed extensively by
FIG. 3. Recovery of “Carb-desensitized” AcChRrich membrane fragments. AcChR (3 x 10 ‘j M w Bgt-binding sites) in Ringer’s solution was incubated for 20 min at 25°C with Carb (1.1 x 10 ” M) followed by a 40-fold dilution into Ringer’s solution without Carb. for recovery. After times indicated (O10 min), the time course of toxin binding was measured upon addition of a-Bgt (5 x 10 ’ M) and Carb (10 Ii M) at the same time. Controls: (A) Dilution ix 40) of AcChR, without preincubation with Carb, into Ringer’s solution without Carb. (B) AcChR diluted into Ringer’s solution containing toxin and Carb (10 ” MI; no preincubation. Insert: Half-time of recovery of “desensitized” AcChR-rich membrane fragments. Initial toxin-binding rates determined as in Fig. 1 insert and plotted versus recovery time.
Rang and Ritter (91, is the cyclical model (Scheme II) where R represents receptor, A is an agonist, AR represents an agonistreceptor complex in the active (open channel) form and R’ represents desensitized receptor.
SCHEME
II
Such a scheme could also account for the metaphilic effect (7, 10) and was considered by Weber et aL.(8) to explain their
RECEPTOR
ACETYLCHOLINE
1
c Oo
I
1 2
3
Time
(mln)
4
AFFINITY
61
CHANGES
polarization in response to agonists. The minimal model which would include the known effects would have the form shown in Scheme III with R denoting receptor in its ground (resting) state, RL an initial agonist-receptor complex, RL’ and R’ the activated (depolarizing or open) receptor conformations, respectively, and RL” and R” the desensitized (high-affinity) forms.
r‘ 5
FIG. 4. Antagonist-induced desensitization toward agonist. In all assays, the concentration of AcChR and l”“I-labeled cx-Bgt were 0.64 x lo-” M and 5.1 x lo-” M, respectively. (1) ‘““I-labeled cu-Bgt binding in the absence of ligands. Membrane fragments were added at time zero to ?-labeled n-Bgt in Torpedo Ringer’s solution, 1 ml total volume. (21 Membrane fragments were added at time zero to lo-” M Carb plus ““I-labeled a-Bgt. (31 Membrane fragments preincubated with lo-” M Dap, diluted 50 x (2 x lo-’ M final Dap concentration), incubated an additional 20 min before simultaneous addition of Carb (to 10 ’ M) and “sI-labeled a-Bgt at time zero. (41 Membrane fragments preincubated as in (31, diluted (50 x 1 into lo-” M Carb plus ““I-labeled LU-Bgt at time zero. (51 Membrane fragments preincubated as in (31, diluted (50 xl into a solution containing lo-” M Carb, and incubated an additional 20 min before addition of ““I-labeled o-Bgt at time zero.
data. Our observation that antagonists, in addition to having a higher affinity for agonist-desensitized AcChR, can themselves cause conversion of the receptor to a high-affinity form clearly suggests that the cyclical model is incomplete in explaining the effects observed in vitro. A twostate scheme (11) is sufficient to explain “desensitization” by both agonists and antagonists, the fractions of AR and AR’ depending on the relative affinities of R and R’ for a given ligand. However, a two-state model is too restricted to include the physiologically important step of membrane de-
SCHEME
III
In the absence of agonist, the ground state of receptor (R) is favored over desensitized (R”) or activated (R’) forms. It is not possible at present to decide the relative contributions of equilibria K, or K,, for agonist-induced desensitization, or of K,, K, compared with K4, K, for depolarization. It should be emphasized that this and related (12,30) schemes do not include reference to the two classes of cr-Bgt- or ligand-binding sites on the AcChR molecule which we have observed for T. californica AcChR both in membrane-bound and purified form (12, 13) or to T. marmorata, T. nobiliana, Narcine braziliensis and Electrophorus electricus AcChR in purified form (29). It is clear that in order to obtain detailed understanding of events leading to receptor desensitization and to depolarization, the role of each class of binding site in a receptor molecule must be investigated since both sites may play important roles in each case. Several reports of high-affinity AcCh binding (K,, = 10P”-lO--’ M) to membrane fragments from T. marmorata (15-18) or to solubilized (19) and purified (20) Torpedo AcChR have appeared. Additionally Hill coefficients of 1.3-2 .O were observed in these studies, and it was suggested (21) that such positive cooperativity of AcCh binding could be responsible for positively
62
LEE
cooperative electrophysiological response. These studies were conducted by equilibrium dialysis and related methods where approach to equilibrium is slow compared with the rates of onset and recovery from conversion to a higher affinity form(s) reported here. Under such conditions ligand affinities would presumably depend on the extent of receptor desensitization, the latter varying in extent depending on ligand concentration, making analysis of equilibrium binding decidedly difficult. AcChR preparations which appear to be in either a high- or low-affinity state, without interconversion, have been reported; thus, membrane preparations from T. cal ifornica, purified following osmotic shock (4, 51, display higher affinity for ligands (13, 221, while Triton X-lOO-solubilized AcChR from such membranes, purified by affinity chromatography in 1% Triton (23-261, displayed only low ligand affinity (12, 27,28). Both of these preparations, frozen in highand low-affinity forms, showed Hill coefficients of unity or less than unity for all ligands tested. It is therefore not yet clear whether productive AcCh binding leading to depolarization has a Hill coefficient of unity or greater. It is evident that T. californica AcChR can exist in (at least) three separate forms of differing ligand affinity in a membraneassociated state, as depicted in Scheme III. Additional forms which are locked in a high-affinity state (13, 22) in membranes or locked in a low-affinity state in solution (12, 27, 28) have been described. It is possible that several factors such as interaction with lipids, ions, other membrane proteins, nonmembrane proteins, or receptorreceptor interactions could affect AcChR conformation further and so influence ligand affinity and physiological function.
J.,
AND
RAFTERY,
Biochem. 52, 349-354. 2. CLARK, D. G., MACMURCHIE. WOLCOTT, R. G., LANDEL, TERY, M. A. (1972)Biochemistry 3. MOOSER, G., SCHULMAN, H.,
AL 5I
M.
A. (1973)
Anal.
D. D., ELLIOT, E., A. M., AND RAF11,1663-1668. AND SIGMAN, D. S. (1972) Biochemistry 11, 1595-1602. 4. DUGUID, J., AND RAFTERY, M. A. (1973) Biochemistry 12, 3593-3597.
REED, AND
K.,
VANDLEN, RAFTERY, M.
phys. 138.
R., BODE, J., DUGUID, A. (1975) Arch. Bioch.
167: 138-144. B., AND THESLEFF,
6. KATZ,
S. (19571
J.
J., Bio-
Physiol.
63-80.
7. RANG,
H. P.. AND RITTER. J. M. (1969) Mol. Pharmacol. 5, 394-411. 8. WEBER, M., DAVID-PFEUTY, T., AND CHANGEUX, J.-P. (1975) Proc. Nut. Acad. Sci. USA 72, 3443-3447.
H. P., AND RITTER, J. M. (1970) Mol. I Pharmacol. 6, 357-382. RANG, H. P., AND RITTER. J. M. (1970) Mol. 10. Pharmacol. 6. 383-390. 11. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 12. RAFTERY, M. A., VANDLEN, R. L., REED, K. E.. AND LEE, 1’. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, 193-202. R. L., CLAUDIO, T., AND RAF13. LEE, T.. VAND~EN. TERY, M. A. (1976) In preparation. 14. SCHIMERLIK. M., QUAST, U., LEE, T.. WITZEMANN, V., BLANCHARD, S., AND RAFTERY, M. A. (1977) Submitted for publication. 15. ELDEFRAWI, M. E., BRITTEN, A. G., AND ELDEFRAWI, A. T. (19711 Science 173, 338-340. 16. ELDEFRAWI, M. E.. AND ELDEFRAWI, A. T. (19711 Proc. Nat. Acad. Sci. USA 69, 1776-1780. 17. WEBER, M.. AND CHANGEUX, J.-P. (1971) Mol. Pharmacol. 10. 15-34. J., AND CHANGEUX, J.-P. (1973) Bio18. COHEN, chemistry 12, 4855-4864. R. D., AND GIBSON, R. E. (1975) Arch. 19. O’BRIEN, Biochrm. Biophys. 169, 458-463. 20. ELDEFRAWI, M. E., AND ELDEFRAWI. A. T. (1973) Biochem. Pharmacol. 22, 3145-3150. 21. ELDEFRAWI. M. E., ELDEFRAWI, A. T., AND SHAMOO. A. E. (1976) Ann. N.Y. Acad. Sci. 264, 9.
RANG,
183-202. 22.
31 MV.
24.
REFERENCES 1. SCHMIDT,
ET
25.
26. 27.
RAFTERY, M. A., BODE, J., VANDLEN, R., CHAO, Y., DEUTSCH, J.. DUGUID. J., REED, K.. AND MOODY, T. (1974) in Proceedings of the Mos-
bath Colloquium, Symposium on the Biochemistry of Sensory Functions (Jaenicke, L., ed.), pp. 25, 541-564, Springer-Verlag, New York. SCHMIDT, J., AND RAFTERY, M. A. (1972) Biothem. Biophys. Res. Commun. 49, 572-578. SCHMIDT, J., AND RAFTERY, M. A. (1973a) Biochemistry 12, 852-856. RAFTERY, M. A., VANDLEN, R., MICHAELSON, D.. BODE, J., MOODY, T., CHAO, Y., REED, K., DEUTSCH, J., AND DUGUID, J. (1974) J. Supramol. Strut. 2, 582-592. VANDLEN, R. L., AND RAFTERY. M. A. (1976) Submitted for publication. MOODY, T., SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 53, 761-772.
ACETYLCHOLINE
RECEPTOR
28. MARTINEZ-CARRION. M., AND RAFTERY, M. A. (1973) Biochenz. Biophys. Res. Commun. 55, 11X-1164. 29. DEUTSCH, J., AND RAFTERY, M. A. (1977) Submitted for publication.
AFFINITY
CHANGES
63
30. CHANGEUX, J.-P., BENEDETTI, L., BOURGEOIS. J.-P., BRISSAR, A.. CARTAND, J., DEVAUX, P.. LGRIINHAGEN, H., MOREAU, M., POPOT, J.-L., SOBEL, A., AND WEBER, M. (1975) Cold Spring Harbor S.vmp. Quant. Bid. 10. 211-230.
OF
BIOCHEMISTRY
Cholinergic
T. LEE,
AND
BIOPHYSICS
183,
57-63
(19771
Ligand-Induced Affinity Acetylcholine V. WITZEMANN,
Changes Receptor’
M. SCHIMERLIK,
in Torpedo
califomica
M. A. RAFTERY
AND
Church Laboratory of Chenzical Biology, Diuisior1 0fChfvnistry rind Chcmiccd Engirwcring.” cazifwnia Institute
of Twhnologv. Received
Pasadena, September
CaIifornicl
9112.5
13, 1976
The binding of cholinergic ligands to Torpedo californica acetylcholine receptor has a-bungarotoxin been studied in vitro by inhibition of the time course of ‘“‘I-labeled receptor complex formation. The extent of inhibition was dependent on the duration of exposure to the ligand, the apparent affinity for ligand increasing with time, and was reversible upon removal of ligand. Ligand concentration, temperature. and Ca’+ ions influenced this effect which is reminiscent of receptor desensitization in uivo. Such effects were observed both for a cholinergic agonist. carbamylcholine, and for an antagonist, bis(3-aminopyridiniuml-1.10.decane diiodide. A minimal model is discussed which can account for these effects and for receptor ligand association leading to postsynaptic depolarization.
The association of AcCh3 with the nicotinic AcChR constitutes the first step in postsynaptic depolarization at the neuromuscular junction. Due to recent advances in AcChR identification and purification, this event can now be studied in vitro by direct binding methods using radiolabeled or fluorescent ligands, or indirectly by ligand-induced inhibition of the kinetics of AcChR-““I-labeled cu-Bgt complex formation. This latter method is relatively uncomplicated due to the irreversible nature of the receptor-toxin complex and is convenient using a simple direct assay procedure (1). In addition, the availability of membrane preparations highly enriched in AcChR from sources such as Torpedo
californica facilitates studies of the binding properties of the AcChR in its native membrane environment. Such studies provide an opportunity to determine various molecular parameters for the binding process, such as the number of ligandbinding sites on a receptor molecule, the possible existence of cooperative site-site interactions, and possible ligand-induced conformational changes related to receptor mechanism. In this communication we report studies of cholinergic ligand binding to T. californica AcChR-enriched membrane preparations and describe the characteristics of time-dependent effects which such ligands have on receptor properties. A minimal model for AcChR-ligand interaction leading to physiological effects is discussed.
’ This research was supported by U.S. Public Health Service Grant NS-10294 and by a grant from the Sloan Foundation. The authors are grateful for an NIH postdoctoral fellowship (to M. S.1, a fellowship from the Deutsche Forschungsgemeinschaft (to V. W.), and for an NIH Research Career Development Award (to M. R.). ” Contribution No. 5336. ‘I Abbreviations used: AcCh. acetylcholinc; AcChR, acetylcholine receptor; cu-Bgt. a-bungarntoxin; Dap, bis(3-aminopyridinium).1, 10.decane diiodide; Carb, carbamylcholinc; DEAE. diethylaminoethyl.
EXPERIMENTAL
PROCEDURES
Materials. Il’or&o californica was obtained live from local sources and was kept in a tank at 16”C, or electric organs were excised and frozen at -90°C. ““I-Labeled u-Bgt was prepared from purified Bgt (2) obtained from Bungurus multicinctus venom (Sigma Chemical Co.). Carbamylcholine tCarb) was obtained from Aldrich Chemical Co. and Dap was synthesized according to published procedures (3). AcChR-rich membrane fragments were prepared by 57
Copyright All rights
8 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
0003~9R61
58
LEE
ET
AL
preincubation a concentration in excess of this (lOPfi M, see Fig. 1) did not cause significant complexation. The half-time for conversion of the AcChR preparation to a high-affinity form for Carb (Figs. 1 and 2) was found to be dependent on ligand concentration (Table I). In all these cases the final (equilibrium) values for the 12”1-labeled cw-Bgt-AcChR complex formed were identical whether in the absence or presence of ligand, in accord with the apparent irreversible nature of this process. A further interesting property was that the conversion of the recepRESULTS tor to a high-affinity form was reversible The effects of preicubation with Car-b on (Fig. 3). Following pretreatment for 20 the kinetics of Y-labeled cY-Bgt-receptor min with 1.8 x lo-” M Carb, a preparation complex formation are shown in Figs. 1 was diluted (40 X) and a solution of Carb and 2. Pretreatment with the cholinergic (1 PM) and 12”1-labeled (Y-Bgt was added at agonist resulted in a dramatic reduction of varying time intervals followed by deterthe time course of receptor-toxin complex mination of the kinetics of receptor-12”1formation in the presence of the agonist. It labeled ~Bgt complex formation. As was demonstrated that the toxin bound shown in Fig. 3, the receptor reverted to a with similar observed rates to ligand-unlow-affinity form for the ligand as a functreated preparations and to ones which tion of time with a half-time of recovery of were treated with ligand for 30 min fol140-160 sec. lowed by dilution (X 40) just prior to lz51Such interconvertible forms of differing labeled (u-Bgt addition (Fig. 1, right). It ligand affinity were not restricted to Carb was therefore evident that (a) ligand such but were also observed with AcCh and as Carb dissociate rapidly from their rewith a variety of other cholinergic comceptor complexes and (b) pretreatment pounds. The most illustrative of these is with ligand does not alter the time course the compound Dap, an antagonist. The of toxin association with ligand-uncomdata in Fig. 4 demonstrate the affinity plexed AcChR. Obviously, pretreatment change observed using this ligand to inwith ligand depleted the amount of uncomhibit ‘““I-labeled a-Bgt-receptor complex plexed AcChR available for reaction with formation. This Dap-induced affinity 12”1-labeled a-Bgt (Scheme I). change was also reversible (Fig. 4) as in T the case of Carb and with a similar rate k, RL RT-k,R + L km, constant for recovery of low ligand afinSCHEME I ity. Thus the change from low- to high(R = AcChR, T = ‘2”I-labeled a-Bgt, L = ligand (Garb); affinity form induced by an antagonist as RT complex formation is irreversible; RL complex formawell as by an agonist and recovery of lowtion is reversible.) affinity binding, upon removal of ligand, are independent of the nature of the liSince we could independently show that gand. RL complex formation was rapid compared It is possible that the time-dependent with RT complex formation (to be pubincrease in affinity for agonist (Car-b) and lished), a reasonable explanation of the (Dap) is due to conversion of results in Figs. 1 and 2 is that a change of antagonist the AcChR from its ground state to the ligand affinity (to higher affinity) occurred species, since Carb upon pretreatment with a ligand such as same high-affinity shows an increased affinity for DapCar-b. The apparent dissociation constants treated AcChR-enriched membrane (Fig. for Car-b from all its binding sites in the high-affinity form range from 1 x lop8 to 5 4) and vice versa (data not shown). Alternatively, different but related high-affinx 10eR M (13). It was evident that without homogenization of electroplax in Ringer’s solution and fractionated by procedures previously detailed (4, 5) using Ringer’s-containing solutions throughout. Methods. ‘Y-Labeled a-Bgt-AcChR complex formation was determined using a DEAE-cellulose tilter-disc assay procedure (11. AcChR-rich membrane preparations (lo-‘-3 x IO-” M cY-Bgt binding sites) were treated with excess 12”I-labeled u-Bgt (8- to 12fold excess) with cholinergic ligand added simultaneously, or were pretreated with the ligand for periods of time varying from 1 to 30 min prior to izaIlabeled ~Bgt addition. The precise conditions for each experiment are given in the figure legends.
ACETYLCHOLINE
RECEPTOR
T,ne :m “1
AFFINITY
CHANGES
59
T,rrc ‘mrl
FIG. 1. Left: Time-dependent desensitization of AcChR-rich membrane fragments with Carb. AcChR (lo-’ M ol-Bgt-binding sites) was incubated in the presence of Carb (lo-” M) in Ringer’s solution at 25°C. At times indicated, n-Bgt (5 x lo-’ M) was added and the time course of toxin binding was determined. (A) Time course of toxin binding in absence of Carb. Final values for all curves were within 3% of 3 x lo4 cpm. Insert: Half-time of “desensitization” process: Toxin binding was plotted as In 2 (C, - C,/C,) (where C, is the toxin binding as measured at given time periods and C, is the toxin binding at equilibrium1 versus time, as described in Ref. (12). To determine half-time of “desensitization,” initial rates were extrapolated (m = In [2 (C, - C,lC,ll/min) and plotted versus incubation times of AcChR with Carb. Right: AcChR stock solution = 3.1 x lo-” M in ol-Bgt-binding sites. Concentration of ““I-labeled cy-Bgt in all assays = 0.3 x lo-” M. (--a--, AcChR diluted 40 x and time course of reaction with ““I-labeled a-Bgt measured. (-0-J AcChR stock solution made 0.9 x lo--” M in Carb, incubated for 30 min, diluted 40 x , and ‘?“I-labeled u-Bgt added. (-0-l AcChR diluted 40 x into Ringer’s solution containing lo-” M Carb and ““I-labeled n-Bgt. (-W--j AcChR stock pretreated for 30 min with 0.9 x lo-” M Carb and diluted 40 x into Ringer’s solution containing lo-” M Carb and ““I-labeled ol-Bgt. Infinity values were 18,000 ? 500 cpm for all time courses (measured after 6 hr of reaction).
ity forms could be involved. These results, coupled with the lack of dependence of recovery of low affinity on the nature of the ligand, are highly indicative of a process involving a change in receptor conformation. The conversion of AcChR to a highaffinity conformation(s) by cholinergic ligands was found to be dependent on additional factors (Table 1) such as ligand (Garb) concentration, temperature, and the presence of Ca’+ ions, all of which affected the rate of conversion to high-afflnity form(s). DISCUSSION
Several features of the in vitro conversion of the AcChR to a higher affinity form(s) in the presence of agonists are suggestive of this phenomenon being an in
vitro parallel of in vivo desensitization. Specifically, the dependence of the rate of onset on the parameters listed in Table 1 and the half-times observed are consistent with this notion. Katz and Thesleff (6) proposed a simple kinetic scheme to account for the phenomenon. In uivo experiments of Rang and Ritter (7) demonstrated that agonist-treated AcChR appeared to have a greater affmity for some antagonists, a phenomenon they termed a metaphilic effect. Recently Weber et al. (8) reported on studies of Torpedo marmorata electroplax membrane-bound AcChR being converted to a high-affinity form by several agonists but not by antagonists such as Dtubocurarine or gallamine. They also showed that hexamethonium behaved in an anomalous manner and suggested that
60
LEE
I ‘Ir -
1 a I ,I,lJii’
:
ET
1 I
FIG. 2. Effect of preincubation of AcChR-rich membrane fragments with Carb on the kinetics of ‘“:I-labeled cu-Bgt-receptor complex formation. Conditions of excess toxin-binding sites over toxin ([Carbl = 10 PM; [toxin-binding sites] = 8 x 10 i M; I toxin] = 5 x IO-’ M). (-0-l Membrane fragments preincubated with 10 pM Carb for 20 min followed by addition of “‘I-labeled cu-Bgt. The reaction followed simple first-order kinetics as expected. The slope, which is the pseudo first-order rate constant. reflects theK,, of the ligand; i.e., the greater the slope, the larger the K,, (C,: cpm of “‘I-labeled cu.Bgtt AcChR complex formed at time t). (-(J-j Reaction started by adding Carb and “‘I-labeled cu-Bgt simultaneously to the membrane fragments (final Carb concentration, 10 @MI. The slope was greater than that given above (-O--i at t = 0 and decreased gradually with time to a final value equal to that of (-0-j yielding evidence for a slow conversion of AcChR caused by Carb from a low-affinity to a highaffinity form. This process was estimated to have a half-time of 47 SW under the conditions used. See Table 1 for additional experiments. TABLE PARAMETERS
AFFECTING
AFFINITY
Solvent Ringer’s Ringer’s Ringer’s Ringer’s Ringer’s (Ca’*-free)
I HALF-TIME
Method” A B B B B
OF LICAND
FOR AcChR
CHANGE
T (“C)
[CarbJ
fl (set)
(/AM)
25 25 25 0 25
1.0 10 50 50 50
AL
80 47 17 37 39
k k -t+ k
8 5 3 3 3
” A, See legend to Fig. 1. B, Receptor concentrations ranged from 8 x 10 i to 2.8 x lo-” M oc-Bgtbinding sites. Initial I25I-labeled cu-Bgt concentrations correspondingly changed from 0.5 x 10 -’ to 1.7 X lom7 M.
its behavior could be explained by the metaphilic effect (7). As shown here, antagonists can also cause a change, for themselves or for agonists, to a conformation of higher affinity with the greatest effect thus far being observed with the antagonist Dap. The simplest model to account for desensitization first proposed by Katz and Thesleff (61, and subsequently discussed extensively by
FIG. 3. Recovery of “Carb-desensitized” AcChRrich membrane fragments. AcChR (3 x 10 ‘j M w Bgt-binding sites) in Ringer’s solution was incubated for 20 min at 25°C with Carb (1.1 x 10 ” M) followed by a 40-fold dilution into Ringer’s solution without Carb. for recovery. After times indicated (O10 min), the time course of toxin binding was measured upon addition of a-Bgt (5 x 10 ’ M) and Carb (10 Ii M) at the same time. Controls: (A) Dilution ix 40) of AcChR, without preincubation with Carb, into Ringer’s solution without Carb. (B) AcChR diluted into Ringer’s solution containing toxin and Carb (10 ” MI; no preincubation. Insert: Half-time of recovery of “desensitized” AcChR-rich membrane fragments. Initial toxin-binding rates determined as in Fig. 1 insert and plotted versus recovery time.
Rang and Ritter (91, is the cyclical model (Scheme II) where R represents receptor, A is an agonist, AR represents an agonistreceptor complex in the active (open channel) form and R’ represents desensitized receptor.
SCHEME
II
Such a scheme could also account for the metaphilic effect (7, 10) and was considered by Weber et aL.(8) to explain their
RECEPTOR
ACETYLCHOLINE
1
c Oo
I
1 2
3
Time
(mln)
4
AFFINITY
61
CHANGES
polarization in response to agonists. The minimal model which would include the known effects would have the form shown in Scheme III with R denoting receptor in its ground (resting) state, RL an initial agonist-receptor complex, RL’ and R’ the activated (depolarizing or open) receptor conformations, respectively, and RL” and R” the desensitized (high-affinity) forms.
r‘ 5
FIG. 4. Antagonist-induced desensitization toward agonist. In all assays, the concentration of AcChR and l”“I-labeled cx-Bgt were 0.64 x lo-” M and 5.1 x lo-” M, respectively. (1) ‘““I-labeled cu-Bgt binding in the absence of ligands. Membrane fragments were added at time zero to ?-labeled n-Bgt in Torpedo Ringer’s solution, 1 ml total volume. (21 Membrane fragments were added at time zero to lo-” M Carb plus ““I-labeled a-Bgt. (31 Membrane fragments preincubated with lo-” M Dap, diluted 50 x (2 x lo-’ M final Dap concentration), incubated an additional 20 min before simultaneous addition of Carb (to 10 ’ M) and “sI-labeled a-Bgt at time zero. (41 Membrane fragments preincubated as in (31, diluted (50 x 1 into lo-” M Carb plus ““I-labeled LU-Bgt at time zero. (51 Membrane fragments preincubated as in (31, diluted (50 xl into a solution containing lo-” M Carb, and incubated an additional 20 min before addition of ““I-labeled o-Bgt at time zero.
data. Our observation that antagonists, in addition to having a higher affinity for agonist-desensitized AcChR, can themselves cause conversion of the receptor to a high-affinity form clearly suggests that the cyclical model is incomplete in explaining the effects observed in vitro. A twostate scheme (11) is sufficient to explain “desensitization” by both agonists and antagonists, the fractions of AR and AR’ depending on the relative affinities of R and R’ for a given ligand. However, a two-state model is too restricted to include the physiologically important step of membrane de-
SCHEME
III
In the absence of agonist, the ground state of receptor (R) is favored over desensitized (R”) or activated (R’) forms. It is not possible at present to decide the relative contributions of equilibria K, or K,, for agonist-induced desensitization, or of K,, K, compared with K4, K, for depolarization. It should be emphasized that this and related (12,30) schemes do not include reference to the two classes of cr-Bgt- or ligand-binding sites on the AcChR molecule which we have observed for T. californica AcChR both in membrane-bound and purified form (12, 13) or to T. marmorata, T. nobiliana, Narcine braziliensis and Electrophorus electricus AcChR in purified form (29). It is clear that in order to obtain detailed understanding of events leading to receptor desensitization and to depolarization, the role of each class of binding site in a receptor molecule must be investigated since both sites may play important roles in each case. Several reports of high-affinity AcCh binding (K,, = 10P”-lO--’ M) to membrane fragments from T. marmorata (15-18) or to solubilized (19) and purified (20) Torpedo AcChR have appeared. Additionally Hill coefficients of 1.3-2 .O were observed in these studies, and it was suggested (21) that such positive cooperativity of AcCh binding could be responsible for positively
62
LEE
cooperative electrophysiological response. These studies were conducted by equilibrium dialysis and related methods where approach to equilibrium is slow compared with the rates of onset and recovery from conversion to a higher affinity form(s) reported here. Under such conditions ligand affinities would presumably depend on the extent of receptor desensitization, the latter varying in extent depending on ligand concentration, making analysis of equilibrium binding decidedly difficult. AcChR preparations which appear to be in either a high- or low-affinity state, without interconversion, have been reported; thus, membrane preparations from T. cal ifornica, purified following osmotic shock (4, 51, display higher affinity for ligands (13, 221, while Triton X-lOO-solubilized AcChR from such membranes, purified by affinity chromatography in 1% Triton (23-261, displayed only low ligand affinity (12, 27,28). Both of these preparations, frozen in highand low-affinity forms, showed Hill coefficients of unity or less than unity for all ligands tested. It is therefore not yet clear whether productive AcCh binding leading to depolarization has a Hill coefficient of unity or greater. It is evident that T. californica AcChR can exist in (at least) three separate forms of differing ligand affinity in a membraneassociated state, as depicted in Scheme III. Additional forms which are locked in a high-affinity state (13, 22) in membranes or locked in a low-affinity state in solution (12, 27, 28) have been described. It is possible that several factors such as interaction with lipids, ions, other membrane proteins, nonmembrane proteins, or receptorreceptor interactions could affect AcChR conformation further and so influence ligand affinity and physiological function.
J.,
AND
RAFTERY,
Biochem. 52, 349-354. 2. CLARK, D. G., MACMURCHIE. WOLCOTT, R. G., LANDEL, TERY, M. A. (1972)Biochemistry 3. MOOSER, G., SCHULMAN, H.,
AL 5I
M.
A. (1973)
Anal.
D. D., ELLIOT, E., A. M., AND RAF11,1663-1668. AND SIGMAN, D. S. (1972) Biochemistry 11, 1595-1602. 4. DUGUID, J., AND RAFTERY, M. A. (1973) Biochemistry 12, 3593-3597.
REED, AND
K.,
VANDLEN, RAFTERY, M.
phys. 138.
R., BODE, J., DUGUID, A. (1975) Arch. Bioch.
167: 138-144. B., AND THESLEFF,
6. KATZ,
S. (19571
J.
J., Bio-
Physiol.
63-80.
7. RANG,
H. P.. AND RITTER. J. M. (1969) Mol. Pharmacol. 5, 394-411. 8. WEBER, M., DAVID-PFEUTY, T., AND CHANGEUX, J.-P. (1975) Proc. Nut. Acad. Sci. USA 72, 3443-3447.
H. P., AND RITTER, J. M. (1970) Mol. I Pharmacol. 6, 357-382. RANG, H. P., AND RITTER. J. M. (1970) Mol. 10. Pharmacol. 6. 383-390. 11. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 12. RAFTERY, M. A., VANDLEN, R. L., REED, K. E.. AND LEE, 1’. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, 193-202. R. L., CLAUDIO, T., AND RAF13. LEE, T.. VAND~EN. TERY, M. A. (1976) In preparation. 14. SCHIMERLIK. M., QUAST, U., LEE, T.. WITZEMANN, V., BLANCHARD, S., AND RAFTERY, M. A. (1977) Submitted for publication. 15. ELDEFRAWI, M. E., BRITTEN, A. G., AND ELDEFRAWI, A. T. (19711 Science 173, 338-340. 16. ELDEFRAWI, M. E.. AND ELDEFRAWI, A. T. (19711 Proc. Nat. Acad. Sci. USA 69, 1776-1780. 17. WEBER, M.. AND CHANGEUX, J.-P. (1971) Mol. Pharmacol. 10. 15-34. J., AND CHANGEUX, J.-P. (1973) Bio18. COHEN, chemistry 12, 4855-4864. R. D., AND GIBSON, R. E. (1975) Arch. 19. O’BRIEN, Biochrm. Biophys. 169, 458-463. 20. ELDEFRAWI, M. E., AND ELDEFRAWI. A. T. (1973) Biochem. Pharmacol. 22, 3145-3150. 21. ELDEFRAWI. M. E., ELDEFRAWI, A. T., AND SHAMOO. A. E. (1976) Ann. N.Y. Acad. Sci. 264, 9.
RANG,
183-202. 22.
31 MV.
24.
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ET
25.
26. 27.
RAFTERY, M. A., BODE, J., VANDLEN, R., CHAO, Y., DEUTSCH, J.. DUGUID. J., REED, K.. AND MOODY, T. (1974) in Proceedings of the Mos-
bath Colloquium, Symposium on the Biochemistry of Sensory Functions (Jaenicke, L., ed.), pp. 25, 541-564, Springer-Verlag, New York. SCHMIDT, J., AND RAFTERY, M. A. (1972) Biothem. Biophys. Res. Commun. 49, 572-578. SCHMIDT, J., AND RAFTERY, M. A. (1973a) Biochemistry 12, 852-856. RAFTERY, M. A., VANDLEN, R., MICHAELSON, D.. BODE, J., MOODY, T., CHAO, Y., REED, K., DEUTSCH, J., AND DUGUID, J. (1974) J. Supramol. Strut. 2, 582-592. VANDLEN, R. L., AND RAFTERY. M. A. (1976) Submitted for publication. MOODY, T., SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 53, 761-772.
ACETYLCHOLINE
RECEPTOR
28. MARTINEZ-CARRION. M., AND RAFTERY, M. A. (1973) Biochenz. Biophys. Res. Commun. 55, 11X-1164. 29. DEUTSCH, J., AND RAFTERY, M. A. (1977) Submitted for publication.
AFFINITY
CHANGES
63
30. CHANGEUX, J.-P., BENEDETTI, L., BOURGEOIS. J.-P., BRISSAR, A.. CARTAND, J., DEVAUX, P.. LGRIINHAGEN, H., MOREAU, M., POPOT, J.-L., SOBEL, A., AND WEBER, M. (1975) Cold Spring Harbor S.vmp. Quant. Bid. 10. 211-230.
