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Studies on a reconstituted acetylcholine receptor system: Effect of agonists
ARCHIVES
OF BIOCHEMISTRY
Studies
AND
BIOPHYSICS
164, 20-29 (1974)
on a Reconstituted
Acetylcholine
Receptor
System:
Effect of Agonists MAHENDRA Department
of Chemistry
and Health
KUMAR JAIN
Sciences, University
Received February
of Delaware,
Newark,
Delaware
197111
22, 1974
An impure preparation of acetylcholinesterase from electroplax of the electric eel can be incorporated into a bimolecular lipid membrane. The acetylcholinesterase-modified bimolecular lipid membrane shows a concentration-dependent increase in membrane conductance elicited by several agonists (acetylcholine, carbamylcholine, phenyltrimethylammonium ion, tetraethylammonium ion, decamethonium ion, and nicotine) added to the compartment opposite that to which acetylcholinesterase was originally added. Affinity and efficacy of the various agonists in generating the conductance increase were measured from dose-response curves; these are in good quantitative agreement with corresponding values observed for depolarization of intact eel electroplax. The ion conduction pathways induced by agonists in the modified bimolecular lipid membrane show a slight cation selectivity, Na 2 K > Cl (3 : 3 : l), similar to that observed for the depolarized electroplax membrane. Evidence is presented that suggests that some components other than acetylcholinesterase induce the acetylcholine receptor response in the bimolecular lipid membrane.
The molecular basis of chemical regulation of electrical activity at the nerve synapse and neuromuscular junction has been subject to extensive study and discussion (1-7). Compounds which initiate changes in membrane potential (such as acetylcholine, carbamylcholine, and decamethonium), and compounds which inhibit these changes (such as tubocurarine, a-bungarotoxin, and atropine) are presumed to bind to a single specific site on the postsynaptic membrane. After interaction with acetylcholine, the postsynaptic membrane permeability and selectivity to ions changes dramatically, leading to a concentration-dependent depolarization of transmembrane potential from about - 70 mV to a minimum of about -15 mV (7). In chemically excitable membranes, such as the innervated face of eel electroplax, the membrane conductance (measured and correlated as depolarization) varies sigmoidally with the logarithm of acetylcholine concentration, suggesting a cooperative in-
teraction between ligand and receptor which mediates the conductance increase. Depolarization of the innervated face of eel electroplax and several other chemically excitable systems can be effected not only by acetylcholine but also by a variety of structurally related compounds termed “activators,” or “lep“agonists,” tocurares.” Some of these compounds are only distantly related to acetylcholine in the structural sense in that they contain a quaternary nitrogen atom but not a hydrolyzable ester group. These observations have been taken to indicate that the interaction of a quaternary group with an anionic site on a receptor causes depolarization of chemically excitable membranes. Study of intact chemically excitable membranes, such as eel electroplax, suffers from several inherent difficulties. It is difficult to separate the contribution of agonist-sensitive components from background components. In addition, membrane potential is not a linear function of
A RECONSTITUTED
ACETYLCHOLINE
RECEPTOR
21
membrane conductance; therefore, depolarization data has limited use for kinetic analysis of the receptor response (see the Discussion below). Furthermore, since the receptor response is an integral membrane property and since the receptor complex is believed to be noncatalytic, the usual analytical techniques for the study of catalytic proteins cannot be used successfully for the study of a receptor. Considerable effort has been devoted to isolation and characterization of the component(s) responsible for binding of agonists and antagonists to eel (8-10) and torpedo (10-14) electroplax, chick embryo muscle (X5), housefly brain (12), and muscle endplate from rat diaphragm (16). However, it is likely that the “binding proteins” isolated from these sources are not the acetylcholine receptor whose in vivo response consists of an increase in membrane ionic permeability in response to agonist binding. Marked differences have been noticed between the affinity of the purified binding proteins for cholinergic agonists and the apparent affinity of the excitable membrane for the same agonist for the depolarization response. These observations are consistent with recent studies on the mode of action of perhydrohistrionicotoxin on neuromuscular endplate which suggest a separate identity for “ion-gating” and a-bungarotoxin binding sites” (17). An alternative approach to identification of receptor components is provided by reconstitution studies. Earlier we have demonstrated that in vitro reconstitution of a model for the acetylcholine receptor system can be achieved by incorporation of an impure acetylcholinesterase preparation from eel electroplax into a black lipid membrane (BLM)’ (18). Addition of a water-soluble commercially available acetylcholinesterase to a BLM prepared from oxidized cholesterol results in ion channel formation in response to acetylcholine and carbamylcholine. The channel activity is apparent only when acetylcholine is added to the tram side (side opposite to which the
receptor protein is added of the membrane. has been reconstitution Similarly, achieved from a detergent-solubilized (Ybungarotoxin binding protein preparation from neuromuscular endplate (19). Although this detergent-solubilized preparation increases the membrane conductance significantly even in the absence of agonists, a 4-fold increase in membrane conductance was observed after acetylcholine addition to the tram side of the membrane. The reconstitution studies with impure acetylcholinesterase from eel electroplax have been confirmed and extended by Goodall et al. (20) who have shown that reconstitution can also be achieved on BLM prepared from soybean lecithin. Furthermore, they have resolved the impure acetylcholinesterase preparation into several components by gel electrophoresis and have convincingly and elegantly demonstrated that the acetylcholine sensitivity of the reconstituted receptor is not due to acetylcholinesterase. Moreover, it has been shown that two of the components obtained by gel electrophoresis reconstitute acetylcholine receptor function on BLM only when present simultaneously. These and other observations on reconstituted systems suggest that the acetylcholine receptor presumably consists of an “agonist binding protein” and an “ion-gating site.” These function together as a complete receptor. In this paper, further characterization of the acetylcholine receptor system reconstituted by incorporation of a crude preparation of acetylcholinesterase from eel electroplax into BLM is described. Various aspects of dose-response relationships for certain known agonists of the acetylcholine receptor are examined. Current-voltage relationships and ionic selectivity for conduction pathways have also been determined from measurement of diffusion potentials in the presence of appropriate ionic gradients.
’ Abbreviations used: BLM, bimolecular lipid membrane; ACh, acetylcholine; AChRJAChE, an impure acetylcholinesterase (Type VI) purchased from Sigma Chemical Co.
Acetylcholine, carbamylcholine, and acetylcholinesterase Type VI of sp act 200-300 pM units were purchased from the Sigma Chemical Company, St. Louis, MO. Decamethonium was purchased from
MATERIAL
AND METHODS
22
MAHENDRA
KUMAR
JAIN
times as much as 25% when Z, is -10-l’ A mm-‘. In Burroughs Wellcome. Tetraethylammonium perchlothe absence of an agonist the noise level rarely exceeds rate was purchased from Aldrich Chemical Company. of data from one set to the next Phenyltrimethylammonium tosylate was prepared by 2-5%. Reproducibility under optimal conditions is about 20%. Part of this methylation of dimethylaniline with methyltosylate. The experimental set-up, electrical circuit, modes may be due to difference in the area of BLM which could not be measured with the accuracy of better of measurement, and general precautions for electrical measurements and BLM formation are described than 10%. elsewhere (21, 22). Other details are given in the RESULTS appropriate sections of this manuscript. All results described herein were obtained from measurements Incorporation of AChRIAChE into BLM made on BLM prepared from a solution obtained’after and the effect of carbamylcholine on the refluxing with vigorous stirring a 4% solution of modified BLM. After thinning of the BLM cholesterol (by weight) in (1: 1) decane plus tetradecwas complete, a known amount of freshly ane (18). Bimolecular lipid membranes were prepared by (within 6 hr) prepared solution of AChR/ blowing a small bubble of air with Pasteur pipets AChE was added to one (cis) compartment precoated inside with a small volume of the lipid bathing the membrane. A constant potensolution. The thinning of the film to the secondary tial of 70 mV (cis-side positive) was mainstage is achieved in less than 30 set under optimum tained across the BLM. Complete equiliconditions (see p. 60 in Ref. 21 for details). The BLM bration of protein between the surrounding thus prepared have a constant (for several hours) medium and the membrane was achieved resistance of >5 x 10’ ohm for a membrane on the in 5-15 min depending upon the concentrahole of area of about 1 mmz. Under optimum condition of the protein, the level of large tions the BLM were stable for several hours; however, transmembrane current fluctuations, and all measurements described herein on a given BLM were completed less than 40-45 min after BLM the presence of various ions and substrates formation. If a BLM broke in the first minute after in the medium. However, these variables formation, it was re-formed; however, if it broke more have not yet been investigated systematithan three to five times in a row, then a fresh cally. Incorporation of AChR/AChE into membrane assembly was used. If the resistance of a the BLM could be followed in the initial BLM was found to be less than 3 x IO9 ohm, a stages by monitoring changes in the memrecleaning of the membrane assembly and a recheck brane resistance. Thus, after the addition of the water supply and source of chemicals was conducted until the desired membrane resistance was of the protein to the aqueous medium fluctuations in the achieved. BLM were never formed in the presence of there are significant transmembrane resistance. Transient added proteins and/or agonists (see below). Impure acetylcholinesterase (Sigma, Type VI) was changes in BLM resistance as large as a used for all the reconstitution studies described. factor of 5-8 have been noted occasionally. However, as demonstrated by Goodall et al. (201, the The BLM are very susceptible to breakage active receptor-forming agent is not acetylcholinesterduring the first 2-6 min after the addition ase itself. Therefore, this impure preparation is reof AChR/AChE. After about 10 min, the ferred to as AChR/AChE in the text. Furthermore, membrane conductance returns to the inisignificant quantitative variations were found in diftial value of the unmodified membrane and ferent batches of AChWAChE in producing a receptor response on BLM. Quantitative reproducibility of the the BLM become more stable. The effect of acetylcholine (ACh) on I,,, response from the same batch of AChR/AChE is fair (*20%). All measurements were conducted in 1 M across the modified BLM at 70 mV (inside NaCl + 10 mM MgCl,, containing 20 mM Tris-chlopositive) is given in Table I. Addition of ride at pH 7.4. Temperature for all measurements was ACh to the trans compartment leads to a maintained at 32-34°C. An error of +3% in concentrapersistent increase in I,,, . However, the tions of various agents added to individual compartaddition of ACh to the cis compartment ments is estimated. Similarly, a considerable increase leads to only a transient and relatively in membrane “noise” after the addition of agonists small increase in the membrane conductmakes it difficult to gather data on membrane conance. This difference in behavior of ACh in ductance with better than 15% accuracy. Furthermore, the noise level in Z, depends significantly upon the cis and trans compartments is consistthe nature of the agonist. Thus, for example, in the ent with the view that the free AChE in the presence of decamethonium the noise level is some- cis compartment rapidly hydrolyzes ACh,
A RECONSTITUTED
ACETYLCHOLINE
RECEPTOR
23
Dose-response relationship for the effect on AChRIAChE-modified ON AChR/AChE” MODIFIED BLM BLM. Conductance increases induced by varying concentrations of ACh added to Additives I ,,, x 10”A the trans side of the modified BLM are (for 70.mV pulse) cis tram shown in Fig. 2. On a linear scale, I, increases sigmoidally with the logarithm of None None 2 the ACh concentration in the trans comNone CBC or ACh 2 partment. At sufficiently high ACh conAChlUAChE None 5+2 centrations, the membrane conductance AChR/AChE + None 15 * 5 decreases gradually to a constant value. CBC or ACh When the concentration of AChR/AChE AChR/AChE CBC or ACh 180 * 50 AChR/AChE + CBC or ACh 200 * 50 in the cis compartment is high (-12 pgl CBC or ACh ml), saturation in the dose-response curve AChR/AChE Nicotine 125 i 5 is observed at much larger values of I,,, . In AChRlAChE + None 20 * 5 this case, however, it is not possible to nitotine increase membrane conductance indefinitely. In fact, the modified BLM become ’ Carbamylcholine (CBC) or acetylcholine (ACh) exceedingly unstable when conductance is when present is at 50 KM; AChR/AChE when present by a factor approaching 103. is at 4 &ml. Nicotine when present is at 1.0 mM. I ,,, increased was measured at least 3 min after the addition of Indeed, saturation of I,,, when the AChR/ additives to either compartment. AChR/AChE-modiAChE concentration was varied at confied BLM was found to be very unstable in the stant ACh concentration in the trans comTABLE
SIDEDNESS
I
OF THE DEPOLARIZING
presence of carbamylcholine in both compartments.
ACTION
or nicotine
OF AGONISTS
of acetylcholine
when present
thus reducing its concentration in the medium (however, see below). A similar difference in response from the cis and trans sides is also elicited by CBC and nicotine. It is, therefore, quite likely that the receptor sites are oriented asymmetrically across the BLM, and the agonist response is elicited principally from the trans side. This possibility has been confirmed by Goodall et al. (20) who have shown that the receptor protein, free from AChE activity, does indeed show sensitivity to acetylcholine only from the trans side. The results also indicate that the addition of CBC to the trans side of BLM significantly alters its conductance. Furthermore, the AChR/ AChE-modified BLM appears to be asymmetrical with respect to the orientation of ACh receptors and their access to CBC. However, as shown in Fig. 1, the currentvoltage relationship for BLM modified with AChR/AChE is almost linear in the presence and absence of carbamylcholine. The slope of the I-Vcurve changes considerably in the presence of carbamylcholine, suggesting that this agonist opens conduction pathways which are symmetrical.
FIG. 1. Current-voltage (1, vs V,,,) relationships for (A) unmodified BLM, (B) BLM modified with 20 rg/ml of AChR/AChE and (C) AChR/AChE-modified BLM with 50 pM carbamylcholine in the trans compartment. Data were obtained by continuously varying the transmembrane potential in the steps of 10 mV from +70 mV to - 70 mV. The sign of V, refers to the cis side, the side containing AChFUAChE.
24
MAHENDRA
KUMAR JAIN
ACh (PM)
FIG. 2. Dependence of I,,, on the concentration of ACh on the trans side of the modified BLM. Protein/AChE concentration in the cis compartment is 5 pg or 1 unit/ml. Each point is the mean of five readings on five different membranes. ACh concentration was increased by successive addition of small volumes of a concentrated ACh solution to the trans compartment. The error bars in this and other figures indicate standard deviation.
partment was never observed. A plot of log Im vs log ACh is shown in Fig. 3. The slope of the curve in the linear region is 4, which suggests that four molecules of ACh are needed to elicit a unit change in conductance across the modified BLM. The effect of uarious agonists on membrane conductance. Several agonists were
tested for ability to increase membrane conductance; the results are shown in Fig. 4. All the curves are sigmoidal with affinity (K, , half-maximal concentration) and efficacy (I,,,), maximal response) characteristic of a given agonist. Values for affinity and efficacy of these agents are given in Table II. Values cited in this Table were derived from separate plots on expanded scales. For the sake of comparison K, values for these agonists for depolarization of intact electroplax are also given in Table II. The log-log plots of various doseresponse relationships show linear regions, the slopes (n) of which are also collected in Table II. The data for carbamylcholine, phenyltrimethylammonium chloride, and tetraethylammonium chloride do not show a large scatter, and the molecularity (nl of the response was found to be four for these agonists. A slope (n) of 1.5-2.5 was observed for decamethonium (Fig. 5). Of all
ACh (/.A41
FIG 3. Plot for the increase in transmembrane current (I,) as a function of ACh concentration in the trams compartment. Protein/AChE concentration was approximately 1 unit/ml. The line is drawn with a slope of 4.
the agonists studied, decamethonium showed the maximum scatter in experimental data. Decamethonium also showed membrane lytic activity at concentrations
A RECONSTITUTED
ACETYLCHOLINE
25
RECEPTOR
30
P 0 X : E h E 9
4o
30
7.0
IO
0
IO
00 Activator
1000
($4)
relationships for various agonists. DECA, decamethonium, ACh, FIG. 4. Dose-response acetylcholine, CBC, carbamylcholine, PTA, phenyltrimethylammonium, TEA, tetraethylammonium. Each point is the mean of Z-lO.measurements on different membranes. The range of scatter in data is not shown here, but the scatter was maximal for decamethonium and least for carbamylcholine. Both ACh and carbamylcholine were added only to the tmns side, and protein/AChE (approximately 1 unit/ml) to the cis side only. Decamethonium, phenyltrimethylammonium chloride, and tetraethylammonium chloride were added to both compartments along with 3 units of protein/AChE per ml. TABLE VARIOUS
Agonist
CONSTANTS
CHARACTERIZING
K,, 33” BLM (wM)
Acetycholine Carbamylcholine Decamethonium Phenyltrimethylammonium Tetraethylammonium Nicotine
24 35 4.0 16 115 650
II
DOSE-RESPONSE
Km, 22”
RELATIONSHIPS
ZmaxC
Electrp. (PM)*
FOR SEVERAL AGONISTS’
Zi”l
Molecularity
20 30 1.2 13
50 57 55 13
30 45 30 10
0.6 0.77 0.54 O.-6
-
40 -
24 -
0.6 0.8
4.1 3.95 1.5-2.5 3.5-4.0 4.0 -
OData were derived from plots similar to those shown in Figs. 1 and 2 but on expanded concentration axis. b From References 3 and 23. ‘Protein/AChE concentration for ACh and carbamylcholine was 1 unit (4 rg)/ml on only one side of the membrane; corresponding concentration for decamethonium, phenyltrimethylammonium, and tetraethylammonium chloride was 3 units/ml on both sides of the membrane.
above 10 PM. The data taken as a whole tential which developed across the BLM suggest that several known agonists of the due to ionic gradients was measured under acetylcholine receptor increase the con- the conditions listed in Table III. The magnitude and sign of the diffusion potenductance of AChIUAChE-modified BLM. tial shows that BLM alone do not show any Ion selectivity of the conduction pathdiscrimination among Na+, K+, or Cl-. ways induced by AChRIAChE incorporation into BLM. BLM were prepared with However, BLM modified with AChR/ appropriate ionic gradients (Table III) AChE show ionic selectivity in the order across the membranes. The membrane po- Na+ N K+ < Cl-. The same selectivity is
26
MAHENDRA
KUMAR
JAIN
FIG. 5. Dose-response relationship on a log-log scale for the increase in transmemhrane current as a function of decamethonium (DECA) concentration. Both protein/AChE and decamethonium were present in both compartments; however, decamethonium was added about 15 min after the addition of protein/AChE. Each data point is the mean of 5-15 independent measurements. TABLE
III
OPEN CIRCUIT POTENTIAL AS A FUNCTION OF IONIC GRADIENT ACROSSBLM MODIFIED WITH AChR/AChE PRESENCE OF CARBAMYLCHOLINE Trans compartment 1 M NaCl 1 M NaCl 1 MNaCl
+ 50 mM CBC
1 MKCl+50mMCBC 1 MKC~ 1 MKC~ 1~KClf 50
mM
Cis compartment
Potential (sign for cis side)
0.1 M NaCl 0.1 MNaCl + AChlUAChE 0.1 M NaCl + AChR/AChE 0.1 M NaCl + AChRJAChE 1 M NaCl 1 MNaCl+ AChlUAChE lMNaCl+ AChWAChE
<+2 mV +5 mV
PNa 7 PO
+21 mV
Pi%7 PC,
+15
P Na =pK 7 PO
mV
IN THE
Relative permabilities”
<+l mv -3 mV -4 mV
CBC
n CBC, carbamylcholine. b Order of permeability approximately: Na 2 K > Cl. BLM permeability does not change significantly presence of carbamylcholine alone. AChR/AChE when present is 15-&ml.
observed in the presence of carbamylcholine. Furthermore, the data show that the ratio of permeabilities for K+, Na+, and Cl- ions is approximately 3: 3: 1. Resolution of receptor actiuity and choline&erase activities from the AChRIAChE preparation by affinity chromatography. An affinity column containing p-trimethyl-
in the
phenylammonium chloride ligand was prepared by the method of Berman and Young (26). Protein absorbed on the column (7 ml gel volume) was eluted with a NaCl gradient (linear, 0.5-2.0 M). The elution profile for AChE activity is shown in Fig. 6. The AChR activity on the BLM was measured in pooled fractions (of 35-ml size) after
A RECONSTITUTED
ACETYLCHOLINE
Volume
RECEPTOR
27
(ml)
FIG. 6. The elution profile for acetylcholinesterase from affinity column (7-ml column volume) containing p-trimethylammoniumphenyl bound to Sepharose by a spacer arm (cf. 26).
lyophilization. Most of the AChR activity (less than 10% of the starting activity) was found in the eluant (135-170 ml) as indicated in Fig. 6. Although this fraction showed a response that was qualitatively similar to that observed for the crude preparation, reproducibility of separation was poor. Affinity chromatography with other ligands and eluant systems is being tried. However, the results do strongly suggest that neither acetylcholinesterase alone, nor in combination with other molecular species is responsible for the receptor activity. DISCUSSION
In the preceding section I have described some of the fundamental features of interaction of acetylcholine and related agonists in BLM modified with an impure commercially available preparation of acetylcholinesterase from eel electroplax. In general, changes in membrane permeability produced by various agonists are concentration dependent. A sigmoidal doseresponse curve with a characteristic affinity for the agonist is exhibited. The efficacy of the agonist depends not only upon the nature of the agonist but also upon the concentration of AChRlAChE on the opposite side of the membrane. All these characteristics would be expected from a reconstituted acetylcholine receptor (cf. 1-7). The affinity and efficacy data for six different agonists are given in Table II. The values of half-maximal activating concen-
trations for the agonists on the AChR/ AChE-modified BLM are listed in column 2. Corresponding values for isolated electroplax obtained by measurement of depolarization of resting potential as reported in the literature (3, 23) are given in column 3. A comparison of the values for K, in these two columns suggest a good correspondence in absolute values of K,. Thus, for example, the values of apparent affinity constants for carbamylcholine are 35 and 30 pM and for PTA are 16 and 13 pM in the modified BLM and isolated electroplax, respectively. It may be noted that the value of K, for acetylcholine in the isolated electroplax system (23) obtained by Voltage Clamp IIX!aSUreInentS, 20 FM, is also in good agreement with the value for K, , 24 pM, obtained on the reconstituted BLM. However, the corresponding values for DECA are 4.0 and 1.2 pM, respectively. The significance of this difference is not known (see below). In column 4 of Table II are given the maximal values of current that could be drawn through the BLM modified by a given concentration of AChR/AChE. The values for both acetylcholine and carbamylcholine are for BLM treated with 6 pglml of AChIUAChE whereas the corresponding values were obtained with BLM treated with 18 pg/ml of the same AChR/ AChE. Thus, qualitatively the agonists for depolarization of AChR/AChE-treated BLM have their efficacies in the following order: carbamylcholine (115) 2 acetylcho-
28
MAHENDRA
line (100) > decamethonium (27) 2 tetraethylammonium chloride > phenyltrimethylammonium chloride. It is pertinent to note that Changeux et al. (3) have observed the following order of efficacies for agonists in the isolated electroplax: carbamylcholine > decamethonium > phenyltrimethylammonium chloride. The value of efficacy given for each agonist is only approximate and is based on the observation that, in this particular range of protein concentration, a 3-fold increase in AChR/AChE concentration leads to approximately a loo-fold increase in I,,, . It is based on a fourth-order dependence of I,,, on AChR/AChE concentration. As noted in column 5 of Table II, the value of current at high agonist concentration (linr) is about 70% of I,,, . Such a “desensitization” has been observed in electrophysiological experiments with muscles and electroplax (7, 24, 25). In column 6 of Table II are given the values of molecularity (n) obtained from the slopes of the plots of log 1, vs log (agonist concentration). Values of n for acetylcholine, carbamylcholine, phenyltrimethylammonium chloride, and tetraethylammonium chloride are approximately 4. In contrast, the value of n for decamethonium is 1.5-2.5. (As noted earlier, there is relatively large scatter in this data.) The significance of this difference may be found in the study of the effect of polymethonium ions on intact chemically excitable membranes (6). This would suggest that, like the natural receptor, the reconstituted system binds to two molecules of decamethonium, instead of four molecules of other monoammonium agonists leading to opening of ionconduction pathways. The values of Hill coefficient -2 have been reported by Changeux et al. (3) for the depolarization of isolated electroplax by carbamylcholine, decamethonium, and phenyltrimethylammonium chloride. However, these authors have plotted log [E - J’-LJIGL,,~ - E,,)] vs log of agonist concentration (Hill plot). E, is the resting membrane potential, Em,, is the maximum possible depolarization, and E is the mem-
KUMAR JAIN
brane potential at any given agonist concentration. Such a plot neither yields a true Hill coefficient nor the molecularity of the overall process. In order to obtain a true value of n from a log response vs log dose curve, the response should be on a truly linear scale. However, membrane potential in the resting state, as well as in the depolarized state, is a complex function of relative ionic permeabilities (27, 28). It is governed not only by the gross membrane conductance and concentrations of ions in the media surrounding the membrane, but also on the relative permeabilities of these ions for the resting and depolarized membranes. It is obvious from the data of Changeux et al. (2, 3) that both the relative and absolute permeabilities of the electroplax membrane change drastically during an agonist-induced depolarization of the cholinergic membrane. Most of the parameters characterizing and relating these changes to membrane potential are not known. This makes a close comparison of our data with the available data on the intact electroplax rather difficult. It is, however, pertinent to note that the slope at the inflexion point is plots of E - E, vs log of agonist concentration (cf. Fig. 3 in Ref. 3) do differ for decamethonium and carbamylcholine. In fact, the slope for carbamylcholine is almost twice as large as that for the decamethonium curve. This is in qualitative accord with the observations presented here as well as with the hypothesis that one molecule of decamethonium has (or can have) depolarizing ability equivalent to that of two carbamylcholine molecules. Furthermore, this relationship is also consistent with the suggestion that the receptor sites for quaternary ammonium groups may be separated by about 14A, the distance between two ammonium groups in decamethonium (6). The data presented in Table III show that the AChlUAChE reconstituted membrane does not show significantdiscrimination between Na+ and K+ ions. However, these cations are about 3-fold more permeable than Cl- ions. These results are in good quantitative accord with data on eel electroplax (29).
A RECONSTITUTED
ACETYLCHOLINE
All these observations show qualitative and quantitative similarities in the behavior of isolated electroplax membrane and the BLM modified with an impure preparation of acetylcholinesterase from eel electroplax. It is now certain that some impurity in the preparation is responsible for the observed effects (20). Purified AChE does not show any receptor-like response on BLM. In contrast, AChR/AChE resolved by electrophoresis on polyacrylamide gel yields bands which are distinctly separate from AChE and show the characteristic AChR response (20, unpublished observations). Furthermore, the data presented herein and elsewhere (18, 20, and Jain, unpublished observations) suggest that the receptor reconstituted from impure AChR/AChE shows both the muscarinic and nicotinic response. Whether this reflects the presence of distinct receptor species or is the consequence of the same receptor species in two different “states,” remains to be resolved. Attempts at biochemical, topographical, and pharmacological characterization of the reconstituted receptor are in progress.
RECEPTOR
29
(1966) Pharm. Reu. 18, 1051. 7. KATZ, B., AND THESLEFF, S. (1957) J. Physiol. 138, 63. 8. KLEDD,R. B., FULPUIS, B. W., COOPER,D., SMITH, M., REICH, E., ANDPOSSANI, L. D. (1973) J. Biol. Chem. 248,684l. 9. KARLSSON, E., HEILBRONN, E., AND WILDLUND, L. (1972) Fed. Eur. Biochem. Sot. Lett. 28, 107. 10. CLARK, D. G., Wo~corr, R. G., AND RAFTERY, M. A. (1972) Biochem. Biophys. Res. Commun. 48, 1061. 11. SCHMIDT, J. S., AND RA~ERY, M. A. (1972) Biothem. Biophys. Res. Commun. 49, 572; (1973) Biochemistry 12,852. 12. ELDEFRAWI, M., E., ELDEFRAWI, A. T., AND O’BRIEN, R. T. (1971) Mol. Pharmacol. 7, 104. 13. MEUNIER, J. C., AND CHANGEUX, J. P., Fed. Eur. Biochem. Sot. Lett. 32, 143. 14. MILEDI, R., MOLINOFF, P., AND POWER, L. T. (1971) Nature (London) 229, 554. 15. SUGIYAMA, H., BENDA, P., MEUNIER, J. C., AND CHANGEUX, J. P. (1973) Fed. Eur. Biochem. Sot. Lett. 35, 125. 16. CHIU, T. H., DOLBY, J. O., AND BARNARD, E. A. (1973) Biochem. Biophys. Res. Commun. C 51, 205. 17. ALBUQUERQUE,E. X., BARNARD, E. A., CHIU, T. H., LAPA, A. J., DOLLY, J. 0.. JANSSON, S., DALY, J., AND WITKOP, B. (1973) Proc. Nat. Acad. Sci. USA 70,949. 18. JAIN, M. K., MEHL, L. E., AND CORDES, E. H. ACKNOWLEDGMENTS (1973) Biochem. Biophys. Res. Commun. 51, I express my appreciation to Professor E. H. Cordes 192. for his encouragement and advice, in whose labora19. KEMP, G., DOLBY, J. O., BARNARD, E. A., AND tory part of this work was done at Indiana University, WENNER, C. E. (1973) Biochem. Biophys. Res. Bloomington, Indiana. I also thank Dr. M. Goodall for Commun. 54, 607. providing his unpublished manuscripts mentioned in 20. GOODALL, M. C., BRADLEY, R. J., SACCOMANI, G., this paper, and to Dr. H. White for comments on the AND ROMINE, W. 0. (1974) Nature (in press); manuscript. also unpublished data. REFERENCES 21. JAIN, M. K. (1972) The Bimolecular Lipid Mem1. EDELMAN, J. S. (1961) Annu. Reu. Phy~iol. 23,37. brane: A System, Van Nostrand-Reinhold, New York. 2. PODLESKI, T. R., AND CHANGEUX, J. P. (1970) in Fundamental Concepts in Drug Receptor In- 22. JAIN, M. K., WILLIAM, E., WHITE, F. P., STRICKHOLM, A., AND CORDES, E. H. (1972) J. Memteractions (Danelli, J. F., Moran, J. F., and brane Biol. 8, 363. Triggle, D. J., eds.), p. 93, Academic Press, New York. 23. RUIZ-MANRESA, F., Personal Communication 3. CHANGEUX, J. P., KASAI, M., HUCHET, M., AND (1973). 24. BENNEW, M. V. L., WURZEL, M., AND GRUNDFEST, MEUNIER, J. C. (1971) in Cholinergic Ligand H. (1961) J. Gen. Physiol. 44, 757. Interactions (Triggle, D. J., Moran, J. F., and Barnard, E. A., eds.), p. 33, Academic Press, 25. GOLDSTEIN, A., ARONOW, L., AND KALMAN, S. (1968) Principles of Drug Action, Harper and New York. Row, New York. 4. LONG, J. P: (1963) in Handbuch der Experimen26. BERMAN, J. B. (1973) Biochemistry 12, 1710. tellen Pharmakologie: Cholinesterases and AnPhenomena, ticholinesterase Agents (Eichler, O., and Fa- 27. PLONSEY, R. (1969) Bioelectric McGraw-Hill, New York. rah, A., eds.), Koelle, G. B., Sub. ed., Vol. 15, 28. HODGKIN, A. L., AND KATZ, B. (1949) J. Physiol. p. 398, Springer-Verlag, Berlin. 108, 37. 5. BARLOW, R. B. (1964) Introduction to Chemical 29. RUIZ-MANRESA, F., AND GRUNDFEST, H. (1971) J. Pharmacology 2nd. Ed., Methuen, London. Gen. Physiol. 57, 71. 6. KHROMOV-BORISOV, N. Y., AND MICHELSON, M. J.
OF BIOCHEMISTRY
Studies
AND
BIOPHYSICS
164, 20-29 (1974)
on a Reconstituted
Acetylcholine
Receptor
System:
Effect of Agonists MAHENDRA Department
of Chemistry
and Health
KUMAR JAIN
Sciences, University
Received February
of Delaware,
Newark,
Delaware
197111
22, 1974
An impure preparation of acetylcholinesterase from electroplax of the electric eel can be incorporated into a bimolecular lipid membrane. The acetylcholinesterase-modified bimolecular lipid membrane shows a concentration-dependent increase in membrane conductance elicited by several agonists (acetylcholine, carbamylcholine, phenyltrimethylammonium ion, tetraethylammonium ion, decamethonium ion, and nicotine) added to the compartment opposite that to which acetylcholinesterase was originally added. Affinity and efficacy of the various agonists in generating the conductance increase were measured from dose-response curves; these are in good quantitative agreement with corresponding values observed for depolarization of intact eel electroplax. The ion conduction pathways induced by agonists in the modified bimolecular lipid membrane show a slight cation selectivity, Na 2 K > Cl (3 : 3 : l), similar to that observed for the depolarized electroplax membrane. Evidence is presented that suggests that some components other than acetylcholinesterase induce the acetylcholine receptor response in the bimolecular lipid membrane.
The molecular basis of chemical regulation of electrical activity at the nerve synapse and neuromuscular junction has been subject to extensive study and discussion (1-7). Compounds which initiate changes in membrane potential (such as acetylcholine, carbamylcholine, and decamethonium), and compounds which inhibit these changes (such as tubocurarine, a-bungarotoxin, and atropine) are presumed to bind to a single specific site on the postsynaptic membrane. After interaction with acetylcholine, the postsynaptic membrane permeability and selectivity to ions changes dramatically, leading to a concentration-dependent depolarization of transmembrane potential from about - 70 mV to a minimum of about -15 mV (7). In chemically excitable membranes, such as the innervated face of eel electroplax, the membrane conductance (measured and correlated as depolarization) varies sigmoidally with the logarithm of acetylcholine concentration, suggesting a cooperative in-
teraction between ligand and receptor which mediates the conductance increase. Depolarization of the innervated face of eel electroplax and several other chemically excitable systems can be effected not only by acetylcholine but also by a variety of structurally related compounds termed “activators,” or “lep“agonists,” tocurares.” Some of these compounds are only distantly related to acetylcholine in the structural sense in that they contain a quaternary nitrogen atom but not a hydrolyzable ester group. These observations have been taken to indicate that the interaction of a quaternary group with an anionic site on a receptor causes depolarization of chemically excitable membranes. Study of intact chemically excitable membranes, such as eel electroplax, suffers from several inherent difficulties. It is difficult to separate the contribution of agonist-sensitive components from background components. In addition, membrane potential is not a linear function of
A RECONSTITUTED
ACETYLCHOLINE
RECEPTOR
21
membrane conductance; therefore, depolarization data has limited use for kinetic analysis of the receptor response (see the Discussion below). Furthermore, since the receptor response is an integral membrane property and since the receptor complex is believed to be noncatalytic, the usual analytical techniques for the study of catalytic proteins cannot be used successfully for the study of a receptor. Considerable effort has been devoted to isolation and characterization of the component(s) responsible for binding of agonists and antagonists to eel (8-10) and torpedo (10-14) electroplax, chick embryo muscle (X5), housefly brain (12), and muscle endplate from rat diaphragm (16). However, it is likely that the “binding proteins” isolated from these sources are not the acetylcholine receptor whose in vivo response consists of an increase in membrane ionic permeability in response to agonist binding. Marked differences have been noticed between the affinity of the purified binding proteins for cholinergic agonists and the apparent affinity of the excitable membrane for the same agonist for the depolarization response. These observations are consistent with recent studies on the mode of action of perhydrohistrionicotoxin on neuromuscular endplate which suggest a separate identity for “ion-gating” and a-bungarotoxin binding sites” (17). An alternative approach to identification of receptor components is provided by reconstitution studies. Earlier we have demonstrated that in vitro reconstitution of a model for the acetylcholine receptor system can be achieved by incorporation of an impure acetylcholinesterase preparation from eel electroplax into a black lipid membrane (BLM)’ (18). Addition of a water-soluble commercially available acetylcholinesterase to a BLM prepared from oxidized cholesterol results in ion channel formation in response to acetylcholine and carbamylcholine. The channel activity is apparent only when acetylcholine is added to the tram side (side opposite to which the
receptor protein is added of the membrane. has been reconstitution Similarly, achieved from a detergent-solubilized (Ybungarotoxin binding protein preparation from neuromuscular endplate (19). Although this detergent-solubilized preparation increases the membrane conductance significantly even in the absence of agonists, a 4-fold increase in membrane conductance was observed after acetylcholine addition to the tram side of the membrane. The reconstitution studies with impure acetylcholinesterase from eel electroplax have been confirmed and extended by Goodall et al. (20) who have shown that reconstitution can also be achieved on BLM prepared from soybean lecithin. Furthermore, they have resolved the impure acetylcholinesterase preparation into several components by gel electrophoresis and have convincingly and elegantly demonstrated that the acetylcholine sensitivity of the reconstituted receptor is not due to acetylcholinesterase. Moreover, it has been shown that two of the components obtained by gel electrophoresis reconstitute acetylcholine receptor function on BLM only when present simultaneously. These and other observations on reconstituted systems suggest that the acetylcholine receptor presumably consists of an “agonist binding protein” and an “ion-gating site.” These function together as a complete receptor. In this paper, further characterization of the acetylcholine receptor system reconstituted by incorporation of a crude preparation of acetylcholinesterase from eel electroplax into BLM is described. Various aspects of dose-response relationships for certain known agonists of the acetylcholine receptor are examined. Current-voltage relationships and ionic selectivity for conduction pathways have also been determined from measurement of diffusion potentials in the presence of appropriate ionic gradients.
’ Abbreviations used: BLM, bimolecular lipid membrane; ACh, acetylcholine; AChRJAChE, an impure acetylcholinesterase (Type VI) purchased from Sigma Chemical Co.
Acetylcholine, carbamylcholine, and acetylcholinesterase Type VI of sp act 200-300 pM units were purchased from the Sigma Chemical Company, St. Louis, MO. Decamethonium was purchased from
MATERIAL
AND METHODS
22
MAHENDRA
KUMAR
JAIN
times as much as 25% when Z, is -10-l’ A mm-‘. In Burroughs Wellcome. Tetraethylammonium perchlothe absence of an agonist the noise level rarely exceeds rate was purchased from Aldrich Chemical Company. of data from one set to the next Phenyltrimethylammonium tosylate was prepared by 2-5%. Reproducibility under optimal conditions is about 20%. Part of this methylation of dimethylaniline with methyltosylate. The experimental set-up, electrical circuit, modes may be due to difference in the area of BLM which could not be measured with the accuracy of better of measurement, and general precautions for electrical measurements and BLM formation are described than 10%. elsewhere (21, 22). Other details are given in the RESULTS appropriate sections of this manuscript. All results described herein were obtained from measurements Incorporation of AChRIAChE into BLM made on BLM prepared from a solution obtained’after and the effect of carbamylcholine on the refluxing with vigorous stirring a 4% solution of modified BLM. After thinning of the BLM cholesterol (by weight) in (1: 1) decane plus tetradecwas complete, a known amount of freshly ane (18). Bimolecular lipid membranes were prepared by (within 6 hr) prepared solution of AChR/ blowing a small bubble of air with Pasteur pipets AChE was added to one (cis) compartment precoated inside with a small volume of the lipid bathing the membrane. A constant potensolution. The thinning of the film to the secondary tial of 70 mV (cis-side positive) was mainstage is achieved in less than 30 set under optimum tained across the BLM. Complete equiliconditions (see p. 60 in Ref. 21 for details). The BLM bration of protein between the surrounding thus prepared have a constant (for several hours) medium and the membrane was achieved resistance of >5 x 10’ ohm for a membrane on the in 5-15 min depending upon the concentrahole of area of about 1 mmz. Under optimum condition of the protein, the level of large tions the BLM were stable for several hours; however, transmembrane current fluctuations, and all measurements described herein on a given BLM were completed less than 40-45 min after BLM the presence of various ions and substrates formation. If a BLM broke in the first minute after in the medium. However, these variables formation, it was re-formed; however, if it broke more have not yet been investigated systematithan three to five times in a row, then a fresh cally. Incorporation of AChR/AChE into membrane assembly was used. If the resistance of a the BLM could be followed in the initial BLM was found to be less than 3 x IO9 ohm, a stages by monitoring changes in the memrecleaning of the membrane assembly and a recheck brane resistance. Thus, after the addition of the water supply and source of chemicals was conducted until the desired membrane resistance was of the protein to the aqueous medium fluctuations in the achieved. BLM were never formed in the presence of there are significant transmembrane resistance. Transient added proteins and/or agonists (see below). Impure acetylcholinesterase (Sigma, Type VI) was changes in BLM resistance as large as a used for all the reconstitution studies described. factor of 5-8 have been noted occasionally. However, as demonstrated by Goodall et al. (201, the The BLM are very susceptible to breakage active receptor-forming agent is not acetylcholinesterduring the first 2-6 min after the addition ase itself. Therefore, this impure preparation is reof AChR/AChE. After about 10 min, the ferred to as AChR/AChE in the text. Furthermore, membrane conductance returns to the inisignificant quantitative variations were found in diftial value of the unmodified membrane and ferent batches of AChWAChE in producing a receptor response on BLM. Quantitative reproducibility of the the BLM become more stable. The effect of acetylcholine (ACh) on I,,, response from the same batch of AChR/AChE is fair (*20%). All measurements were conducted in 1 M across the modified BLM at 70 mV (inside NaCl + 10 mM MgCl,, containing 20 mM Tris-chlopositive) is given in Table I. Addition of ride at pH 7.4. Temperature for all measurements was ACh to the trans compartment leads to a maintained at 32-34°C. An error of +3% in concentrapersistent increase in I,,, . However, the tions of various agents added to individual compartaddition of ACh to the cis compartment ments is estimated. Similarly, a considerable increase leads to only a transient and relatively in membrane “noise” after the addition of agonists small increase in the membrane conductmakes it difficult to gather data on membrane conance. This difference in behavior of ACh in ductance with better than 15% accuracy. Furthermore, the noise level in Z, depends significantly upon the cis and trans compartments is consistthe nature of the agonist. Thus, for example, in the ent with the view that the free AChE in the presence of decamethonium the noise level is some- cis compartment rapidly hydrolyzes ACh,
A RECONSTITUTED
ACETYLCHOLINE
RECEPTOR
23
Dose-response relationship for the effect on AChRIAChE-modified ON AChR/AChE” MODIFIED BLM BLM. Conductance increases induced by varying concentrations of ACh added to Additives I ,,, x 10”A the trans side of the modified BLM are (for 70.mV pulse) cis tram shown in Fig. 2. On a linear scale, I, increases sigmoidally with the logarithm of None None 2 the ACh concentration in the trans comNone CBC or ACh 2 partment. At sufficiently high ACh conAChlUAChE None 5+2 centrations, the membrane conductance AChR/AChE + None 15 * 5 decreases gradually to a constant value. CBC or ACh When the concentration of AChR/AChE AChR/AChE CBC or ACh 180 * 50 AChR/AChE + CBC or ACh 200 * 50 in the cis compartment is high (-12 pgl CBC or ACh ml), saturation in the dose-response curve AChR/AChE Nicotine 125 i 5 is observed at much larger values of I,,, . In AChRlAChE + None 20 * 5 this case, however, it is not possible to nitotine increase membrane conductance indefinitely. In fact, the modified BLM become ’ Carbamylcholine (CBC) or acetylcholine (ACh) exceedingly unstable when conductance is when present is at 50 KM; AChR/AChE when present by a factor approaching 103. is at 4 &ml. Nicotine when present is at 1.0 mM. I ,,, increased was measured at least 3 min after the addition of Indeed, saturation of I,,, when the AChR/ additives to either compartment. AChR/AChE-modiAChE concentration was varied at confied BLM was found to be very unstable in the stant ACh concentration in the trans comTABLE
SIDEDNESS
I
OF THE DEPOLARIZING
presence of carbamylcholine in both compartments.
ACTION
or nicotine
OF AGONISTS
of acetylcholine
when present
thus reducing its concentration in the medium (however, see below). A similar difference in response from the cis and trans sides is also elicited by CBC and nicotine. It is, therefore, quite likely that the receptor sites are oriented asymmetrically across the BLM, and the agonist response is elicited principally from the trans side. This possibility has been confirmed by Goodall et al. (20) who have shown that the receptor protein, free from AChE activity, does indeed show sensitivity to acetylcholine only from the trans side. The results also indicate that the addition of CBC to the trans side of BLM significantly alters its conductance. Furthermore, the AChR/ AChE-modified BLM appears to be asymmetrical with respect to the orientation of ACh receptors and their access to CBC. However, as shown in Fig. 1, the currentvoltage relationship for BLM modified with AChR/AChE is almost linear in the presence and absence of carbamylcholine. The slope of the I-Vcurve changes considerably in the presence of carbamylcholine, suggesting that this agonist opens conduction pathways which are symmetrical.
FIG. 1. Current-voltage (1, vs V,,,) relationships for (A) unmodified BLM, (B) BLM modified with 20 rg/ml of AChR/AChE and (C) AChR/AChE-modified BLM with 50 pM carbamylcholine in the trans compartment. Data were obtained by continuously varying the transmembrane potential in the steps of 10 mV from +70 mV to - 70 mV. The sign of V, refers to the cis side, the side containing AChFUAChE.
24
MAHENDRA
KUMAR JAIN
ACh (PM)
FIG. 2. Dependence of I,,, on the concentration of ACh on the trans side of the modified BLM. Protein/AChE concentration in the cis compartment is 5 pg or 1 unit/ml. Each point is the mean of five readings on five different membranes. ACh concentration was increased by successive addition of small volumes of a concentrated ACh solution to the trans compartment. The error bars in this and other figures indicate standard deviation.
partment was never observed. A plot of log Im vs log ACh is shown in Fig. 3. The slope of the curve in the linear region is 4, which suggests that four molecules of ACh are needed to elicit a unit change in conductance across the modified BLM. The effect of uarious agonists on membrane conductance. Several agonists were
tested for ability to increase membrane conductance; the results are shown in Fig. 4. All the curves are sigmoidal with affinity (K, , half-maximal concentration) and efficacy (I,,,), maximal response) characteristic of a given agonist. Values for affinity and efficacy of these agents are given in Table II. Values cited in this Table were derived from separate plots on expanded scales. For the sake of comparison K, values for these agonists for depolarization of intact electroplax are also given in Table II. The log-log plots of various doseresponse relationships show linear regions, the slopes (n) of which are also collected in Table II. The data for carbamylcholine, phenyltrimethylammonium chloride, and tetraethylammonium chloride do not show a large scatter, and the molecularity (nl of the response was found to be four for these agonists. A slope (n) of 1.5-2.5 was observed for decamethonium (Fig. 5). Of all
ACh (/.A41
FIG 3. Plot for the increase in transmembrane current (I,) as a function of ACh concentration in the trams compartment. Protein/AChE concentration was approximately 1 unit/ml. The line is drawn with a slope of 4.
the agonists studied, decamethonium showed the maximum scatter in experimental data. Decamethonium also showed membrane lytic activity at concentrations
A RECONSTITUTED
ACETYLCHOLINE
25
RECEPTOR
30
P 0 X : E h E 9
4o
30
7.0
IO
0
IO
00 Activator
1000
($4)
relationships for various agonists. DECA, decamethonium, ACh, FIG. 4. Dose-response acetylcholine, CBC, carbamylcholine, PTA, phenyltrimethylammonium, TEA, tetraethylammonium. Each point is the mean of Z-lO.measurements on different membranes. The range of scatter in data is not shown here, but the scatter was maximal for decamethonium and least for carbamylcholine. Both ACh and carbamylcholine were added only to the tmns side, and protein/AChE (approximately 1 unit/ml) to the cis side only. Decamethonium, phenyltrimethylammonium chloride, and tetraethylammonium chloride were added to both compartments along with 3 units of protein/AChE per ml. TABLE VARIOUS
Agonist
CONSTANTS
CHARACTERIZING
K,, 33” BLM (wM)
Acetycholine Carbamylcholine Decamethonium Phenyltrimethylammonium Tetraethylammonium Nicotine
24 35 4.0 16 115 650
II
DOSE-RESPONSE
Km, 22”
RELATIONSHIPS
ZmaxC
Electrp. (PM)*
FOR SEVERAL AGONISTS’
Zi”l
Molecularity
20 30 1.2 13
50 57 55 13
30 45 30 10
0.6 0.77 0.54 O.-6
-
40 -
24 -
0.6 0.8
4.1 3.95 1.5-2.5 3.5-4.0 4.0 -
OData were derived from plots similar to those shown in Figs. 1 and 2 but on expanded concentration axis. b From References 3 and 23. ‘Protein/AChE concentration for ACh and carbamylcholine was 1 unit (4 rg)/ml on only one side of the membrane; corresponding concentration for decamethonium, phenyltrimethylammonium, and tetraethylammonium chloride was 3 units/ml on both sides of the membrane.
above 10 PM. The data taken as a whole tential which developed across the BLM suggest that several known agonists of the due to ionic gradients was measured under acetylcholine receptor increase the con- the conditions listed in Table III. The magnitude and sign of the diffusion potenductance of AChIUAChE-modified BLM. tial shows that BLM alone do not show any Ion selectivity of the conduction pathdiscrimination among Na+, K+, or Cl-. ways induced by AChRIAChE incorporation into BLM. BLM were prepared with However, BLM modified with AChR/ appropriate ionic gradients (Table III) AChE show ionic selectivity in the order across the membranes. The membrane po- Na+ N K+ < Cl-. The same selectivity is
26
MAHENDRA
KUMAR
JAIN
FIG. 5. Dose-response relationship on a log-log scale for the increase in transmemhrane current as a function of decamethonium (DECA) concentration. Both protein/AChE and decamethonium were present in both compartments; however, decamethonium was added about 15 min after the addition of protein/AChE. Each data point is the mean of 5-15 independent measurements. TABLE
III
OPEN CIRCUIT POTENTIAL AS A FUNCTION OF IONIC GRADIENT ACROSSBLM MODIFIED WITH AChR/AChE PRESENCE OF CARBAMYLCHOLINE Trans compartment 1 M NaCl 1 M NaCl 1 MNaCl
+ 50 mM CBC
1 MKCl+50mMCBC 1 MKC~ 1 MKC~ 1~KClf 50
mM
Cis compartment
Potential (sign for cis side)
0.1 M NaCl 0.1 MNaCl + AChlUAChE 0.1 M NaCl + AChR/AChE 0.1 M NaCl + AChRJAChE 1 M NaCl 1 MNaCl+ AChlUAChE lMNaCl+ AChWAChE
<+2 mV +5 mV
PNa 7 PO
+21 mV
Pi%7 PC,
+15
P Na =pK 7 PO
mV
IN THE
Relative permabilities”
<+l mv -3 mV -4 mV
CBC
n CBC, carbamylcholine. b Order of permeability approximately: Na 2 K > Cl. BLM permeability does not change significantly presence of carbamylcholine alone. AChR/AChE when present is 15-&ml.
observed in the presence of carbamylcholine. Furthermore, the data show that the ratio of permeabilities for K+, Na+, and Cl- ions is approximately 3: 3: 1. Resolution of receptor actiuity and choline&erase activities from the AChRIAChE preparation by affinity chromatography. An affinity column containing p-trimethyl-
in the
phenylammonium chloride ligand was prepared by the method of Berman and Young (26). Protein absorbed on the column (7 ml gel volume) was eluted with a NaCl gradient (linear, 0.5-2.0 M). The elution profile for AChE activity is shown in Fig. 6. The AChR activity on the BLM was measured in pooled fractions (of 35-ml size) after
A RECONSTITUTED
ACETYLCHOLINE
Volume
RECEPTOR
27
(ml)
FIG. 6. The elution profile for acetylcholinesterase from affinity column (7-ml column volume) containing p-trimethylammoniumphenyl bound to Sepharose by a spacer arm (cf. 26).
lyophilization. Most of the AChR activity (less than 10% of the starting activity) was found in the eluant (135-170 ml) as indicated in Fig. 6. Although this fraction showed a response that was qualitatively similar to that observed for the crude preparation, reproducibility of separation was poor. Affinity chromatography with other ligands and eluant systems is being tried. However, the results do strongly suggest that neither acetylcholinesterase alone, nor in combination with other molecular species is responsible for the receptor activity. DISCUSSION
In the preceding section I have described some of the fundamental features of interaction of acetylcholine and related agonists in BLM modified with an impure commercially available preparation of acetylcholinesterase from eel electroplax. In general, changes in membrane permeability produced by various agonists are concentration dependent. A sigmoidal doseresponse curve with a characteristic affinity for the agonist is exhibited. The efficacy of the agonist depends not only upon the nature of the agonist but also upon the concentration of AChRlAChE on the opposite side of the membrane. All these characteristics would be expected from a reconstituted acetylcholine receptor (cf. 1-7). The affinity and efficacy data for six different agonists are given in Table II. The values of half-maximal activating concen-
trations for the agonists on the AChR/ AChE-modified BLM are listed in column 2. Corresponding values for isolated electroplax obtained by measurement of depolarization of resting potential as reported in the literature (3, 23) are given in column 3. A comparison of the values for K, in these two columns suggest a good correspondence in absolute values of K,. Thus, for example, the values of apparent affinity constants for carbamylcholine are 35 and 30 pM and for PTA are 16 and 13 pM in the modified BLM and isolated electroplax, respectively. It may be noted that the value of K, for acetylcholine in the isolated electroplax system (23) obtained by Voltage Clamp IIX!aSUreInentS, 20 FM, is also in good agreement with the value for K, , 24 pM, obtained on the reconstituted BLM. However, the corresponding values for DECA are 4.0 and 1.2 pM, respectively. The significance of this difference is not known (see below). In column 4 of Table II are given the maximal values of current that could be drawn through the BLM modified by a given concentration of AChR/AChE. The values for both acetylcholine and carbamylcholine are for BLM treated with 6 pglml of AChIUAChE whereas the corresponding values were obtained with BLM treated with 18 pg/ml of the same AChR/ AChE. Thus, qualitatively the agonists for depolarization of AChR/AChE-treated BLM have their efficacies in the following order: carbamylcholine (115) 2 acetylcho-
28
MAHENDRA
line (100) > decamethonium (27) 2 tetraethylammonium chloride > phenyltrimethylammonium chloride. It is pertinent to note that Changeux et al. (3) have observed the following order of efficacies for agonists in the isolated electroplax: carbamylcholine > decamethonium > phenyltrimethylammonium chloride. The value of efficacy given for each agonist is only approximate and is based on the observation that, in this particular range of protein concentration, a 3-fold increase in AChR/AChE concentration leads to approximately a loo-fold increase in I,,, . It is based on a fourth-order dependence of I,,, on AChR/AChE concentration. As noted in column 5 of Table II, the value of current at high agonist concentration (linr) is about 70% of I,,, . Such a “desensitization” has been observed in electrophysiological experiments with muscles and electroplax (7, 24, 25). In column 6 of Table II are given the values of molecularity (n) obtained from the slopes of the plots of log 1, vs log (agonist concentration). Values of n for acetylcholine, carbamylcholine, phenyltrimethylammonium chloride, and tetraethylammonium chloride are approximately 4. In contrast, the value of n for decamethonium is 1.5-2.5. (As noted earlier, there is relatively large scatter in this data.) The significance of this difference may be found in the study of the effect of polymethonium ions on intact chemically excitable membranes (6). This would suggest that, like the natural receptor, the reconstituted system binds to two molecules of decamethonium, instead of four molecules of other monoammonium agonists leading to opening of ionconduction pathways. The values of Hill coefficient -2 have been reported by Changeux et al. (3) for the depolarization of isolated electroplax by carbamylcholine, decamethonium, and phenyltrimethylammonium chloride. However, these authors have plotted log [E - J’-LJIGL,,~ - E,,)] vs log of agonist concentration (Hill plot). E, is the resting membrane potential, Em,, is the maximum possible depolarization, and E is the mem-
KUMAR JAIN
brane potential at any given agonist concentration. Such a plot neither yields a true Hill coefficient nor the molecularity of the overall process. In order to obtain a true value of n from a log response vs log dose curve, the response should be on a truly linear scale. However, membrane potential in the resting state, as well as in the depolarized state, is a complex function of relative ionic permeabilities (27, 28). It is governed not only by the gross membrane conductance and concentrations of ions in the media surrounding the membrane, but also on the relative permeabilities of these ions for the resting and depolarized membranes. It is obvious from the data of Changeux et al. (2, 3) that both the relative and absolute permeabilities of the electroplax membrane change drastically during an agonist-induced depolarization of the cholinergic membrane. Most of the parameters characterizing and relating these changes to membrane potential are not known. This makes a close comparison of our data with the available data on the intact electroplax rather difficult. It is, however, pertinent to note that the slope at the inflexion point is plots of E - E, vs log of agonist concentration (cf. Fig. 3 in Ref. 3) do differ for decamethonium and carbamylcholine. In fact, the slope for carbamylcholine is almost twice as large as that for the decamethonium curve. This is in qualitative accord with the observations presented here as well as with the hypothesis that one molecule of decamethonium has (or can have) depolarizing ability equivalent to that of two carbamylcholine molecules. Furthermore, this relationship is also consistent with the suggestion that the receptor sites for quaternary ammonium groups may be separated by about 14A, the distance between two ammonium groups in decamethonium (6). The data presented in Table III show that the AChlUAChE reconstituted membrane does not show significantdiscrimination between Na+ and K+ ions. However, these cations are about 3-fold more permeable than Cl- ions. These results are in good quantitative accord with data on eel electroplax (29).
A RECONSTITUTED
ACETYLCHOLINE
All these observations show qualitative and quantitative similarities in the behavior of isolated electroplax membrane and the BLM modified with an impure preparation of acetylcholinesterase from eel electroplax. It is now certain that some impurity in the preparation is responsible for the observed effects (20). Purified AChE does not show any receptor-like response on BLM. In contrast, AChR/AChE resolved by electrophoresis on polyacrylamide gel yields bands which are distinctly separate from AChE and show the characteristic AChR response (20, unpublished observations). Furthermore, the data presented herein and elsewhere (18, 20, and Jain, unpublished observations) suggest that the receptor reconstituted from impure AChR/AChE shows both the muscarinic and nicotinic response. Whether this reflects the presence of distinct receptor species or is the consequence of the same receptor species in two different “states,” remains to be resolved. Attempts at biochemical, topographical, and pharmacological characterization of the reconstituted receptor are in progress.
RECEPTOR
29
(1966) Pharm. Reu. 18, 1051. 7. KATZ, B., AND THESLEFF, S. (1957) J. Physiol. 138, 63. 8. KLEDD,R. B., FULPUIS, B. W., COOPER,D., SMITH, M., REICH, E., ANDPOSSANI, L. D. (1973) J. Biol. Chem. 248,684l. 9. KARLSSON, E., HEILBRONN, E., AND WILDLUND, L. (1972) Fed. Eur. Biochem. Sot. Lett. 28, 107. 10. CLARK, D. G., Wo~corr, R. G., AND RAFTERY, M. A. (1972) Biochem. Biophys. Res. Commun. 48, 1061. 11. SCHMIDT, J. S., AND RA~ERY, M. A. (1972) Biothem. Biophys. Res. Commun. 49, 572; (1973) Biochemistry 12,852. 12. ELDEFRAWI, M., E., ELDEFRAWI, A. T., AND O’BRIEN, R. T. (1971) Mol. Pharmacol. 7, 104. 13. MEUNIER, J. C., AND CHANGEUX, J. P., Fed. Eur. Biochem. Sot. Lett. 32, 143. 14. MILEDI, R., MOLINOFF, P., AND POWER, L. T. (1971) Nature (London) 229, 554. 15. SUGIYAMA, H., BENDA, P., MEUNIER, J. C., AND CHANGEUX, J. P. (1973) Fed. Eur. Biochem. Sot. Lett. 35, 125. 16. CHIU, T. H., DOLBY, J. O., AND BARNARD, E. A. (1973) Biochem. Biophys. Res. Commun. C 51, 205. 17. ALBUQUERQUE,E. X., BARNARD, E. A., CHIU, T. H., LAPA, A. J., DOLLY, J. 0.. JANSSON, S., DALY, J., AND WITKOP, B. (1973) Proc. Nat. Acad. Sci. USA 70,949. 18. JAIN, M. K., MEHL, L. E., AND CORDES, E. H. ACKNOWLEDGMENTS (1973) Biochem. Biophys. Res. Commun. 51, I express my appreciation to Professor E. H. Cordes 192. for his encouragement and advice, in whose labora19. KEMP, G., DOLBY, J. O., BARNARD, E. A., AND tory part of this work was done at Indiana University, WENNER, C. E. (1973) Biochem. Biophys. Res. Bloomington, Indiana. I also thank Dr. M. Goodall for Commun. 54, 607. providing his unpublished manuscripts mentioned in 20. GOODALL, M. C., BRADLEY, R. J., SACCOMANI, G., this paper, and to Dr. H. White for comments on the AND ROMINE, W. 0. (1974) Nature (in press); manuscript. also unpublished data. REFERENCES 21. JAIN, M. K. (1972) The Bimolecular Lipid Mem1. EDELMAN, J. S. (1961) Annu. Reu. Phy~iol. 23,37. brane: A System, Van Nostrand-Reinhold, New York. 2. PODLESKI, T. R., AND CHANGEUX, J. P. (1970) in Fundamental Concepts in Drug Receptor In- 22. JAIN, M. K., WILLIAM, E., WHITE, F. P., STRICKHOLM, A., AND CORDES, E. H. (1972) J. Memteractions (Danelli, J. F., Moran, J. F., and brane Biol. 8, 363. Triggle, D. J., eds.), p. 93, Academic Press, New York. 23. RUIZ-MANRESA, F., Personal Communication 3. CHANGEUX, J. P., KASAI, M., HUCHET, M., AND (1973). 24. BENNEW, M. V. L., WURZEL, M., AND GRUNDFEST, MEUNIER, J. C. (1971) in Cholinergic Ligand H. (1961) J. Gen. Physiol. 44, 757. Interactions (Triggle, D. J., Moran, J. F., and Barnard, E. A., eds.), p. 33, Academic Press, 25. GOLDSTEIN, A., ARONOW, L., AND KALMAN, S. (1968) Principles of Drug Action, Harper and New York. Row, New York. 4. LONG, J. P: (1963) in Handbuch der Experimen26. BERMAN, J. B. (1973) Biochemistry 12, 1710. tellen Pharmakologie: Cholinesterases and AnPhenomena, ticholinesterase Agents (Eichler, O., and Fa- 27. PLONSEY, R. (1969) Bioelectric McGraw-Hill, New York. rah, A., eds.), Koelle, G. B., Sub. ed., Vol. 15, 28. HODGKIN, A. L., AND KATZ, B. (1949) J. Physiol. p. 398, Springer-Verlag, Berlin. 108, 37. 5. BARLOW, R. B. (1964) Introduction to Chemical 29. RUIZ-MANRESA, F., AND GRUNDFEST, H. (1971) J. Pharmacology 2nd. Ed., Methuen, London. Gen. Physiol. 57, 71. 6. KHROMOV-BORISOV, N. Y., AND MICHELSON, M. J.