Eur. J. Biochem. 80, 225-242 (1977)

Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane-Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence Hans-Heinrich GRUNHAGEN, Motohiro IWATSUBO, and Jean-Pierre CHANGEUX Laboratoire de Neurobiologie Moleculaire, Institut Pasteur, Paris, and Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette (Received April 12, 1977)

Rapid changes of fluorescence intensity take place after fast mixing of quinacrine-labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 - 1000 ms, accessible to the stopped-flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d-tubocurarine, flaxedil or Naja nigricollis cx-toxin. In addition, preincubation of the membrane fragments with N . nigricollis a-toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the a-toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible. Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme A + B AB A*B, when

3

k2

k4

k, % k , , if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4=(1.5$-0.5) s p l , k3=(60f40) s-', k2>5 x 10' s-' and kl 3 lo71.mol - s ', K1 = k ~ / k= l (7 k 3) x 1O-' M, K= K 1 . k4/k3= (2 k 1) x 10 - 6 M ;for carbamylkI, , = ( l O + 5 ) s-', k,3102 s-l and k,>3 x lo5 s-', Kl = ( 3 f l ) x M choline: k4=(0.2+0.1) SK and R=(4+3) x lop6 M. The values of the apparent dissociation constants determined from the variation of the amplitudes of the fast effect for acetylcholine and carbamylcholine are respectively (1.010.5) x M and ( 5 + 2 ) x M. Some variability in the absolute values of the constants is noticed from one preparation to another and, for a given preparation, changes with age. Sets of kinetic constants have also been determined in the case of choline and suberyldicholine. At 20 "C, the Q,, is about 2.5 in the presence of 0.03 mM carbamylcholine and 1.3 in the presence of 0.01 M carbamylcholine; it is 1.3 with 0.05 M acetylcholine. The data are interpreted in terms of a three-state model and their physiological significance is discussed.

'

In a typical chemical synapse, such as the neuromuscular junction or the electroplaque synapse, the transmission of the nerve impulse is mediated by a chemical signal, the neurotransmitter, which after being liberated by the nerve terminal causes a transient and selective increase of permeability of the postsynapTrivial name. Tetram, O,O'-diethyl-S-(2-diethylamino)ethyl phosphorothiolate.

tic membrane to cations. Such fast opening of the ionic channels or 'activation' takes place in the millisecond range. When the neurotransmitter is applied to the subsynaptic membrane in a prolonged and continuous manner rather than as a brief pulse, a much slower process, lasting seconds or minutes, may follow. It manifests itself as a decrease of membrane permeability which is distinct from the reversal of the 'activation' reaction and is usually referred to as

226

‘pharmacological desensitization’. These phenomena have been investigated by means of sophisticated electrophysiological techniques principally in the case of the cholinergic synapse (for reviews cJ [l,21) leading, for instance, to the determination of the kinetic parameters of the permeability changes [3,4] and to indirect estimations of the transient increase of acetylcholine concentration during the physiological transmission of the impulse in vivo [5,6]. Despite their elegance these methods in vivo present several drawbacks. For instance, concentrations of effector can hardly be monitored ad libitum in a welldefined environment and direct determinations of the exact quantities of effector bound to the receptor sites are yet impossible. Systems in vitro, recently developed in this laboratory [7 - 91, now permit the simultaneous measurement, on the same membrane preparation, of the permeability to ions [ l l - 131, of the binding of cholinergic ligands [14] and of the structural transitions of membrane macromolecules and in particular of the acetylcholine receptor protein [lo, 15 - 191. The receptor-rich membrane fragments from Torpedo marmorata [9] appear particularly convenient to carry on such structural studies, since as much as 40% of their protein consist of the cholinergic receptor [9] (and Sobel, Weber and Changeux [59]). Interaction of cholinergic ligands with the receptor protein present in these fragments has been followed by fluorescence spectroscopy either with extrinsic probes [lo, 17 - 191 or by recording intrinsic changes of protein fluorescence [lo, 15,161. This last signal may be of some use to gain insight into the kinetics of the activation process [15,16] but the amplitude of the changes recorded, which remain within 1% of the total fluorescence, seriously limits the resolution of the kinetic analysis. This is why extrinsic labels, since they give much larger signals, yet appear more convenient for a physicochemical approach. Early studies on the structural transitions of the membrane-bound acetylcholine receptor were carried out with Dns-chol, 1 - ( 5 - dimethylaminonaphthalene - 1 - sulfonamino) propane-3-trimethylammonium iodide, a compound with mixed pharmacological properties which binds both to the acetylcholine binding site and to ‘secondary’ sites related to the local anesthetic binding sites [17]. More recently, fluorescent labels have been developed which, like quinacrine [lo] or ethidium bromide [20], bind preferentially to this last class of sites while others, like some acylcholine derivatives, act as agonists and interact selectively with the acetylcholine receptor site [18]. This paper deals with the fast kinetics of the fluorescence response as observed in T. marmorata receptor-rich membrane fragments labelled with quinacrine. As shown previously, quinacrine acts in vivo on Electrophorus electroplaque as a local anesthetic and, accordingly [lo], does not compete in vitro with

Fast Acetylcholine Receptor Kinetics in vitro: Quinacrine Fluorescence

agonists or antagonists for the acetylcholine receptor site but on the contrary, enhances the affinity of this site for agonists [lo]. Binding of quinacrine to receptor-rich membrane fragments leads to an enhanced fluorescence intensity [lo], which increases further upon addition of agonists and some competitive antagonists. This last effect is blocked by snake a-toxins, which by themselves do not change the fluorescence level at rest. The variation of the amplitude of fluorescence intensity as a function of cholinergic effector concentration yielded equilibrium dissociation constants which are in close agreement with those determined by following directly the binding of radioactive ligands at equilibrium [lo]. Under conditions of fluorescence energy transfer from protein to quinacrine the equilibration processes after addition of cholinergic effectors revealed a qualitative difference between agonists and antagonists [19]. Addition of an agonist to yield a high concentration of about 100 times the equilibrium dissociation constant causes a fast increase of the fluorescence intensity, which is followed by a decreasing phase in the minute range, before a stable final level is reached. A stepwise addition of small quantities of agonists leads to the same final level, but instead of the transient overshoot of the fluorescence intensity only a slow increase in the minute range is observed. This transient overshoot was never observed with known antagonists, not even at a high concentration. It was therefore attributed to the physiological process of receptor ‘activation’ [19], the slow subsequent processes in the minute range being possibly related to the ‘desensitization’ phenomenon. To account for these data a three-state model of the receptor was proposed according to which the receptor.ionophore complex may exist in discrete states in reversible equilibrium, corresponding respectively to a ‘resting’, ‘active’ and ‘desensitized’state of the system [19]. These states would differ, in particular, by their affinities for agonists: the binding of acetylcholine to the pre-existing resting state with a low affinity transforming the receptor towards states of higher affinity, first to the active state with an intermediate affinity and finally to the desensitized state with the highest affinity [19]. According to this threestate model, the slow desensitization process should be paralleled by an increase of affinity. Indeed, during the first minutes of contact of the membrane-bound receptor with an agonist such an increase of affinity was independently shown to take place using an cc-toxin binding technique [21]. Also the ‘desensitizazation’ of the permeability response in vivo [I31 and in vitro [11,121is known to occur in Torpedo marmorata electroplaque [I31 or microsacs in the second to minute range. Whereas the slow fluorescence changes in the minute range could be analyzed by means of a static

221

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

spectrofluorimeter, the increase of fluorescence intensity observed in the presence of high concentrations of an agonist is too fast to be resolved by that technique. This paper presents a quantitative analysis of the changes of quinacrine fluorescence recorded in the millisecond and second time range after rapid mixing of agonists with receptor-rich membrane fragments by means of the stopped-flow technique. Part of the data has been reported in preliminary communications [22,23].

MATERIALS AND METHODS Receptor-rich membrane fragments from Torpedo marmorata electric organ were prepared by the method of Cohen et al. [9] modified as follows. Fresh electric tissue was homogenized in its volume of 10 mM CaC1, in distilled water, centrifuged 10 min at 5 000 x g ; then the pellet was rehomogenized in an equal volume of 10 mM CaCl, and centrifuged for 10 min at 5 000 x g. The collected supernatants were passed through cheesecloth and added on top of a layer of 1.2 M sucrose (20 ml crude supernatant over 5 ml sucrose solution in a Beckman 30 rotor or 50 ml of crude supernatant over 15 ml of sucrose solution in a Beckman 35 rotor). After centrifugation at 100000 x g for 90 min at 4 "C the pellets were collected and resuspended in a sucrose solution to yield a sucrose concentration of approximately 0.6 M. The membrane suspensions were stored at 4 "C. Proteins were assayed by the method of Lowry et al. [24] with bovine serum albumin as standard. The concentration of acetylcholine receptor sites was estimated by a Millipore filtration assay following the binding of the a,- [3H]isotoxin from Nu@ nigricollis [25,26]. Specific activities were in general close to 1000 nmol a-toxin sites/g protein. The exact values are given in the legends of the figures or tables. DL-Quinacrine (dihydrochloride dihydrate salt) was a gift from Rh8ne Poulenc Inc. and was used without further purification. The analytical data were (calculated values in parentheses): N, 8.1 % (8.25%); C1, 21.1% (20.9%); H,O (Fischer), 7.1% (7.1%); melting point approx. 245 "C (decomp.). Stock solutions were prepared in Torpedo physiological saline solution (250 mM NaC1, 5 mM KCl, 4 mM CaCl,, 2 mM MgCl,, 5 mM phosphate buffer pH 7). Quinacrine solutions were made weekly and kept in the dark. The purified a,-isotoxin of N. nigricollis was a gift of Dr P. Boquet and was tritiated by Drs A. Menez, J. Morgat and P. Fromageot. Ceruleotoxin was a gift of Dr C. Bon [27]. Cholinergic effectors were used without further purification : acetylcholine iodide (Eastman), carbamylcholine chloride (K K), phenyltrimethylam-

+

monium chloride (Eastman), choline chloride (Eastman), hexamethonium bromide (K + K), flaxedil (gallamine triethyliodide) (Specia), decamethonium bromide (K K), d-tubocurarine chloride (Sigma). To avoid hydrolysis, aqueous solutions of acetylcholine were prepared immediately before use. In some cases ethanolic stock solutions of acetylcholine were used. In these cases the final concentration of ethanol in the sample containing membrane fragments was less than 1%. Control experiments without ethanol revealed no significant alteration of the kinetics by this low dose of ethanol. Where indicated, the membrane fragments were incubated with Tetram, 0,O'-diethyl-S-(2-diethylamino)ethyl phosphorothiolate, a potent acetylcholine esterase inhibitor [28]. Suberyldicholine iodide [29] was a gift from Drs B. Sakmann and J. Heesemann. The stopped-flow experiments were carried out in a Durrum-Gibbson rapid-mixing spectrometer equipped with fluorescencedetection. Except for therneasurement of the temperature dependence, the sample-containing system was therinostated at 20 "C. Fluorescence was excited at 295 nm using a 450-WOsram xenon lamp and a grating monochromator (JobinYvon HRS 2). 90" fluorescence in the observation cell (quartz tube of 2-mm diameter and 18-mm length) was monitored by an Hamamatsu R 376 photomultiplier after having passed a MTO 538b filter and a cut-off filter at 310 mm. Unless otherwise specified, two equal volumes of a suspension of membrane fragments and of a solution of cholinergic effector were mixed, each containing the same total concentration of quinacrine in Torpedo physiological saline solution. The dead time of the mixing device was 2.5 ms as determined by oxidizing reduced cytochrome c by Fe3 . Single-shot fluorescence signals were digitally stored in a Tracor NS 570 (12 bits, 1024 points) and plotted with an X - Y recorder. The experimental traces were analyzed by means of graphical methods.

+

+

Analysis of the Kinetic Data The occurrence of first-order kinetics in a bimolecular reaction may be based on a simple reversible equilibrium :

A+B~'.c kb

( k f ,k, being the forward and backward kinetic constants) as long as the concentrations of the reactants are such that cB% cA. In this case, the apparent firstorder rate constant kobs will depend linearly on cB:

A deviation from linearity indicates a more complex mechanism of the reaction.

228

Fast Acetylcholine Receptor Kinetics in vitro: Quinacrine Fluorescence

A reaction scheme involving a binding equilibrium and a subsequent isomerization equilibrium is frequently observed in enzymology: A + B & AB 3 A*B

(3)

k4

k2

( k l , k 2 , k , , k 4 : kinetic constants, A*: isomer of A). If the binding step reaches equilibrium much faster than the isomerization step, i.e.

(4)

k2 % k3

and if the signal to be monitored parallels the isomerization from A to A*, the observed kinetics are of pseudo-first-order and the perfectly linear semilogarithmic plots of the experimental traces show no lag phase or initial burst [30]. An initial lag phase is expected if k , % k , (steady-state condition). An initial burst of the binding reaction would appear, if instead of the reaction in Scheme (3) an inverse sequence with a pre-existing equilibration between A and A' was the basis of the reaction: A

A'

SA ~ B ,

(5)

-B

where A' is an isomer of A and the signal parallels the formation of A'B. If, on the other hand, the signal parallels the isomerization from A to A', the limiting rate at high cB would be independent of the nature of the ligand B. In contrast, the reaction scheme in Eqn (3) can account for the occurrence of different limiting rates at high concentrations of different ligands. The kinetics of the reaction scheme in Eqn (3) can be analyzed, if one applies the steady-state condition to the species AB. One obtains [30]: klcB ( k 3 + kobs

k4)

k , cB k,

=

k1k3cB

kt cB

+ k2

Having determined the kinetic constants according to Eqns (6) and (7) or (8) and (9), the thermodynamic dissociation constant of the binding equilibrium in Scheme (3) can be evaluated. The ratio of slope to intercept yields (10)

k21kl = K , .

The crucial assumption of this analysis, k,%k3 (cf. Eqn 4), allows us to evaluate on the basis of k , a lower limit of the value of k , . This lower limit of k , may be used to estimate a lower limit for the kinetic binding constant k , :

Furthermore, an overall thermodynamic dissociation constant K may be defined, which describes the binding of B to A* in equilibrium with A (cf. Eqn 3):

+ k4.

A plot of the apparent first-order rate constant kobsas a function of cB yields a non-linear function, which levels off at high c B . The intersection with the ordinate axis at cB=O gives k,. In general, a doublereciprocal plot of (kobs)-lversus (cB)-l will be nonlinear, too. If cB+ cA, (kobs)-llevels off at high values of (cB)-' and extrapolates to (k4)-I. At low values of (cB)-' extrapolation of the function to its intersection with the ordinate axis gives ( k 3 + k 4 ) - ' . Only in cases where k4 is zero or negligibly small does a plot of (kobs)-' versus (cB)-l become linear and intersect the ordinate axis at: (l/ k o b s ) ( l , c r , =O)

and from the slope one obtains:

'

The equilibrium restriction imposed by Eqn (4) reduces this dependence to : kobs

In the general case where k4 is finite, the non-linear plot of (kobs)-' versus (cB)-' can be linearized by a secondary treatment of the experimental first-order constants [30], i.e. by plotting (kobs- k4)-' versus (cB)-'. Besides the criterion of linearity in the fitting procedure, there are two additional aides to estimate the value of k,: the determination of (a) the intercept in the plot kobsversus cB and (b) the reciprocal value of (kobs)-' for which the apparent first-order constant reaches an asymptotic value at high (cB)-l. In addition to the value of k4 this secondary plot yields k , , which is given by the intersection with the ordinate axis:

+ k2k4

+ +k ,

=

its slope is:

= 11'3

(6)

If the signal to be monitored parallels the isomerization of A to A* and if the isomerized state A* is only populated after binding of a ligand B to A, the overall dissociation constant K , as determined by means of kinetic methods, should be related to the apparent dissociation constant Kappas evaluated on the basis of the signal amplitude, i.e.: Kapp

(CB)balf-maximum

amplitude 9

(13)

where Kappis defined as the concentration of ligand B giving rise to a half-maximum signal amplitude.

229

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

+acetylcholine

(1)

H 20ms

-_

+phenyltrirnethylammonium

(2)

1 !-

H 500rns

-D c

5

x

_ -

c Y)

H lOOrns

+ d -tubocurarine a,-

) 3 (>

c

~

Time

H 100ms +f laxedil l

I/

(

Time

4

)

-

Fig. 1. Single-shot traces of stopped-flow experiments: change of quinacrine fluorescence intensity after mixing of T. marmorata receptor-rich membrane fragments with cholinergic effectors. Excitation at 295 nm, emission monitored at 538 nm (cf: Materials and Methods) 1: 1 mixing of a suspension of receptor-rich membrane in sites, 0.5 mg/ml protein) in fragments (0.4 pM ~ - [ ~ H ] t o xbinding Torpedo physiological saline solution,4pM quinacrine, with Torpedo physiological saline solution, 4pM quinacrine, containing (from top): (1) 20 mM acetylcholine iodide, (2) 1 mM phenyltrimethylammonium chloride, ( 3 ) 0.1 mM d-tubocurarine chloride, (4) 2 mM flaxedil (gallamine triethyliodide). Dead time at the beginning of all traces 2.5 ms. Ordinate scale: 5.5 "/,/divisionchange ofthe fluorescence intensity. The experiments with phenyltrimethylammonium, d-tubocurarine and flaxedil were carried out the same day with the same preparation, the acetylcholine experiment with a different preparation

RESULTS QUALITATIVE OBSERVATIONS

Fig. 1 shows experimental traces reflecting the time dependance of the changes of quinacrine fluorescence intensity after mixing receptor-rich membrane fragments with various cholinergic effectors. Mixing with the agonists : acetylcholine or phenyltrimethylammonium causes an increase of the fluorescence intensity in the time range accessible to the stoppedflow technique, i.e. the millisecond and second time range. Such an increase of the fluorescence is also found with other agonists, such as carbamylcholine (cJ: Fig. 2 and 3), choline, and suberyldicholine. As seen in Fig. 1, no fast increase of fluorescence intensity takes place after mixing of the receptor-rich membranes with the antagonists d-tubocurarine or flaxedil. Finally, the mixing procedure as such does

-

Fig. 2. Single-shot traces of stopped-$ow experiments. Fast increase and subsequent slow decrease of quinacrine fluorescence intensity after binding of carbamylcholine to T . marmorata receptor-rich membrane fragments. Excitation at 295 nm, emission monitored at 538 nm. 1 : 1 mixing of the following components in Torpedo physiological saline solution, 4 pM quinacrine: receptor-rich membrane fragments (0.4 pM ~ - [ ~ H ] t o xbinding in sites, 0.4 mg/ml protein) and 1 mM carbamylcholine chloride. Final concentrations: 4 pM quinacrine, 0.2 pM ~ - [ ~ H ] t o x ibinding n sites, 0.5 mM carbamylcholine. Ordinate scale: 3.1 %:division change of the fluorescence intensity. Dead time: 2.5 ms. The two traces represent two separate experiments

not alter the membrane fragments or affect quinacrine binding to cause a measurable fluorescence signal in the absence of effector [22]. These experiments demonstrate a qualitative distinction between agonists and antagonists. The bisquaternary compounds decamethonium and hexamethonium, however, give a particular response. No resolvable fluorescence response takes place in the stopped-flow time range [22], but an analysis of fluorescence amplitudes indicates that a fluorescence increase exists but is too fast to be resolved by the mixing technique. In the case of acetylcholine or carbamylcholine a detailed analysis of the experimental amplitudes has shown that any possible fast fluorescence increase of this sort, preceding the resolved effect in the millisecond time range, does not exceed 20% of the amplitude of the resolved signal. On the basis of equilibration studies in the minute range it had been concluded that binding of an agonist to the membrane-bound receptor gives rise first to a fast increase of intensity of quinacrine fluorescence before a subsequent slow decrease shows up [19]. Fig. 2 demonstrates indeed that the stopped-flow technique allows us to analyze the fast fluorescence increase preceding the partial slow decrease in the minute range.

230

Fast Acetylcholine Receptor Kinetics in uitro : Quinacrine Fluorescence

I I

tcarbarnylcholine

t I

1

0

I

I

200

I

L

.. w

1

I

400

1

I

600 Time (ms)

1

800

,

I

1000

Fig. 3. Single-shot traces of stopped-jlow experiments: blockinz effect o f t h e a-toxin from Naja nigricollis on quinacrinefluorescence increase, caused by carbamylcholine or acetylcholine binding to receptor-rich membrane fragments. Excitation at 295 nm, emission monitored at 538 nm. 1 : 1 mixing of the following components in Torpedo physiological saline solution, 4 pM quinacrine (from top): (I) receptor-rich in sites, 0.3 mg/ml protein) with 1 mM carbamylcholine chloride; (2) receptor (as above), membrane fragments (0.4 pM ~ e [ ~ H ] t o x binding incubated for 40 min in N . nigricollis u-toxin 1.2 pM, with 1 mM carbamylcholine chloride; ( 3 ) receptor (as above) with 0.1 mM acetylcholine iodide; (4) receptor (as above) incubated for 15 min in 1.2 pM a-toxin, with 0.1 mM acetylcholine iodide; (5) receptor (as above), incubated for 30 min in 1.2 pM a-toxin, with 0.1 mM acetylcholine iodide, 1.2 pM a-toxin. Ordinate scale: 3.0%/division change of the fluorescence intensity. Dead time: 2.5 ms

Incubation of the membrane fragments with a saturating concentration of a-toxin before mixing with an agonist gives a complete blocking of the fast increase of fluorescence intensity caused by agonists (cf. Fig. 3). The a-toxin also blocks the slow decrease of fluorescence intensity in the minute range [19]. Ceruleotoxin is a toxin purified from the venom of Bungarus caeruleus [27], which blocks the postsynaptic response to acetylcholine in a manner distinct from the a-toxins. In particular, at variance with the atoxins, it does not displace acetylcholine from the cholinergic receptor site. Fig. 4 shows that preincubation with ceruleotoxin modifies the fluorescence response to carbamylcholine : in particular, the apparent amplitude of the resolved signal decreases. Interestingly, this effect can be observed under conditions where the concentration of ceruleotoxin is smaller than the concentration of cr-toxin binding sites present in the membrane suspension.

It has been shown earlier that acetylcholinesterase is a minor component of the receptor-rich membrane fragment [9] and that in a subcellular fractionation of electric organ the quinacrine fluorescence signal parallels the content of receptor rather than that of esterase [lo]. Fig. 5 confirms that the presence of an esterase blocking agent does not affect the fast increase of fluorescence intensity. For instance in the presence of 0.05 mM tetram the kinetics of the fast fluorescence signal initiated by either 2.5 pM or 0.05 M acetylcholine do not differ significantly from the control trace. An important difference was, however, noticed in the minute range: in the absence of tetram acetylcholine is hydrolyzed and the elevated fluorescence intensity slowly decreases. In the presence of 0.05 M acetylcholine it reaches a final fluorescence level far below the initial one (lower part in Fig. 5 ) probably because of pH changes consecutive to acetylcholine hydrolysis in the poorly buffered

231

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

0

I

0

1

3

2

4

5

Time ( s )

4

(4)control

.e 01

1/

$ c

0

I

I

E

0

I

I

200

400

I

100 Time (rns)

200

I

600

Time (rns)

Fig. 4. Single-shot traces of stopped-$ow experiments: effect of ceruleotoxin on quinacrine Juorescence, caused by carbamylcholine binding to the receptor. Excitation at 295 nm, emission monitored at 538 nm. 1 : 1 mixing of receptor-rich membrane fragments (0.4 pM ~c-[~H]toxin binding sites, 0.4 mg/ml protein) with 1 mM carbamylcholine chloride, each component in Torpedo physiological saline solution, 4 pM quinacrine. The receptor component suspensions were pretreated in the following way (from top): (1) no ceruleotoxin; (2) incubation in 0.2 pM ceruleotoxin for 2.5 min; (3) incubation in 0.2 pM ceruleotoxin for 20 min (final concentrations in (2) and sites, 0.1 pM ceruleotoxin; (4) no ceruleo(3): 0.2 pM ~c-[~H]toxin toxin; ( 5 ) incubation in 0.03 pM ceruleotoxin for 2.0 min; (6) incubation in 0.03 pM ceruleotoxin for 60 min (final concentrations in ( 5 ) and ( 6 ) :0.2 pM receptor binding sites, 0.015 pM ceruleotoxin). Ordinate scale (from top): (1) 3.9%/division; (2) and (3) 3.3%/division; (4), (5) and (6) 3.9%/division change of the fluorescence intensity. Dead time: 2.5 ms

physiological saline solution. This pH effect adds up on reversal of the agonist response consecutive to the removal of acetylcholine from the solution. Fig. 6 shows semilogarithmic plots of the experimental traces taken from Fig. 1 and 3. All experimental traces analyzed by the graphical method yield straight lines without any significant deviation within the limits of experimental error. The half-life time zljz has been taken as a quantitative measure for these first-order processes. The apparent first-order rate constant kobsis then : kobs

= (In

.

2)/zl /Z

(14)

Experimental traces in Fig. 5 and the semilogarithmic plots for acetylcholine in Fig. 6 demonstrate that

0

20

40

60 Time(s)

80

Fig. 5. Single-shot truces of stopped-$ow experiments: acetylcholine esterase activity and its blocking by Tetrum. Effects on the fast increase and subsequent slow decrease of quinacrine fluorescence after mixing of the receptor-rich membrane fragments with acetylcholine. 1 :Imixing of receptor-rich membrane fragments (0.4 pM ~c-[~H]toxin binding sites, 0.3 mg/ml protein), preincubated where indicated in 0.1 mM Tetram with acetylcholine, each component in Torpedo physiological saline solution, 4 pM quinacrine. Preincubation conditions in Tetram and acetylcholine component concentrations were as follows (from top): (1) 60 min in Tetram, 5 pM acetylcholine; (2) no Tetram, 5 pM acetylcholine; (3) 120 min in Tetram, 0.1 M acetylcholine; (4) no Tetram, 0.1 M acetylcholine; (5) 120 min in Tetram, 0.1 M acetylcholine; (6) no Tetram, 0.1 M acetylcholine. Ordinate scale (from top): (1) 2.4O/,/division; (2) 2.2%/division; (3) 3.4%/division; (4) 3.8%/division; (5) 3.4%/division; (6) 3.8 %/division change of the initial fluorescence intensity. Dead time: 2.5 ms

the observed kinetics vary with the concentration of agonist. On the other hand it can be shown that under the present experimental conditions (concentration of agonist 9 concentration of receptor sites) the kinetics do not significantly vary with the concentration of receptor sites [23]. In order to examine the influence of quinacrine on the kinetics of the fluorescence signal, the concentration of quinacrine was varied over the whole accessible

232

Fast Acetylcholine Receptor Kinetics in uitro: Quinacrine Fluorescence B

me

-

Fig. 6. Semi-logarithmic plots of experimental traces: fast increase of quinacrine fltlorescence after binding of ( A ) 0.5 m M carbamylcholine or ( B ) 0.01 M ond 0.05 m M acetylcholine to the vereptov. The original experimental traces are shown in (from left): (A) Fig. 3 (trace 1); (B) pig. 1 (trace 1); Fig. 3 (trace 3 )

Table 1. Fast increase of quinacrine Jluorescence after rapid mixing of T. marmorata receptor-rich membrane fragments with acetylcholine Dependence of the observed half-life times T ~ on, the ~ concentration of quinacrine. 1 :1 mixing of receptor-rich membrane fragments (0.4 pM toxin binding sites, 0.3 mg/ml protein) with acetylcholine iodide 5 pM or 0.01 M, respectively, both components in Torpedo physiological saline solution. Both component solutions were supplemented 15 min before mixing with quinacrine, 0.5 pM or 0.01 mM, respectively. Each half-life time represents the mean value of four experiments, the error range is estimated on the basis of the experimental deviations in these experiments. All the experiments in the table have been carried out with the same membrane fragment preparation Quinacrine concn

tllZ with

1

010-5

1

I

I

I

I

3.10-5 5.10-5 [Acetylcholine] ( M )

2.10.~

10-

Fig. 7. Fast increase of quinacrine jluorescence after binding of an agonist :plot of the apparent first-order rate eonstant k,,, as a function of the final concentration of acetylcholine. 1 : 1 mixing of receptorbinding sites, 0.5 rich membrane fragments (0.4 pM ~t-[~H]toxin mg/ml protein) with acetylcholine iodide twice as concentrated as indicated in the figure, both components in Torpedo physiological saline solution, 4 pM quinacrine

acetylcholine concn of

2.5 pM

5 mM

ms 0.5 10

0

*+

300 50 260 30

16&3

17*3

concentration range [lo] from 0.5 pM to 0.01 mM. Neither the slow kinetics observed at a low concentration of acetylcholine nor the fast ones at a higher concentration are significantly affected by the variation of quinacrine concentration (Table 1). On the basis of pharmacological [ l o ]and optical criteria, a standard concentration of 4 pM quinacrine was selected for the routine kinetic experiments. QUANTITATIVE ANALYSIS OF THE KINETIC DATA AS A FUNCTION OF LIGAND CONCENTRATION

Acetylcholine and Carbamylcholine

Fig. 7 represents a plot of the apparent kinetic constants kobsas a function of acetylcholine concentration. Since the value of kobslevels off at high concentrations of acetylcholine, a simple one-step equilibration

process [cf. Eqns ( 1 and 2)] cannot account for these experimental data. A double-reciprocal plot of the same data is shown in Fig. 8. Again it is found that the value of ljkobs levels off at high values of ljc. According to the reaction scheme given in Eqn (3) the concentration dependence shown in Fig. 8 suggests a finite value of k,. Fig. 9 is a secondary plot of the data from Fig. 8. Instead of ljkob,, lj(kobs-k4) is plotted as a function of the reciprocal concentration of acetylcholine. Taken into consideration the intercept from Fig. 7 and an indicated asymptotic value of ljkobsin Fig. 8 it is found that a value of k, = 1 s-l gives a linear plot over about a 10000 times range of acetylcholine concentration. This concentration dependence, together with the basic characteristics of the experimental traces, i.e. linearity of the semilogarithmic plots and lack of an initial burst or a lag phase, is therefore in full agreement with the reaction scheme of Eqn (3) with the additional restriction of Eqn (4). According to Eqn (S), from the intercept in Fig. 9 a value of k,=(100*50) s-' can be derived and according to Eqns (8) and (9) from the ratio of slope to intercept the value for the thermodynamic dissociation constant: Kl =(1.1 f0.7) x l o p 4 M.

233

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

[Carbamylcholine]

(M)

/

1

1/ [pcetylcholine] (M-')

Fig. 8. Double-reciprocal plot of the apparent first-order rate constant kobsas a funct ion of the final concentration of acetylcholine. Data from Fig. 7

0

1o5

2.10~

1/ [Carbamylcholine] (M")

Fig. 10. Fast increase of quinacrineJIuorescence: plot of the apparent first-order rate constant kobsas a function of the final concentration of carbamylcholine. Main part: secondary double-reciprocal plot with k=k,,,-k,; k4=0.1 s-'. Inset: direct plot ofthe experimental data. 1 :1 mixing of receptor-rich membrane fragments (0.4 pM a-[3H]toxin binding sites, 0.4 mg/ml protein) with carbamylcholine chloride twice as concentrated as indicated in the figure, both components in Torpedo physiological saline solution, 4 pM quinacrine

0

1o6 1/[ Acetylcholine] (M-')

Fig. 9. Secondary double-reciprocal plot of the data in Fig. 8: instead of the apparent first-order rate constant k , the diflerence k = k,, is plotted as a function of the final concentration of acetylcholine. A value k4 = 1 s-l has been assumed. For experimental conditions c$ Fig. 7

The error ranges in k, and K, are estimated on the basis of the continuous and dashed lines in Fig. 9, which both correspond to possible fits of the experimental points with their error ranges. Each experimental point represents the mean value of three or more kinetic experiments and the error range of the

experimental points has been estimated on the basis of scattering of half-life times from one experiment to another with the same preparation. Knowing the values of k , , k4 and K , , one can calculate for acetylcholine the overall thermodynamic dissociation constant R as defined in Eqn (12): M. R=(1.1 f0.8) x A graphical analysis of a set of carbamylcholine kinetics is shown in Fig. 10. It can be seen from the inset that k is not a linear function of the concentration of carbamylcholine, and to linearize the double-reciprocal plot, a finite value of k4=0.1 s-l has to be assumed. From this linear secondary plot the following values can be deduced (cf. Eqns 8 - 10,12) : k, = (10f3) s - , , K1=(1.9+o.8) x M, R=(1.9+1.0) x lop6 M. Analysis of the amplitudes of the experimental kinetic traces provides further insight into the thermodynamics of the interaction between receptor and agonist. Since the fast fluorescence increase is completed in milliseconds or at most a few seconds, whereas the subsequent slow fluorescence decrease has a time constant of the order of a minute (cf. Fig. 2 and [19]), the amplitudes of the fast effect can easily

234

Fast Acetylcholine Receptor Kinetics in oitro : Quinacrine Fluorescence

be read. They give an apparent dissociation constant for the molecular process underlying the fast increase of fluorescence intensity [cf. Eqn (13)]. Fig. 11 shows a double-reciprocal plot of the variation of these intermediate amplitudes as a function of acetylcholine

I

I

I

1o6

0

I

I

2.106

1/ [Agonist] (M-')

Fig. 11. Fast increase of quinacrine fluorescence after binding of acetylcholine ( 0 ) or carbamylcholine (A) to receptor-rich membrane fragments: double-reciprocal plot of the amplitude of the transient fluorescence increase as a function of the Jinal concentration of the agonist. The amplitudes have been obtained from the kinetic experiments represented in Fig. 7 - 10

Table 2. Kinetic constants for the interaction of acetylcholine and carbamylcholine with T. marmorata receptor-rich membrane fragments labelled with quinacrine Mean values from different preparations. k , , k z , k , , k4: kinetic constants with regard to the reaction scheme in Eqn (3). The concentration of quinacrine was always 4 pM. See text

1 . mol-'

S-l

Acetylcholine Carbamylcholine

. s-1

60+40 10i5

1.5+0.5 2 5 x 10' 0.2k0.1 >loz

310'

> 3 x lo5

.

and carbamylcholine concentration. The data have been derived from the experiments, the kinetics of which are presented in Fig. 7 - 10. The apparent dissociation constants and the error ranges which can be evaluated from Fig. 11 are : for acetylcholine Kapp= (5f2) x M, for carbamylcholine KapP=(5f2) x M. The kinetic analyses carried out with a series of membrane preparations and with the same preparation after aging yielded a non-negligible variation of the kinetic parameters [22,23]. It has been found that the kinetics become faster during the first day after preparation and that there may exist a significant difference of the kinetics from one preparation to another immediately after preparation, even if performed with the same standard preparation procedure. Tables 2 and 3 summarize the results of kinetic analyses, 4 for acetylcholine and 2 for carbamylcholine, carried out under different conditions [22,23] (and this paper). The error ranges take into account the variations observed between different analyses, too. The kinetic constants are given in Table 2. In addition to k, and k4,which can be read from the secondary reciprocal plot, lower limits for k, and k, are given. They are estimated on the basis of Eqns (4) and (1 1). In Table 3 are given the values of the thermodynamic constants. Kl and K, calculated from the kinetic constants [cf. Eqns (8 - 10,12)], Kappis derived from the amplitude of the fast fluorescence increase [cf. Eqn (13)], &luxis given by the dose-response curves obtained by measuring ',Na+ efflux from a preparation of receptor-rich microsacs from Torpedo [ l l ] and Kequis the dissociation constant as measured after complete equilibration in the minute range with the toxin binding and Dns-chol fluorescence technique [14] or by means of quinacrine fluorescence [lo] at equilibrium. Choline

A kinetic analysis of the interaction of choline with quinacrine-labelled membrane fragments is presented in Fig. 12. Since choline exerts its pharmacological

Table 3. Thermodynamic constantsfor the interaction of acetylcholine and carbamylcholine with T. marmorata receptor-rich membrane fragments labelled with quinacrine Mean values from different preparations. K1 : dissociation constant for the preexisting state of the receptor [cf. Eqn (lo)]; K : overall dissociation constant for the binding to the isomerized state in equilibrium with the preexisting state, evaluated on the basis of kinetic data [cf.Eqn(l2)l; K a p p apparent : dissociation constant derived from the amplitudes of the fast fluorescence increase [cf. Eqn (13)]; &lux: apparent dissociation constant estimated from the increase of "Na+ flux in oitro [ ll ] ; K,,,: equilibrium dissociation constant [14]. The concentration of quinacrine was always 4 pM. See text. Kflurvalues from Popot et al. [Ill, Keq,,values from Cohen et al. [14]

M Acetylcholine Carbamylcholine

(7k3) x 10-5 (3 +_I)x 10-4

(2

* 1)

x

(4+3)x

(1.0k0.5) x (5 f2) x

2x

s x 10-5

sx 10-~ 5 x 10-7

235

H.-H. Grunhagen, M. Iwatsubo, and J.-P. Changeux

4 l

01

I

0

100

I

I

I

I

500

400

200 300 1 / [ Choline] (M-')

Fig. 12. Fast increase of quinacrine Jluorescence after binding of choline to receptor-rich membrane fragments: double-reciprocal plot of the apparent first-order rate constant k,,, as a function of the final concentration of choline. 1 :1 mixing of receptor-rich membrane fragments (0.4 pM ~+[~H]toxin binding sites, 0.4 mg/ml protein) with choline chloride twice as concentrated as indicated in the figure, both components in Torpedo physiological saline, 4 pM quinacrine

I

T

l/[Suberyld~choline](M-')

Fig. 14. Fast increase of quinacrine Jluorescence after binding of suberyldicholine to receptor-rich membrane fragments: double-reciprocalplot of the rate constant k (primary plot (0) k = kobs,secondary plot (+) k = k o b s - h , kq=0.4s-l; kobs=apparmt,first-orderrateconstant) as a function of the final concentration of suberyldicholine. 1 : 1 mixing of receptor-rich membrane fragments (0.4 pM u-[~H]toxin binding sites, 0.3 mg/ml protein), preincubated 60 min in 0.05 mM Tetram, with suberyldicholine iodide twice as concentrated as indicated in the figure, both components in Torpedo physiological saline solution, 4 pM quinacrine

T

7M. A plot of the corresponding signal amplitudes is

01

0

I

001

I

I

002 003 [Choline] (M)

I

004

I

, I shown in Fig. 13. From this dependence the apparent

005

010

Fig. 13. Fast increase of quinacrine Jluorescence after binding of choline to receptor-rich membrane fragments: direct plot of the amplitudes as a function of the final concentration of choline. The amplitudes have been obtained from the kinetic experiments represented in Fig. 12

thermodynamic dissociation constant as defined in Eqn (13) can be derived: Ka,,=(1.3f0.6)x lop3 M. Since the value of k4 is not known, I? cannot be calculated for choline [cf. Eqn (12)].

Suberyldicholine

action on the receptor protein only at comparably high concentrations, the concentration range studied is less extended than in the case of acetylcholine and carbamylcholine. A double-reciprocal plot of the experimental kinetic constant as a function of choline concentration is found to be fairly linear. A secondary plot to determine ki is therefore not reasonable and k4 is concluded to be very small. According to Eqn (6) one can calculate from the intercept in Fig. 12 the value ofk3=(1.7f0.5) s-'. The ratio of slope to intercept yields KI [cf. Eqns (6,7,10)]: K1=(9+4)x

A few experiments have been carried out with suberyldicholine. Fig. 14 shows a double-reciprocal plot of the kinetics at concentrations ranging from 1 pM to 0.01 M. The limited number of experiments does not allow us to distinguish definitely between a negligible or finite value of k,. Nevertheless the plot demonstrates the range of the k4 value, which is Odk,d0.4 s-'. The intersection with the ordinate yields (cf. Eqn 6 or 8): k,=(25+5) sC1 and the ratio of slope to intercept [cfi Eqns (10 and 6,7 or 8,9)]: Kl =(3+2) x lo-' M. With k4=0.4 s-', from K,, k, and k4 an upper limit for K can be calculated [c$ Eqn (12)]: K s 4 . M.

236

Fast Acetylcholine Receptor Kinetics in uitro : Quinacrine Fluorescence

1

7 10

20

8

/O

-Y)

6 -

12

0 1 7

/O

O

6

5 -log

4

3

N

-

-

0

2

[ Suberyldicholine] / M

Fig. 15. Fast increase of quinacrine fluorescence after suberyldicholine to receptor-rich membrane fragments: of the amplitudes as a function of the final concentration dicholine. The amplitudes have been obtained from the periments represented in Fig. 14

-t

0

4

binding of direct plot of suberylkinetic ex-

Fig. 15 represents the dependence of the amplitudes on the concentration of suberyldicholine. It can be estimatedthat [cjEqn(l3)] 1 0 - 7 M d Kappd 1OP6M. At a high concentration, the signal amplitude decreases with increasing concentration. Besides a possible fluorescence quenching by iodide this may reveal a partial local anesthetic character of suberyldicholine [29], which gives rise to a competition between quinacrine and suberyldicholine.

TEMPERATURE DEPENDENCE

To gain insight into the activation energies of the several reaction steps of the mechanism studied, the kinetics were measured at several different temperatures. Although the complete kinetic analysis was not carried out in a systematic manner, the temperature dependence of the kinetics was studied in two characteristic concentration domains : at low concentrations of agonist the kinetics are found to be highly concentration dependent, and the observed events reflect primarily the binding process. At high concentrations of agonist, limiting rates are observed which are essentially due to k,. Fig. 16 shows the temperature dependence of the apparent kinetic constants after addition of carbamylcholine. In the presence of 0.03 mM carbamylcholine, representative for the binding equilibration [cJ: Eqn (3)], a Qlo of about 2.5 is found around 20 "C. In the presence of 0.01 M carbamylcholine the Q,, around 20 "C is close to 1.3. The corresponding activation energies from these carbamylcholine data would be [23] : E0,03mM = 15.4 kcal/ mol(64.4 kJ/mol), EO,OIM= 5.1 kcal/mol(21.3 kJ/mol). For acetylcholine at a concentration of 0.05 M a Ql0 of 1.3- 1.4 was found around 20 "C and an activation energy of E0,05M acetylcholine = 5.0 kcal/mol (20.9 kJ/mol) was determined [22,23].

/;

/. . I

10

2

0

20

t ("C) Fig. 16. Fast increase of quinacrine fluorescence after binding of carbamylcholine to receptor-rich membrane fragments: temperature dependence of the apparent rate constants after high and low doses ofcarbamylcholine. 1 : 3 mixing of receptor-rich membrane fragments binding sites, 0.3 mg/ml protein) with 0.02 M (0.4 pM ~i-[~H]toxin (0) or 0.06 mM (m) carbamylcholine chloride respectively, both components in Torpedo physiological saline solution, 4 pM quinacrine

THE SIGNIFICANCE OF QUINACRINE FLUORESCENCE SIGNALS WITH REGARD TO STRUCTURAL TRANSITIONS OF THE MEMBRANE-BOUND ACETYLCHOLINE RECEPTOR

The fast changes of quinacrine fluorescence caused by cholinergic ligands might be accounted for either by a direct interaction between these ligands and quinacrine at the receptor level or by a structural transition of the membrane-bound receptor which mediates an indirect interaction between the two compounds of distinct pharmacological specificity. The last interpretation is supported by the following observations. a) Agonists and antagonists trigger qualitatively different responses : the fast increase of fluorescence intensity which takes place in the millisecond range is observed only with agonists but not with antagonists; moreover the amplitude of the maximal response is about the same for compounds such as acetylcholine, choline or phenyltrimethylammonium (not shown in detail), which differ chemically but act all as agonists. Nevertheless the two bisquaternary compounds trigger a rather different fluorescence response : no equilibration in the time range of the stopped-flow but a submillisecond increase of fluorescence. This behaviour is of interest, since decamethonium (like hexamethonium) behaves apparently as an antagonist in vitro [ l l ] on the receptor-rich microsacs but as an

237

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

agonist, in vivo, with T. marmorata electroplaque [13]. No simple chemical correlation, therefore, exists between single structural features of a cholinergic effector and the characteristics of the fluorescence signal, which may account for a direct interaction between quinacrine and effector. b) Neurotoxins from snake venom block specifically the permeability response of the postsynaptic membrane and. reduce or abolish the fluorescence signal otherwise recorded after addition of agonist. A block is expected from the a-toxins known to compete for the agonist binding site [7,8]. Ceruleotoxin, which does not show binding competition with the a-toxins [27], has nevertheless an effect on the fluorescence response. It modifies the kinetics and reduces the apparent amplitude. One may speculate that these changes parallel or implicate the destruction of the proper function of the ionophore. Interestingly the effect of ceruleotoxin develops rather soon in the minute range after an addition of only 0.03 pM ceruleotoxin (based on a molecular weight of 38 000) to a membrane fragment suspension containing 0.4 pM a-toxin binding sites. Assuming a binding of ceruleotoxin to the receptor (rather than an enzymatic action) the submolar ratio would suggest that ceruleotoxin affects a functional unit comprising several a-toxin binding sites. A comparable observation has been made recently with a quaternary derivative of the local anesthetic dimethisoquin [31]. In any case, both the pharmacological specificity of the cholinergic ligands which trigger the fluorescence response, and the selective effect of snake venom toxins on this response cannot be accounted for by any direct interaction between cholinergic ligands and quinacrine. The observed fast increase of quinacrine fluorescence intensity should therefore reflect some structural transition of the macromolecular complex which carries distinct sites for the several categories of ligands concerned. Two alternative hypotheses then may account for the change of quinacrine fluorescence : a re-equilibration of quinacrine binding to the receptor-rich membrane fragments or a change of quantum yield of quinacrine molecules already bound to the receptor membrane. It seems extremely difficult to prove in the millisecond time range a binding event by means of a separation assay. Nevertheless, considering the first hypothesis, a binding re-equilibration is expected for local anesthetics which like quinacrine cause an increase of affinity of the acetylcholine receptor for agonists [14] under equilibrium conditions. Such a reciprocal interaction between an agonist and the monomethyl quaternary derivative of the local anesthetic dimethisoquin has indeed recently been demonstrated at equilibrium [31]. Assuming that the agonists cause an ‘indirect’ increase of quinacrine binding in a one-step reaction, the

kinetics of the fluorescence response should then become faster with increasing quinacrine concentration. It can be seen from Table 1 that increasing the quinacrine concentration docs not modify significantly the kinetics of the fluorescence changes observed after addition of acetylcholine. In particular the limiting rate in the presence of a high concentration of acetylcholine is independent of quinacrine concentration. A one-step quinacrine-binding mechanism therefore cannot limit the observed kinetics. On the other hand, a binding of quinacrine via a multi-step mechanism (including quinacrine-concentration-independent steps) could explain the limiting rate for acetylcholine in Table 1 but not the occurrence of different limiting rates with different agonists. Although a relaxation of quinacrine binding after addition of an agonist to the receptor is possible, it is concluded that such changes in quinacrine binding are not sufficient to account for, and, most likely, do not limit, the observed fluorescence kinetics. As an alternative to a relaxation of quinacrine binding a change of fluorescence quantum yield of already bound quinacrine may be considered. Quinacrine molecules being and remaining bound when agonist molecules attach to the receptor site may be subjected to an intrinsic change of quantum yield and/or to a modification of the non-radiative energy transfer from the receptor protein. Under the present experimental conditions these two possibilities cannot be distinguished. In any case, a global change of fluorescence yield of already bound quinacrine molecules guarantees a prompt response in the millisecond range. DISCUSSION The experimental results presented in this paper confirm and further extend previous studies done at equilibrium with T. marmorata receptor-rich membrane fragments labelled with quinacrine. Cholinergic ligands trigger characteristic changes of fluorescence intensity which are now resolved in the time scale of the millisecond. As can be seen from Table 1, the observed kinetics do not depend on the concentration of quinacrine in the range studied (from 0.5 pM-0.01 mM). In addition, different limiting fast kinetics are found for different agonists. Therefore it is concluded that the process of interaction between quinacrine and the receptor docs not limit the kinetics measured under the present experimental conditions. Accordingly, quinacrine does not occur in the kinetic schemes discussed below. It is however possible, and even expected, that quinacrine modifies the kinetics of the structural changes of the receptor in the presence of agonists and/or the equilibria between states. Two alternative mechanisms may then account for the fluorescence response :

238

(a) the change of fluorescence intensity reflects changes of the fluorescence parameters (quantum yield or efficiency of energy transfer) of already bound quinacrine molecules or (b) the structural changes lead to a binding relaxation of quinacrine, which is paralleled by a change of quantum yield; in this case the binding relaxation has to proceed without measurable delay even for the fastest kinetics observed. (Recent unpublished data of Steinbach and Neher of individual noise events recorded in the presence of a particular local anesthetic tend to support this last interpretation. According to these authors the blocking events observed within a single agonist burst would result from the rapid and multiple binding of the local anesthetic directly to the ion gate.) Reaction Mechanism and Kinetics A one-step reaction mechanism [cf Eqn (l)] cannot account for the observed kinetics, since the corresponding concentration dependance [Eqn (2)] is not observed (cf. Fig. 7 and 10). A two-step mechanism with a preequilibration between two isomeric states [Eqn (5)] can be ruled out because of: (a) accurate first-order kinetics and (b) different limiting rates at high concentrations of different agonists. All the experiments and results including the concentration dependences are therefore explained by the mechanism given in Eqn (3) under equilibrium conditions [cf.Eqn(4)I. This reactionmechanismcomprises a fast binding equilibration and a subsequent isomerization of the receptor.ligand complex. The agreement between experimental data and theoretical predictions confirms our former conclusion that the changes of fluorescence intensity measured under conditions of energy transfer parallel the isomerization of the receptor molecule rather than the binding of effectors to its receptor site. A basic assumption of the kinetic model used in the analysis is an interconversion between structural states of the receptor protein driven by the agonist. There is no hint from the kinetic studies for the alternative possibility of pre-existing independent and stable populations of receptor sites with different affinities. Finally, the interconversion between states of increasing affinitieswas postulated on the basis of kinetics in the minute range done concurrently by fluorescence spectroscopy and direct ligand binding [8, 19,211 and has been confirmed meanwhile [31,33]. Other mechanisms of higher complexity may also account for the same kinetic data but the two-step mechanism discussed here, which is also often found with enzymes [30], still is the simplest one and also accounts satisfactorily for a detailed analysis of the experimental data. The kinetic constants resulting from this analysis may be characterized as follows : (a) The forward isomerization constant k, differs for

Fast Acetylcholine Receptor Kinetics in uitro : Quinacrine Fluorescence

different agonists and ranges from about 100 s-' (acetylcholine) to about 1 sC1 (choline). (b) The different backward isomerization constants k, are of the order of 1 s-' or smaller. In all cases the values of k, have been found to be at least 10 times smaller than k, . Hence the isomerization equilibrium is shifted towards the isomerized state A*B [cf Eqn (3)]. (c) The estimated lower limit of the forward binding constant k , is lo7 M-'S-' for acetylcholine and 3 x 1O5 M - ' S-' for carbamylcholine. This value is reasonable for the binding of a small ligand to a protein and, indeed, the kinetic analysis of acetylcholine binding to detergentsolubilized receptor protein has yielded a forward binding constant of 2.4 x lo7 M-l s-' [34]. The fast fluorescence kinetics presented in this paper are fully described by the reaction scheme of Eqn (3); however, in addition, a slow partial decrease of fluorescence intensity is observed in the minute range (cf. Fig. 2 and [19]). A more complete description of the kinetics caused by agonists in the quinacrine receptor system in vitro should therefore be A+B

+

AB 3 A*B

A**B,

(15)

k4

where the first two equilibration steps refer to Eqn (3) and the transformation from A*B to A**B accounts for the subsequent slow fluorescence decrease [19]. In vivo, and from a strictly phenomenological point of view, acetylcholine is engaged in two distinct processes : the fast opening of the Na', K + gate or ionophore, i.e. the 'activation', followed, after prolonged application, by a slow decrease of permeability or 'desensitization'. To account altogether for these elementary functional properties and for the biochemical and structural data available at present a three-state model for the membrane-bound acetylcholine receptor [8] was proposed [19]. The receptor.ionophore complex was assumed to exist under at least three discrete structural states, resting (R), active (A) and desensitized (D), which differ by their affinity for cholinergic ligands and by the state of opening of the ionophore. These states are postulated to exist, in reversible equilibria, prior to the binding of cholinergic effectors, the resting state being spontaneously favoured in the absence of agonists. If the affinity for agonists increases from R to A to D, then, the binding of agonist shifts the equilibrium in the same direction to the active and finally to the desensitized state. If the ionophore is open only in the A state this would result in a transient opening of the gate corresponding to the population of the A state. Out of the complete equilibration scheme [19] the following reactions are of particular interest for this paper: R+E

p RE kn

RE

kRE A E

RE,R

AE,RE

AE

+DE, k A E DE

DE,AE

(16)

H.-H. Griinhagen, M. Iwatsubo, and J.-P. Changeux

239

where E is an agonist binding to the acetylcholine receptor site. In addition to the desensitization reaction from RE to DE via AE [cf. Eqn(l6)], a direct desensitization step may take place: RE

+DE. k R E DE

DE,RE

(17)

Furthermore it is possible that the activation step in the presence of an agonist proceeds via a pre-equilibration between the resting and the active state: +E

ReA-AE. -E

(18)

The result of the kinetic analysis [c$ Eqn(l5)I is formally equivalent to the postulated equilibration scheme of the receptor after binding of an agonist corresponding to Eqn (16). The alternative activation sequence corresponding to Eqn (18) has been considered as a possible reaction mechanism a priori for the fast equilibration [cf. Eqn (5)]. However, it could be neglected because of the lack of an initial burst in the reaction (as long as A and AE have different fluorescence yields) and the occurrence of different limiting rates for different agonists. Comparison between Kinetic Data in vivo and in vitro The kinetics in vivo of the endplate potential and of the miniature endplate potentials (for a review cf. [2]) indicate that the postsynaptic membrane responds to the release of acetylcholine within about 50 - 500 ps, but apparently the growth kinetics of the endplate currents are limited by the diffusion of actylcholine in the cleft. The endplate potential decay occurs in a few milliseconds. Electric-field-jump [35, 36,551 and noise analysis [37,38,55] experiments in vivo have led to comparable results. If one assumes that the 'activation reaction' in vivo is based on a structural change of the receptor protein, isomerization processes with a kinetic constant > l o 3 s-' are expected. On the other hand, desensitization develops within seconds at the muscle endplate, if the agonist is applied iontophoretically [39] or in the order of minutes [2,13] after bath application of the agonist. Recently, kinetic data for receptor-rich membrane fragments in vitro have been evaluated on the basis of intrinsic fluorescence signals [15,16]. These data could also be fitted to a binding-isomerization mechanism [cf. Eqn (3)], but the kinetic constants found differ significantly from those reported in this paper : e.g. for acetylcholine k,=0.16 s-' and k4=0.27 s-'; k, was estimated to be > lo6 M-' s I . Preliminary results from direct binding studies with a fast mixing technique suggest an isomerization of the membranebound receptor within seconds [31]. Also work in progress with the fluorescent agonist C,DAChol[18] hasrevealed a fast kinetic process of the order of a few milliseconds, an intermediate and a slow one in the

second time range [32]. Yet, it is a common feature of the studies carried out so far in vitro both in the presence or in the absence of quinacrine, that the observed isomerization reactions take place within a significantly longer time range as the activation process in vivo. Also, the value of the backward kinetic constant k4 appears much lower than observed in vivo [2,36,52]. Preliminary data about the temperature dependence of the kinetics obtained in the presence of quinacrine (cf. Fig. 16 and [23]) may help to correlate kinetics in vivo and in vitro. In vivo a Q,, of 1.2 characterizes the growth phase' of miniature endplate potentials (for a review [2]). This is in agreement with a Q,, of about 1.3 for the isomerization reaction as caused by acetylcholine in vitro. However, one cannot rule out the possibility that the activation kinetics in vivo are limited by diffusion processes which lead to a low value of Q,,. Several possibilities then may have to be considered to account for the quantitative differences noticed between the kinetics in vivo and in vitro. a) The activation process (isomerization or direct modification of permeability, e.g. by means of a binding reaction) does not give rise to a change of extrinsic and/or intrinsic fluorescence. b) The amplitude of the fluorescence change accompanying the activation is small and the kinetics too fast for being resolved with the stopped-flow technique. c) The binding of quinacrine to the receptor modifies the kinetics. From experiments with the neuromuscular junction, it is known that local anesthetics alter the falling phase of endplate currents. Instead of a monophasic decay a rapid initial phase followed by a prolonged long-lasting decay is observed [41-461. This may explain the low value of k4 found in vitro in the presence of quinacrine. d) The activation kinetics with isolated membrane fragments in vitro are considerably slower than with the intact cell in vivo, for instance as a consequence of a change in the structure or/and the environment of the excitable membrane during isolation and purification. It is known that particular -SH groups are critical for receptor function [47 - 491. Modification of sulfhydryl groups also affects the amplitude of the fast fluorescence change [22] and may also account for the acceleration of the kinetics during the first days after preparation [23]. Among other physical parameters the electric field strength seems to be most important. The electrical potential across the membrane is certainly much smaller in vitro than in vivo. However, the membrane potential does not seem to The onset of desensitization around 20 "C shows a slightly higher Q,, of 1.9 [40]. For the closing of synaptic channels, a priori another candidate for a molecular isomerization process in a few milliseconds, higher Q,, values of 2 - 5 have been recorded [2].

240

modify the growth kinetics of voltage-clamped miniature endplate currents and depolarization may fasten the growth phase and the decay of the endplate currents (for a review cf. [2] and [50-551). Therefore, a low value of the membrane potential is unlikely to slow down the kinetics in vitro. Finally, significant changes of pharmacological properties have been noticed between the permeability response in uitro of the isolated microsacs and that of Torpedo electroplaque. For instance: the absolute values of the apparent dissociation constants appear systematically higher in vitro than in viuo [l 1,8] and decamethonium, which acts as an agonist in vivo, becomes an antagonist in vitro [11,8]. These differences may indicate that a structural reorganisation of the excitable membrane takes place during homogeneization and purification. Further studies done in parallel in viuo and in vitro should lead to a distinction between these various possibilities. Thermodynamic Dissociation Constants and Structural States of the Membrane-Bound Receptor Protein

If quantitative differences still exist between the kinetic data resulting from experiments in uivo and in vitro, closer relationships are found when thermodynamic dissociation constants are compared. The analysis of the kinetic data in terms of Eqn (3) and (1 5) yielded a set of thermodynamic dissociation constants for cholinergic ligands, which, from a strictly phenomenological point of view, may be assigned to the membrane-bound receptor at rest, to a transient state being populated in the millisecond range in the presence of agonists, and to a final state reached after equilibration in the minute range [19]. First of all an internal consistency exists between the kinetic results obtained in the presence of quinacrine and the equilibrium data obtained with the same membrane preparation. The dissociation constants determined in the minute range by fluorescence spectroscopy in the presence of quinacrine are close to those measured with radioactive ligands in the M for absence of quinacrine, i.e. in the order of M for carbamylcholine [lo]. acetylcholine and 4 x Moreover analysis of the fluorescence amplitude data [cf.Table 3 and Eqn (13)] yields values of the apparent dissociation constants for the transient state Kappof M for acetylcholine and of 5 x M for carbamylcholine which are almost identical to the overall constant IT resulting from the kinetic analysis’. Such Concerning the interpretation of the kinetic data in terms of an interconversion of states it should be recalled that the thermodynamic constants R and Kappfor the isomerized state and Kequfor the high-affinity equilibrium state are overall constants and therefore do not represent microscopic dissociation constantsfor discrete states of the receptor protein. A more extensive analysis of the data in terms of the proposed three-state model [19] should lead to the determination of these constants.

Fast Acetylcholine Receptor Kinetics in uitro : Quinacrine Fluorescence

an excellent agreement confirms the attribution of the fluorescence change to the isomerization rather than to the binding step [Eqn (3)]. The plausibility of the interconversion hypothesis and of the values of the constants determined on this basis is further supported by the results of binding studies in vitro. In vitro, the affinity of the receptor protein for cholinergic ligands has been shown to vary reversibly with detergent concentration [561. In the case of acetylcholine and with T. marmorata receptor-rich membrane fragments, states with Kd% M and & > l o p 6 M were found to exist, in addition to the high-affinity state (Kd= 3 x M), upon dissolution by sodium cholate. Elimination of sodium cholate by dilution leads to a reassociation of the protein and to a recovery of the high-affinity sites. An interconversion between binding states of the receptor protein takes place when varying the detergent concentration. The receptor protein may therefore exist spontaneously under discrete states of affinity which, in that case, obviously pre-exist the binding of cholinergic ligands. In vivo, in the case of the neuromuscular junction [5,6] and Electvophoruselectroplaque[35,57], apparent dissociation constants for the physiological transmitter, acetylcholine, have been evaluated on the basis of electrophysiological experiments. Depending on the kinetic model chosen, they range from about lo-’ M to the order of M for acetylcholine. Transmitter concentrations have been evaluated to be close to l o p 4 M or even more in the synaptic cleft [5,57,58]. Hence a dissociation constant of 7 x M for acetylcholine binding to the pre-existing resting state (cf. Table 3 ) would represent a reasonable value for physiological function. Furthermore, the overall dissociation constant for the isomerized state (Kand Kapp)is close to the concentration range (Kflux),where ion flux can be stimulated in the same preparation of membrane fragments (cf. Table 3 ) . This is expected if functional ‘activation’ results from a conformational change (isomerization) rather than from direct agonist binding. A general consistency therefore exists between the thermodynamic binding constants derived from our kinetic analysis and the concentration or ‘apparent’ constants observed in vivo. In another laboratory [15,16] and in an independent manner, a kinetic analysis based on the intrinsic fluorescence signals has yielded a dissociation constant ‘K1’ of 4.1 x M for acetylcholine binding to the pre-existing state and an overall dissociation constant ‘K’for the isomerized state [cf. Eqn (12)] of 6.9 x M [15]. These data have been interpreted as ‘Kl’ being related to the activation reaction and ‘ K to the desensitization one. Several possibilities may account for the differences of results and interpretations as, for instance, the presence of more than three conformational states or, as already discussed, any

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modification of the kinetics and/or the states in equilibria related to the presence of quinacrine. CONCLUSIONS The fast changes of fluorescence intensity observed after mixing of a cholinergic agonist with receptorrich membrane fragments labelled by quinacrine are not due to a direct interaction between the agonist and quinacrine. They rather reveal structural transitions of the membrane-bound acetylcholine receptor. These changes of structure may be analyzed in terms of reaction mechanisms based on the interconversion between discrete states of the receptor protein : binding of an agonist to a low-affinity state already existing at rest would cause, in the millisecond time range, an isomerization of the receptor protein to a state with higher affinity. Subsequently, in the minute time range a further transformation to a high-affinity equilibrium state takes place [19]. Within the limits of the accuracy of our experiments, the recorded traces and their variation with the concentration of ligand are accounted for by the simple reaction mechanism of Eqns (3) and (15). Minor deviations from the exact fit by these equations (e.g. in Fig.9) might become significant in a more sophisticated analysis of the data involving, for instance, cooperative or sequential binding. The kinetic constant derived from this analysis for the fast isomerization process (k, in Table 2) is lower than that expected for the activation process in vivo. First of all, one cannot exclude that a very fast isomerization process in uitro does not manifest itself by a measurable change of fluorescence. On the other hand, the millisecond isomerization may represent the activation reaction slowed down as compared to conditions in viuo. In this case the changes of quinacrine fluorescence in the minute time range could reflect the desensitization reaction from AE to DE [cJ Eqns (14 and 19)13.Finally, the possibility has to be considered that the millisecond isomerization represents an accelerated desensitization phenomenon, whereas the kinetics in the minute range represent a receptor transition occurring only in the presence of local anesthetics and related compounds [31]. A definite interpretation hinges upon eventual modifications of the intrinsic properties of the preparation in vitro or by the labelling of the receptor with quinacrine, which have been discussed in detail. In any case, several major facts support the view that the millisecond isomerization observed in the presence of quinacrine is, directly or indirectly, related If the millisecond kinetics reflect the activation process but the normal desensitization pathway [Eqn (16)] is blocked in the presence of quinacrine: only an alternative pathway in the minute range, e.g. via a mechanism according to Eqn. (17), RE-DE, would then become accessible for desensitization.

to the activation reaction : (a) the strict specificity for the agonists which cause in vitro the permeability change; (b) the agreement between the values of the thermodynamic dissociation constants and the concentration ranges for the physiological effects of activation and (c) the low value of the PI,,. Further insight into the activation reaction will result from the kinetic analysis of the direct interaction of fluorescent agonists with the acetylcholine receptor site [32] and from the use in parallel of labels specific for the ionic gate or ionophore, which is under the command of the acetylcholine receptor site. Although, it is yet possible that quinacrine (as a typical local anesthetic) may prove to bind specifically to the ionophore and monitor its opening. We wish to thank Drs T. Heidmann and V. Teichberg for stimulating discussions, Drs B. Sakmann and J. Heesemann for a gift of suberyldicholine, Drs P. Boquet and C. Bon for providing respectively cc-toxin and ceruleotoxin, Profs Boisseau and Cazaux for their supply of Torpedo, Mrs N. Courtinat for her excellent secretarial assistance and Mrs H. Gartenhof and Mr J. Hartmann for having prepared the figures. H. H. G. was recipient of a longterm fellowship from the European Molecular Biology Organization (1974/1975) and a fellowship from the Deutsche Forschungsgemeinschafi (197511976).

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H.-H. Grunhagen, Institut fur Physiologische Chemie, Universitat des Saarlandes, Bau 44, D-6650 Homburg/Saar, Federal Republic of Germany M. Iwatsubo, Centre de Genetique Molkculaire du C.N.R.S., F-91190 Gif-sur-Yvette, France J.-P. Changeux*, Laboratoire de Neurobiologie Molkculaire, Institut Pasteur, 25/28 Rue du Docteur-Roux, F-75724 Paris-Cedex-15, France

* To whom correspondence should be addressed