Chi Shing Sum et al- Effects of N-Ethylmaleimide on Conformational Equilibria in Purified Cardiac Muscarinic Receptors

8/3/2019 Chi Shing Sum et al- Effects of N-Ethylmaleimide on Conformational Equilibria in Purified Cardiac Muscarinic Recept

1/16

Effects ofN-Ethylmaleimide on Conformational Equilibria inPurified Cardiac Muscarinic Receptors*

Received for publication, February 20, 2002, and in revised form, July 5, 2002Published, JBC Papers in Press, July 15, 2002, DOI 10.1074/jbc.M201731200

Chi Shing Sum, Paul S.-H. Park, and James W. Wells

From the Department of Pharmacology and Faculty of Pharmacy, University of Toronto,Toronto, Ontario M5S 2S2, Canada

Muscarinic receptors purified from porcine atria anddevoid of G protein underwent a 927-fold decrease intheir apparent affinity for the antagonists quinuclidinylbenzilate, N-methylscopolamine, and scopolamine whentreated with the thiol-selective reagent N-ethylmaleim-ide. Their apparent affinity for the agonists carbacholand oxotremorine-M was unchanged. Conversely, therate of alkylation by N-ethylmaleimide, as monitored bythe binding of [3H]quinuclidinyl benzilate, was de-creased by antagonists while agonists were without ef-

fect. The receptor also underwent a time-dependent in-activation that was hastened by N-ethylmaleimide butslowed by quinuclidinyl benzilate and N-methylscopol-amine. The destabilizing effect ofN-ethylmaleimide wascounteracted fully or nearly so at saturating concentra-tions of each antagonist and the agonist carbachol. Sim-ilar effects occurred with human M

2receptors differen-

tially tagged with the c-Myc and FLAG epitopes,coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was un-changed by N-ethylmaleimide, which therefore waswithout discernible effect on oligomeric size. The dataare quantitatively consistent with a model in which thepurified receptor from porcine atria interconverts spon-taneously between two states (i.e. R R*). Antagonists

favor the R state; agonists and N-ethylmaleimide favorthe comparatively unstable R* state, which predomi-nates after purification. Occupancy by a ligand stabi-lizes both states, and antagonists impede alkylation byfavoring R over R*. Similarities with constitutively ac-tive receptors suggest that R and R* are akin to theinactive and active states, respectively. Purified M

2re-

ceptors therefore appear to exist predominantly in theiractive state.

Sulfhydryl-specific reagents have been useful probes of the

relationship between structure and function in muscarinic and

other G protein-coupled receptors (e.g. Refs. 15). In recent

studies, such reagents have been tagged with environmentally

sensitive fluorescent probes or spin labels and used to detect aconformational change linked to activation (13). That change

is thought to involve the rotation or tilting of the sixth trans-

membrane helix (1, 35). The hydrophilic nature of some re-

agents has been exploited to suggest that the conformational

change leads to increased accessibility of the reactive residue

(e.g. Refs. 4 and 5).

N-Ethylmaleimide is among the most widely used sulfhydryl-

specific reagents. In the case of muscarinic receptors, as with

many others, it has been shown to affect receptor-G protein

coupling and to have a differential effect on the binding prop-

erties of agonists and antagonists. With receptors in native

membranes, the Hill coefficients for the binding of muscarinic

agonists are increased from characteristically low values to

values near or indistinguishable from 1; moreover, the shift inpotency brought about by guanyl nucleotides is reduced or

abolished (e.g. Refs. 69). The affinity of muscarinic antago-

nists generally is not affected by N-ethylmaleimide (712),

whereas the affinity of agonists can be either increased (8,

1014) or decreased (6, 9).

It has been suggested that N-ethylmaleimide reacts prefer-

entially with one of two spontaneously interconverting states of

the receptor, thereby driving the equilibrium toward the reac-

tive conformation (1012). Further support for the existence of

multiple interconverting states, only one or comparatively few

of which are functionally active, derives from the occurrence of

constitutive activity as seen, for example, in mutants of the 2adrenergic receptor (15) or the M5 muscarinic receptor (16). In

the latter case, it was demonstrated that the ligand-regulatedactivity of 13 constitutively active mutants, differing only at

position 465 in helix number 6, can be accounted for by shifts in

a single equilibrium between an inactive and an active state

(16). Also, the notion of spontaneously interconverting states is

consistent with the constitutive activity and related properties

of M1M4 muscarinic receptors overexpressed in Chinese ham-

ster ovary cells (17), and of M5 muscarinic receptors coex-

pressed with comparatively large amounts of Gq (18).

In the present study, N-ethylmaleimide has been used to

probe for multiple states of cardiac muscarinic receptors puri-

fied from porcine atria and devoid of G protein. The effects of

the reagent on the binding properties of the receptor, and the

countervailing effects of muscarinic ligands on the action of

N-ethylmaleimide, have been studied with the system under

both thermodynamic and kinetic control. The data are quantita-

tively consistent with a model in which the receptor interconverts

spontaneously between two states that are intrinsic to the recep-

tor alone. N-Ethylmaleimide appears to favor the state that is of

weaker affinity for antagonists and also undergoes a compara-

tively rapid inactivation, a pattern that is shared by constitu-

tively active receptors (e.g. Refs. 4, 19, and 20).

MATERIALS AND METHODS

Ligands, Detergents, Antisera, and Other MaterialsN-[3H]Methyl-scopolamine was obtained as the chloride salt from PerkinElmer LifeSciences (lot 3406009, 83.5 Ci/mmol) and as the bromide salt from

Amersham Biosciences (batch 27, 78.3 Ci/mmol). ()-[3H]Quinuclidinylbenzilate was obtained from PerkinElmer Life Sciences (lot 3329907,

* This work was supported by Heart and Stroke Foundation of On-tario Grants T3769 and T4506 and Canadian Institutes of HealthResearch Grants MT14171 and MOP43990. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Faculty of Phar-

macy, University of Toronto, 19 Russell St., Toronto, Ontario M5S 2S2,Canada. Tel.: 416-978-3068; Fax: 416-978-8511; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 39, Issue of September 27, pp. 3618836203, 2002 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org36188


2/16

42.0 Ci/mmol; lot 3363333, 42.0 Ci/mmol) and from Amersham Bio-sciences (batch 44, 48 Ci/mmol). Both radioligands were supplied as asolution in ethanol. Both were devoid of synthetic precursors, as indi-cated by mass spectra provided by the manufacturers and, in the caseof N-[3H]methylscopolamine, obtained at the Molecular Medicine Re-search Centre of the University of Toronto. The absence of scopolaminefrom lot 3406009 of N-[3H]methylscopolamine was confirmed by thinlayer chromatography carried out by the manufacturer.

N-Ethylmaleimide was obtained from Sigma (ultra grade). Digitoninwas obtained from Wako Bioproducts at a purity near 100% and at high

purity from Roche Molecular Diagnostics and Calbiochem. The productfrom Wako generally was used to solubilize the receptor, and that fromother sources was used to prepare and wash the columns used in

various procedures. Sodium cholate was purchased from Sigma at apurity of 99% or better. Murine antibodies to the c-Myc epitope (9E10)conjugated to agarose were purchased from Santa Cruz Biotechnology,Inc. Murine antibodies to the FLAG epitope conjugated to horseradishperoxidase were from Sigma. Other materials were obtained from thesources identified previously (21).

Muscarinic ReceptorsPurified M2 receptor solubilized in digitonin/cholate (0.1% digitonin, 0.02% sodium cholate) was prepared from por-cine atria essentially as described previously (22). Receptor was ex-tracted from the sarcolemmal fraction of the sucrose gradient accordingto the two-step procedure of Peterson and Schimerlik (23), except thatthe first step of the extraction was carried out with 0.36% digitonin and0.07% sodium cholate. Subsequent passage through DEAE-Sepharose(Amersham Biosciences), 3-(2-aminobenzhydryloxy)tropane-Sepha-

rose, and hydroxyapatite (CHT-II, Bio-Rad) was carried out as de-scribed previously (21, 22). The buffer used to elute the purified recep-tor from hydroxyapatite was exchanged for buffer D (20 m M KH2PO4, 20mM NaCl, 1 mM Na2EDTA, 0.1 mM PMSF, 0.1% digitonin, and 0.02%sodium cholate, adjusted to pH 7.40 with KOH) (21) to obtain a stocksolution that was divided into aliquots sufficient for one experiment andstored at 75 C.

The specific activity of the purified preparation was 5.4 mol/g ofprotein, as estimated from the maximal binding of [3H]quinuclidinylbenzilate (QNB).1 The corresponding purity was 28%, based on themolecular weight of 51,673 calculated from the primary sequence (24,25). The purified receptor migrated with the mobility of monomers,dimers, and larger oligomers during electrophoresis on polyacrylamidegels. Staining with silver nitrate and subsequent densitometry indi-

cated that those bands represented 29% of all proteins visible on the gelin each of two such experiments. The identity of the bands was con-firmed by means of controls that were blotted and then screened with

receptor-specific antibodies. The purified receptor was devoid of the subunits of Go, G i1, G i2, Gq/11, and Gs, as shown previously by immuno-blotting with G protein-specific antibodies (22) and confirmed in thepresent investigation. Immunoblots with a nonspecific anti-caveolinantibody (BD Biosciences) demonstrated that the purified receptor alsowas devoid of caveolins 1, 2, and 3.2

Human M2 muscarinic receptors tagged with the c-Myc or FLAGepitope were co-expressed in Sf9 cells and extracted in digitonin/cholate

(26). The final composition of the buffer was 15.7 m M KH2PO4, 15.7 mMNaCl, 0.78 mM Na2EDTA, 0.078 mM PMSF, 0.86% digitonin, and 0.17%sodium cholate, adjusted to pH 7.60 with KOH. The extract was storedat 75 C and used without further purification in subsequent assays.Complementary DNA coding for the wild-type and c-Myc-tagged humanM2 muscarinic receptor and cloned into a baculoviral vector was ob-tained from Biosignal, Inc. (Montreal). The construction of the baculo-

virus coding for FLAG-tagged human M2 receptor has been describedelsewhere (26).

Reaction with N-EthylmaleimideTo prepare samples for use in thebinding assays, an aliquot of purified receptor from porcine atria or the

solubilized extract from Sf9 cells (28 65 l) was thawed and diluted toabout 450 l in buffer A (20 mM HEPES, 20 mM NaCl, 5 mM MgSO4, 1mM Na2EDTA, 0.1 mM PMSF, 0.1% digitonin, and 0.02% sodiumcholate, adjusted to pH 7.40 with NaOH) supplemented with N-ethyl-maleimide. A fresh solution ofN-ethylmaleimide was prepared for eachreaction. Controls were prepared in the same manner except that N-ethylmaleimide was omitted. The final concentration of N-ethylmale-imide inthe reaction mixture was 10 mM except where stated otherwise;the concentration of receptor was 6.510 nM, as estimated from thebinding of [3H]quinuclidinyl benzilate to controls from which N-ethyl-

maleimide was omitted. The reaction mixture was kept in an ice bathfor 24 h, unless stated otherwise, and aliquots then were introduceddirectly into the binding assays. N-Ethylmaleimide was without effecton the binding of [3H]quinuclidinyl benzilate and therefore was notremoved in most experiments. All volumes were recorded, and theconcentration of receptor was corrected as required in subsequent

calculations.When the reaction mixture was to contain N-ethylmaleimide to-

gether with either carbachol or N-methylscopolamine, the ligand waspreincubated with the undiluted thawed receptor in an ice bath for 30

min.N-Ethylmaleimide then was added, dissolved in deionized water at10 times the final concentration of 10 mM. The total volume of thereaction mixture was 20 200 l. Controls were prepared in the samemanner except for the omission of N-ethylmaleimide or the muscarinicligand, as required. After the desired period of incubation in the icebath, the volume of the reaction mixture was adjusted to 200 l withbuffer A, and the sample was desalted on a column of Sephadex G-50

(fine) (0.8 5.0 cm) previously equilibrated with buffer A. The eluatefrom the Sephadex column was used directly in the binding assays. Totest for any effect ofN-ethylmaleimide on the binding of [3H]quinuclidi-nyl benzilate, samples prepared with the reagent were kept in an icebath for 24 h, diluted with buffer A, and then desalted on SephadexG-50 as described above.

ReconstitutionNative and alkylated receptors were reconstitutedwith G proteins according to a procedure adapted3 from that describedby Haga et al. (27). The G proteins were obtained as a mixture ofisoforms purified from bovine brain (Calbiochem). Following incubation

of the purified receptor for 24 h at 0 C with or without N-ethylmale-imide, as described above, the native or alkylated product (3.6 7.3 pmolin 36 80 l) was incubated with carbachol (10 mM) for about 15 min,also at 0 C. The receptor then was mixed (1:1) with a suspension oflipids (0.6 mg/ml L--phosphatidylcholine, 0.6 mg/ml -L-phosphatidyl-L-serine, and 60 g/ml cholesterol; Sigma) prepared in buffer B (20 mMHEPES, 160 mM NaCl, 6 mM MgCl2, 0.8 mM Na2EDTA, and 1 mMdithiothreitol, adjusted to pH 8.0 with KOH) supplemented with 0.18%sodium deoxycholate and 0.04% sodium cholate. The G proteins wereadded to the mixture (32 40 pmol of Go, 8 16 pmol Gi-1, 8 16 pmolof Gi-2, 8 pmol of Gi-3, and 64 pmol of G), which then was appliedto a column of Sephadex G-50 (0.8 5 cm) pre-equilibrated with buffer

B. The column was eluted with buffer B, and the reconstituted receptorsemerged in the void volume (450 l) at a concentration of 1.6 4.9 nM.The vesicles were uniform in size with a mean diameter of 30 11 nm(n 6), as determined previously by electron microscopy.3 Bindingassays were performed on aliquots taken directly from the eluate of the

Sephadex column. Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western

Blotting, and ImmunodetectionThe co-immunoprecipitation of c-Myc-and FLAG-tagged M2 receptors extracted from Sf9 membranes wascarried out and monitored as described previously (26). Polyacrylamidegels that were to be silver-stained were fixed for 30 min in a solution of

50% methanol and 10% acetic acid. The solution was rinsed off, and thegel was warmed in a microwave oven (300 watt) for 1.5 min. The gel wasincubated further with dithiothreitol (5 g/ml) for 5 min, rinsed, andstained for 30 min with a solution of silver nitrate (0.1%) (British DrugHouses). Bands were developed with 1.8% formaldehyde prepared in3% sodium carbonate solution. As soon as the bands appeared, thedevelopment was terminated by incubating the gel with sodium citrate(2.3 M) for 10 min. Densitometric scanning was performed at a resolu-tion of 300 dots per inch, and the intensity was determined using 1DImage Analysis software (Kodak Digital Science). Further details havebeen described elsewhere (26).

Binding AssaysBinding was measured essentially as describedpreviously (21). Solutions of the radioligand and any unlabeled ligandswere prepared in buffer A or buffer C (25 mM KH2PO4, 4 mM HEPES,230 mM NaCl, 10 mM MgCl2, 0.8 mM Na2EDTA, and 0.1 mM PMSF,adjusted to pH 7.60 with KOH) for assays with solubilized or reconsti-tuted receptor, respectively. An aliquot (5 l) then was added to thereceptor (48 l) in polypropylene microcentrifuge tubes pretreated withtrimethylchlorosilane. The reaction mixture was incubated at 30 C forthe required period of time, and the bound radioligand was separatedon columns of Sephadex G-50 (fine) (0.8 6.5 cm). In assays with[3H]quinuclidinyl benzilate, the time of incubation was 2 h unless

indicated otherwise. Data obtained at graded concentrations of theradioligand (e.g. Fig. 1) were superimposable after incubation of thereaction mixture for either 2 or 6 h. Binding therefore was independent1 The abbreviations used are: QNB, quinuclidinyl benzilate; GMP-

P(NH)P, 5-guanyly-5-yl imidodiphosphate; NEM, N-ethylmaleimide;NMS, N-methylscopolamine; PMSF, phenylmethylsulfonyl fluoride.

2

A. Ma, A. B. Pawagi, and J. W. Wells, unpublished observations.3

G. Kaur and J. W. Wells, unpublished observations.

N-Ethylmaleimide and Purified Muscarinic Receptors 36189


3/16

of time under the conditions of the experiments. In assays withN-[3H]methylscopolamine, the time of incubation was as stated in thetables or figures. To follow the time dependence of binding, an aliquot ofthe radioligand (552 673 l) in buffer A was added to the receptor(58 78 l) in polypropylene microcentrifuge tubes pretreated with tri-methylchlorosilane. The reaction mixture was incubated at 30 C, and

aliquots (50 l) were removed at the desired times and applied toSephadex G-50 as described above. Assays were performed in duplicateor triplicate, and the receptor was kept in an ice bath until mixed withthe ligand and incubated at 30 C.

Nonspecific binding was taken throughout as total binding in thepresence of 1 mM unlabeled N-methylscopolamine. The value increasedlinearly with the concentration of unbound radioligand. Expressed asthe fraction NS, as defined in Equations 1 and 4 below, the meanestimate of nonspecific binding from several representative experi-ments was 0.000080 0.000009 for [3H]quinuclidinyl benzilate (n 19)and 0.000018 0.000002 for N-[3H]methylscopolamine (n 16).

In assays with N-[3H]methylscopolamine, ethanol that accompaniedthe radioligand accelerated the inactivation of receptors pretreatedwith N-ethylmaleimide. Most or all of the ethanol therefore was evap-orated under argon before the radioligand was taken up in buffer. Insuch cases,the concentration of ethanol in the reaction mixture waslessthan 1% (v/v) at the highest concentration of N-[3H]methylscopolamineand decreased proportionately at lower concentrations. Removal of eth-anol had no effect on the binding of N-[3H]methylscopolamine to un-treated receptors or on the binding of [3H]quinuclidinyl benzilate toeither preparation. The radioligand therefore was used as supplied by

the manufacturer in those assays.Analysis of DataAll data were analyzed with total binding taken as

the dependent variable (Bobsd) and with the total concentrations of allligands taken as the independent variables. Subsequent manipulationsdid not alter the relationship between the data and the fitted curve.Except where stated otherwise, estimates of binding (Bsp, Bmax) andtotal receptor ([R]t) denote the concentration in the binding assay (pM).The concentrations of ligands denote the total molar concentration inthe binding assay.

Empirical analyses of the data were based on the Hill equation(Equations 1 or 2) and on Equation 3. The variables [P]t and [A]trepresent the total concentrations of the radiolabeled probe and an

unlabeled ligand, respectively. In Equation 1, Bsp represents specificbinding at the corresponding value of [P]t, andBmax represents maximalspecific binding; the parameter EC50 denotes the concentration of un-bound radioligand that yields half-maximal occupancy, and nH is theHill coefficient. The parameter NS represents the fraction of unbound

radioligand that appears as nonspecific binding (cf. Equation 14 in Ref.28). Equation 1 was solved numerically (28). In Equations 2 and 3, theparameters B[A]0 andB[A]3 represent the asymptotic levels of bindingwith respect to the concentration of unlabeled ligand; IC50 is the con-centration of unlabeled ligand that reduces specific binding by one-half.In Equation 3, Fj represents that fraction of specific binding defined by

the inhibitory potency IC50(j).

Bobsd Bmax([P]tBsp)

nH

EC50nH([P]tBsp)

nHNSPtBsp) (Eq. 1)

BobsdBA3 BA0BA3IC50

nH

IC50nH At

nH (Eq. 2)

Bobsd BA3 (BA0BA]3)j1

nFjIC50j

IC50j

A t(Eq. 3)

Semi-empirical and mechanistic descriptions of the data were basedon Schemes IIII, in which there are one or more classes of mutuallyindependent sites that bind either the radioligand (P) or an unlabeledligand (A) in a mutually exclusive manner. It is assumed in Schemes Iand II that the system is at thermodynamic equilibrium, whereas theformulation of Scheme III includes time as an explicit variable. In eachcase, the model was fitted to the data by means of Equation 4, in which

the variables and parameters are as described above. An appreciablefraction of the radioligand bound to the receptor under at least someconditions in most experiments. Depletion therefore was accommodatedin the calculation of B

sp.

Bobsd BspNS[P]tBsp (Eq. 4)

Scheme I comprises a mixture of distinct sites (Rj, j 1, 2, . . . , n)

wherein those of type j bind P and A with the equilibrium dissociation

constants KPj and KAj, respectively, and constitute the fraction Fj of allsites (i.e. Fj [Rj]t/[R]t, where [Rj]t [Rj] [ARj] [PRj], and [R]t j1n [Rj]t). Total specific binding of the probe was calculated according to

Equation 5, and the required values of [PRj] were obtained as describedbelow. Values of EC50 were calculated from the fitted estimates of KLj(L P or A) and Fj (n 1), as described previously (21).

Bspj1

n

PRj (Eq. 5)

In Scheme II, each receptor of type j can exist in two spontaneouslyinterconverting states designated Rj and Rj

*. The ligands P and A bindto Rj with equilibrium dissociation constants KPj and KAj (e.g. [P][Rj]/[PRj] KPj), and the relative affinity of the ligand for the two states ofthe receptor is designated Pj and Aj, respectively (i.e. Pj KPj*/KPj,

KPj* [P][Rj*]/[PRj*]; Aj KAj*/KAj

*, KAj* [A][Rj*]/[ARj*]). The pa-

rameter KRj represents the relative concentrations of Rj and Rj* atequilibrium in the absence of ligand (i.e. [Rj]/[Rj*] KRj). Sites of type

j constitute the fraction Fj of all sites, as in Scheme I (i.e. Fj [Rj]t/[R]t,

where [Rj]t [Rj] [Rj*] [ARj] [ARj*] [PRj] [PRj*]). The specificbinding of the probe was calculated according to Equation 6, and therequired values of [PRj] and [PRj*] were obtained as described below.

Bspj1

n

([PRj] [PRj*]) (Eq. 6)

In Scheme III, the receptor can exist in two spontaneously intercon-verting states (R1 and R2) that convert irreversibly to a third and fourthstate (R3 and R4, respectively). The equilibrium between R1 and R2 isanalogous to that between Rj and Rj* for receptors of type j in SchemeII (i.e. [R1]/[R2] KR, [PR1]/[PR2] KPR). Analyses in terms of SchemesII and III required two classes of noninterconverting sites to accommo-date both native and alkylated receptors. That heterogeneity is notshown explicitly in Scheme III, which depicts multiple states within asingle class of sites, but it was accommodated in the model used in the

calculations. The parameters kP(j) and kP(j) in Scheme III denote thefirst- and second-order rate constants for the binding of a radioligand(P) to receptors in state j, and KPj is the corresponding equilibriumdissociation constant (i.e. kP(j)/kP(j) KPj). Similarly, the rate ofinterconversion between R1 and R2 is determined by kR() and kR() (i.e.kR()/kR() KR), and that between PR1 and PR2 is determined by kPR()and kPR() (i.e. kPR()/kPR() KPR). The rate of conversion of Rj and PRj(j 1 o r 2 ) t o Rj2 and PRj2, respectively, is determined by kRj and kPRj.The specific binding of the probe at any time t was calculated accordingto Equation 7, and the required values of [PRj] were obtained as de-scribed below.

Bsp [PR1][PR2][PR3][PR4] (Eq. 7)

The value of Bsp in Equations 5 and 6 was calculated from the

expansions in terms of the total concentration of receptor and the freeconcentrations of both ligands (i.e. [P] and [A]). The required values of

[P] and [A] were obtained by solving the pair of implicit equations

SCHEME 1

SCHEME 2

N-Ethylmaleimide and Purified Muscarinic Receptors36190


4/16

derived from the equations of state for P and A (e.g. [P]t [P] Bsp 0). Solutions were obtained according to the n-dimensional Newton-Raphson procedure (28).

The value ofBsp in Equation 7 was calculated by numerical integra-tion of the nine differential equations that define the system, whichcomprises eight states of the receptor and the unbound radioligand.Solutions were obtained according to the Bulirsch-Stoer method asimplemented by Press et al. (29). The initial step size at each interval

was taken as 1/100th of the half-time of the fastest process in thesystem or 1/100th of the interval to the next point, whichever was less.The values of [R1], [R2], and [P] at t equals zero were taken as [R]tKR/(1 KR), [R]t/(1 KR), and [P]t, respectively; those of [R3], [R4], and all[PR

j

] were taken as zero. The values of kP(

j)

were provided by thefitting procedure, and those of kP(j) were computed as kP(j)KPj; the

values of KP3 and KP4 were selected arbitrarily, and the values of KP1and KP2 were estimated in terms of Scheme II from data acquired atequilibrium (i.e. KPj and PjKPj, respectively, in Scheme II). To ensuremicroscopic reversibility, the value ofKPR was taken as KRKP2/KP1, anda fitted value was obtained for either kPR() or kPR(). With data ac-

quired at a given value of t and graded values of [P]t (i.e. Figs. 3B and12), each point on the binding curve was treated as a separate timecourse beginning at t equals zero.

Statistical and Related Procedures All parameters were estimatedby nonlinear regression, and values at successive iterations of thefitting procedure were adjusted according to the algorithm of Mar-quardt (30). Equilibrium constants and related parameters were opti-mized throughout on a logarithmic scale (i.e. log KR, log KPj, log Lj,etc.). Rate constants were optimized on a linear scale. Most analysesinvolved multiple sets of data, and data from different experiments

were assigned separate values of [R]t and NS. Other details regardingthe assignment of shared parameters are described in the legends to thefigures and tables. The data from such analyses are presented withreference to a single fitted curve. To obtain the values plotted on the yaxis, individual estimates of Bobsd were adjusted for any differences inthe parametric values from one set of data to another (i.e. Equation 6 inRef. 31). Weighting of the data, tests for significance, and other statis-tical procedures were carried out as described previously (28, 31, 32).

RESULTS

Binding of Radiolabeled Antagonists and the Stability of

Native and Alkylated ReceptorsUpon treatment with N-eth-

ylmaleimide for 24 h at 0 C, M2 receptors purified from porcine

atria underwent a 20-fold reduction in their affinity for

[3H]quinuclidinyl benzilate and a decrease of 10 30% in the

corresponding capacity. The results from several experiments

on two preparations of receptor are summarized in Table I, andrepresentative data are illustrated in Fig. 1. There is no appar-

ent relationship between the concomitant changes in EC50 on

the one hand and Bmax on the other. The changes in affinity

appear to arise from alkylation per se, as described below,

whereas the reduction in capacity may derive from the reduced

stability of receptors treated with N-ethylmaleimide. Alkyla-

tion had similar effects on human M2 receptors in digitonin-

solubilized extracts from Sf9 cells (Table I). There was a 7-fold

reduction in affinity as the apparent capacity decreased by

almost 70%.

The affinity of the alkylated receptor for [3H]quinuclidinyl

benzilate was the same regardless of whether or not unreacted

N-ethylmaleimide was removed on Sephadex G-50 immedi-

ately prior to the binding assay (Table I). It follows that no

further alkylation occurred during the assay, assuming that

the change in affinity reflects the progress of the reaction with

the alkylating agent; also, N-ethylmaleimide appears to have

no direct effect on the binding of the radioligand. Upon removal

of the reagent, the capacity decreased on average from about 80

to 64% of that prior to alkylation. The further loss of sites may

be because of incomplete recovery of the receptor from the

desalting column. In most experiments, binding assays on the

treated receptor were performed without removing unreacted

N-ethylmaleimide. The effects of alkylation on affinity and

capacity were essentially the same when the time of incubation

at 0 C was extended from 24 h to 4 days. The reaction there-

fore was complete after 24 h, which was the interval used in

most of the experiments described below. Finally, the effects of

N-ethylmaleimide were the same when the concentration was

reduced from 10 to 1 mM over 24 h at 0 C (Table I).

The time-dependent binding of [3H]quinuclidinyl benzilate to

native and alkylated receptors from porcine atria is illustrated

in Fig. 2. Binding to the untreated receptor was faster at higher

concentrations of the radioligand, becoming virtually instanta-

neous at saturating concentrations. Binding to the alkylated

receptor was instantaneous at all concentrations of the radio-

ligand used in the assays. Under all conditions, binding re-

mained stable for up to 4 h after the maximal level wasattained.

Alkylation reduced the apparent affinity of purified receptors

for N-[3H]methylscopolamine (Table II, Fig. 3),4 consistent

with the effect on [3H]quinuclidinyl benzilate, but the magni-

tude of the change increased with the time of incubation with

the radioligand. Incubation for 15 min caused a 10-fold in-

crease in EC50, from 2.7 nM with the native receptor to 28 nM

after alkylation; incubation for 3 h caused a 30-fold increase,

from 2.7 to 85 nM. The relative capacity for N-[3H]methylsco-

polamine and [3H]quinuclidinyl benzilate was about 95% with

the native receptor and decreased appreciably, to about 77%,

after treatment with N-ethylmaleimide (Table II, Fig. 3). The

decrease was not sensitive to the time of incubation with

N-[

3

H]methylscopolamine. Also, it was due entirely to a de-crease in the absolute capacity for N-[3H]methylscopolamine,

because the binding of [3H]quinuclidinyl benzilate was stable

for up to 4 h (Fig. 2). If ethanol was not removed from N-[3H]

methylscopolamine, the relative capacity of alkylated receptors

for N-[3H]methylscopolamine and [3H]quinuclidinyl benzilate

was 0.51 0.02 after incubation for 45 min (n 5), 0.37 0.02

after 2 h (n 3), and 0.31 after 3 h (n 1).

In assays with N-[3H]methylscopolamine, the time-depend-

ent nature of the effect on EC50 suggests that the alkylated

receptor was unstable under those conditions. To confirm that

the increase in EC50 was not wholly a consequence of instabil-

ity, but derived at least in part from a decrease in affinity per

se, the binding of N-[3H]methylscopolamine was measured af-

ter equilibration for 24 h at 0 C. In three such experiments,

with native and treated receptor taken in parallel, the meanvalue of log EC50 increased upon alkylation from 9.02 0.07

to 8.14 0.42. The corresponding estimates of capacity are

551 97 and 481 100 pM for the native and alkylated

4 The data shown in Figs. 3 and 7 were analyzed in terms of SchemeI, and the parametric values are listed in Tables II and III, respectively.The data in Fig. 3B also were combined with those in Fig. 4 andanalyzed in terms of Scheme III, as described under Discussion and inthe legend to Fig. 3. Similarly, the data represented in Fig. 7 also werecombined with those in Fig. 9A and analyzed in terms of Scheme II, asdescribed under Discussion and in the legends to Figs. 7 and 9. Theuse of Scheme I is essentially empirical, because the model offers noexplanation for the interplay between N-ethylmaleimide on the onehand and muscarinic ligands on the other. In contrast, Schemes II and

III describe the system in mechanistically explicit terms.

SCHEME 3



5/16

receptor, respectively.N-Ethylmaleimide therefore reduced the

affinity for both [3H]quinuclidinyl benzilate and N-[3H]meth-

ylscopolamine by a similar amount, the relative instability of

the receptor in the presence of the latter notwithstanding.

N-[3H]Methylscopolamine bound rapidly to native and alky-

lated receptors at all concentrations of the radioligand, but the

initial burst was followed by a loss of binding upon continued

incubation (Fig. 4). The latter effect was irreversible, as de-

scribed below, and such a loss is in contrast to the stability

observed in the binding of [3H]quinuclidinyl benzilate (Fig. 2).

The decay was more rapid at lower concentrations of N-[3H]-

methylscopolamine, which therefore appears to protect the re-

ceptor from inactivation. The decay also was more rapid after

alkylation, as illustrated in Fig. 4 where the concentrations of

the radioligand were selected to achieve comparable levels of

occupancy in the two preparations.

To examine their intrinsic stability, native and alkylated

receptors were preincubated at 30 C for different periods of

time prior to the addition of a saturating concentration of

[3H]quinuclidinyl benzilate. With the alkylated receptor, ap-

proximately one-half of the initial capacity was lost within 5

min, and virtually all activity was lost within 1 h (Fig. 5). With

the native receptor, the half-time of inactivation varied from at

least 100 min to much longer; in some experiments, binding

was essentially stable for at least 3 h. When the alkylated

receptor was preincubated in the presence of 3.9 n M [3H]quinu-clidinyl benzilate, binding declined rapidly but leveled off at a

value that was consistent with the degree of labeling attained

during preincubation (Fig. 5). Bound [3H]quinuclidinyl benzi-

late therefore protected the receptor from the inactivation that

otherwise occurred in its absence, in accord with the stability

evident in Fig. 2. There was a similar stabilization of the native

receptor when [3H]quinuclidinyl benzilate was present during

preincubation.

The inactivation that occurred at 30 C was irreversible, as

indicated by the failure to regain binding when the tempera-

ture was returned to 0 C. Alkylated receptor was preincubated

for 20 min at 30 C and then transferred to an ice bath for a

further 24 h, all in the absence of ligand. At each step, binding

was measured at graded concentrations of [3H]quinuclidinylbenzilate. The capacity decreased from 477 pM initially to 42.4

pM after preincubation; 24 h later, it was essentially unchanged

at 31.3 pM. Lowering the temperature therefore prevented fur-

ther inactivation but failed to retrieve those sites already lost.

The instability caused by N-ethylmaleimide was prevented

by the agonist carbachol, which had a protective effect similar

to that of N-methylscopolamine or quinuclidinyl benzilate.

When the alkylated receptor was preincubated at 30 C for 15

min, the capacity for [3H]quinuclidinyl benzilate was reduced

to 19% of that in a control preincubated on ice (Fig. 6, cf. Fig. 5).

In contrast, the capacity was virtually identical to that of the

control (p 0.25) when 10 mM carbachol was included during

the preincubation at 30 C and then removed prior to the bind-

ing assays.

TABLE IEffect of N-ethylmaleimide on the specific binding of [3H]quinuclidinyl benzilate to M2 muscarinic receptor purified from porcine atria or

extracted from Sf9 cells

Binding was measured at graded concentrations of [3H]QNB in experiments on two batches of receptor purified from porcine atria and one batchof extract from Sf9 cells. The number of experiments is shown in parentheses. Data from individual experiments were analyzed according toEquations 1, 4, and 5 (n 2) to obtain estimates ofnH, log EC50, and Bmax (21). Individual estimates of log EC50 and nH were averaged to obtainthe mean ( S.E.) listed in the table. Individual estimates ofBmax were adjusted for dilutions prior to the binding assay to obtain an imputed valuefor the stock solution. The mean is shown relative to that for unreacted receptor from the same batch taken as 100. The absolute values ofBmax(nM) for the two batches of purified receptor are as follows ( S.E.): 1, 12.7 0.52 (6), 8.63 0.23 (4); 2, 11.1 0.46 (13), 6.57 0.14 (9); 3, 8.15 1.82 (2), 5.51 (1); 4, 9.54 (1), 5.85 (1); 5, 7.43 (1). The values for receptor from Sf9 cells are as follows: 1, 5.07 0.01 (3); 2, 1.71 0.23 (3).

Condition Treatment with NEM Log EC50 nH Bmax

(%)

Purified M2 receptor from porcine atria1 None (10) 9.36 0.05 0.90 0.08 1002 10 mM NEM, 24 h (22) 8.09 0.04 0.84 0.02 88, 763 10 mM NEM, 24 h; then removed (3) 8.15 0.04 0.77 0.03 64, 644 10 mM NEM, 4 days (2) 7.92 0.07 0.74 0.02 75, 685 1 mM NEM, 24 h (1) 8.24 0.71 86

Solubilized human M2 receptor from Sf9 cells1 None (3) 9.44 0.04 1.06 0.14 1002 10 mM NEM, 24 h (3) 8.60 0.03 0.82 0.03 34

FIG. 1. Effect ofN-ethylmaleimide on thebindingof [3H]quinu-clidinyl benzilate to M

2receptor purified from porcine atria.

Alkylated receptor (, , ) and a control from which NEM was omitted(E, , ) were prepared as described under Materials and Methods.Binding to the two preparations was measured in parallel at gradedconcentrations of the radioligand, either alone (upper curves) or to-gether with 1 mM NMS (baseline). Different symbols denote data fromthree different experiments (E, ; , ; , ) carried out on samplesfrom two batches of purified receptor. The lines represent the best fit ofEquations 4 and 5 (Scheme I) to all of the data taken together. One classof sites was sufficient throughout. With the native receptor, there wasno appreciable increase in the sum of squares with 1 rather than 3

values of KP1

(p 0.25) (log KP1

9.43 0.03). With the alkylatedreceptor, separate values of KP1 were required for data from differentexperiments (p 0.05) [log KP1 8.67 0.04 (), 8.28 0.04 (),and 8.38 0.05 ()]. The mean value of [R]t for the native receptor is519 48 pM, and the ratios of [R]t after and before alkylation are asfollows: 0.77 (), 0.72 (), and 0.91 (). Values plotted on the y axis werenormalized as described under Materials and Methods.



6/16

Binding of Unlabeled LigandsNative and alkylated recep-

tors were examined for the inhibitory effect of five unlabeled

ligands, three antagonists and two agonists, on the binding of[3H]quinuclidinyl benzilate (Fig. 7). To define all parameters in

subsequent analyses and to test for internal consistency, bind-

ing also was measured at graded concentrations of [3H]quinu-

clidinyl benzilate and N-[3H]methylscopolamine (Fig. 7, A and

D). In assays with the alkylated receptor and N-[3H]methylsco-

polamine, the time of incubation was 15 min to avoid or at least

to minimize inactivation.

The data represented in all panels of Fig. 7 were pooled and

analyzed in terms of Scheme I (n 2), and the parametric

values are listed in Table III.4 Although the fit was better with

two classes of sites rather than one, the difference is not readily

discernible in the fitted curves. Like [3H]quinuclidinyl benzi-

late and N-[3H]methylscopolamine, both scopolamine and un-

labeled N-methylscopolamine bound more weakly to alkylated

receptors than to the native preparation (i.e. log EC501.3,

Table III). A substantial reduction in affinity therefore appears

to be a common effect of N-ethylmaleimide on the binding of

antagonists. In contrast, there was little if any change in the

affinities of the agonists carbachol and oxotremorine-M (Fig. 7,

C and F; Table III).

The decreased affinity of unlabeled antagonists indicates

that the corresponding decrease in the affinity of [3H]quinu-

clidinyl benzilate andN-[3H]methylscopolamine is not a kinetic

artifact of destabilization. Because of the protection afforded by

muscarinic ligands, one of which is always present in assays at

near saturating concentrations of [3H]quinuclidinyl benzilate,

the inhibitory potency is expected to be unaffected by the in-

stability of the vacant receptor after alkylation. Furthermore,

the affinity of quinuclidinyl benzilate was the same when esti-

mated from binding at graded concentrations of [3H]quinuclidi-

nyl benzilate (Fig. 7D) and when inferred from the inhibitory

effect of the unlabeled analogue (Fig. 7E) (Table III). Although

there was some discrepancy between labeled and unlabeled

N-methylscopolamine, N-ethylmaleimide reduced the affinities

of both analogues by a similar amount. These observations

support the conclusions that emerge from Scheme I and the

parametric values listed in Table III: namely, that the effect of

N-ethylmaleimide on the binding of antagonists derives from a

change in affinity per se, whereas the affinity of agonists is

essentially unchanged.

The binding of oxotremorine-M was sensitive to GMP-P(NH)P when purified M2 receptor was reconstituted with Go/iin phospholipid vesicles (Fig. 8). Reconstitution alone led to a

more pronounced heterogeneity, and the Hill coefficient for the

native receptor decreased from 0.86 0.05 in solution (n 3,

Fig. 7C) to 0.58 0.04 in vesicles (n 2, Fig. 8). With the

alkylated receptor, nH decreased from 0.88 0.02 (n 3) in

solution to 0.59 0.02 (n 2) in vesicles. There was no effect

of Go/i on the binding of oxotremorine-M when receptors and G

proteins were mixed in solution.3 GMP-P(NH)P increased the

Hill coefficient to 0.77 0.15 and 0.68 0.08 with native and

alkylated receptors, respectively. The data required two classes

of sites when described empirically in terms of Equation 3, and

the fitted curves are illustrated in Fig. 8. The overall effect of

GMP-P(NH)P was estimated as the area between the curves

FIG. 2. Effect of N-ethylmaleimide on the time course for binding of [3H]quinuclidinyl benzilate to M2 receptor purified fromporcine atria. Receptor was incubated at 0 C for 24 h in the absence of reagent and in the presence of 10 m M NEM. The native (A) and alkylated(B) preparations then were mixed with [3H]QNB at different concentrations of the radioligand, and binding was measured at the times shown onthe abscissa. The concentration of [3H]QNB was as follows: panel A, 0.837 (E), 4.19 (), 74.2 (), and 129 nM (); panel B, 3.54 (E), 10.2 (), 102(), 115 (), and 120 nM (R). The four traces in panel A are from two experiments, and the five traces in panel B are from three experiments.Material from the same batch of purified receptor was used throughout. The lines represent the best fit of Equation 4, with the value of Bspcalculated as described elsewhere (i.e. Equation 194 in Ref. 28). The required value of K1 was taken as 9.42 (native receptor) or 8.08 (alkylatedreceptor). Values plotted on the y axis were normalized as described under Materials and Methods.

TABLE IIEffect of N-ethylmaleimide on the specific binding of

N-[3H]methylscopolamine to M2 muscarinic receptor purified fromporcine atria

The binding of [3H]NMS and [3H]QNB was measured in parallel ineach experiment. The number of experiments is shown in parentheses,and the data are illustrated in Fig. 3. The values listed in the tablepertain to [3H]NMS, which was incubated with the receptor for differ-ent times as shown. The time of incubation with [3H]QNB was 2 hthroughout. The values of nH are the mean ( S.E.) of the individual

values estimated separately for each set of data (Equation 1). Theestimates of log EC50 and relative capacity are from the best fit ofEquations 4 and 5 (Scheme I) to the data shown in the same panel takentogether (21). [3H]NMS required two classes for the native receptor and

one class after alkylation. [3

H]QNB required two classes of sitesthroughout. The values of log EC50 for [

3H]QNB are 9.41 (n 4) and8.03 (n 11) for native and alkylated receptor, respectively. The

value of [R]t for [3H]NMS was estimated relative to that for [3H]QNB in

the same experiment (i.e. fR,NMS [R]t,NMS/[R]t,QNB), and the individual values offR,NMS were averaged to obtain the mean ( S.E.).

ReceptorTime of

incubation (h)Log EC50 nH

Relativecapacity

Native 0.75 (4) 8.57 0.95 0.04 0.95 0.01 Alkylated 0.25 (3) 7.56 0.81 0.04 0.77 0.03

0.75 (2) 7.37 0.83 0.04 0.73 0.073.0 (1) 7.07 1.04 0.81



7/16

obtained in the absence and presence of the nucleotide, and the

value after alkylation was 68% of that obtained with the native

receptor.

Control of Alkylation by Muscarinic LigandsThe effect of

N-ethylmaleimide on binding suggests that muscarinic ligands

might act to regulate alkylation. Purified M2 receptors there-

fore were reacted with N-ethylmaleimide in the presence of

either N-methylscopolamine or carbachol, and the progress of

the reaction was monitored by measuring the affinity of the

receptor for [3H]quinuclidinyl benzilate. Binding assays were

carried out prior to the addition of the alkylating agent and

after incubation of the reaction mixture for different times up

to 24 h. Samples with and without the muscarinic ligand were

processed in parallel.

FIG. 3. Effect of N-ethylmaleimide on the binding of [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine to M2

receptorpurified from porcine atria. Binding to native (A) and alkylated receptor (B) was measured in parallel assays with [3H]QNB (E) and [3H]NMS(, , ). Different symbols for [3H]NMS denote different times of incubation as follows: 0.25 (), 0.75 (), and 3 h (). The data shown in eachpanel were analyzed in terms of Scheme I to obtain the parametric values listed in Table II. The data shown in panel B and in both panels of Fig.4 were combined and analyzed in terms of Scheme III, as described in the legend to Fig. 4. The lines in panel A represent the best fit of Equations4 and 5 (Scheme I). Values plotted on the y axis were normalized to the mean value of [R]t obtained for [

3H]QNB (719 29 pM, n 4). The lines

in panel B represent the best fit of Equations 4 and 7 (Scheme III). Values plotted on the y axis were normalized to the mean value of [R]t obtainedfor [3H]QNB (570 46 pM, n 11). The value of [R]t for [3H]NMS was optimized as a fraction of that for [3H]QNB in each experiment (i.e.

fR,NMS [R]t,NMS/[R]t,QNB), and there was a significant increase in the sum of squares when the ratio was fixed at 1 throughout ( p 0.00001). Themean values of fR,NMS (mean S.E.) are as follows: 0.81 0.02 (0.25 h), 0.73 0.06 (0.75 h), and 0.89 (3 h). The corresponding values for acomparable fit according to Scheme I (n 1) are as follows: 0.78 0.02 (0.25 h), 0.74 0.07 (0.75 h), and 0.87 (3 h).

FIG. 4. Effect of N-ethylmaleimide on the time course for binding of N-[3H]methylscopolamine to M2

receptor purified fromporcine atria. Binding to native (A) and alkylated (B) receptors was measured over time at different concentrations of [3H]NMS as follows: panel

A, 2.22 (E), 16.8 (), and 188 nM (); panel B, 20.0 (E), 165 (), and 777 nM (). The lines in both panels represent the best fit of Equations 4and 7 (Scheme III) to the data shown here and in Fig. 3B taken together. Single values of kP(j), kP(j), kR1, kPR1, and kPR2 were common to all dataacquired with native and alkylated receptors, which therefore differed only in the values of kR2 and the rate constants that define KR and KPR. Toensure consistency with the results obtained at equilibrium, the values of log KP1 and log KP2 were fixed at 9.87 and 7.69, respectively (i.e. log

KL and log (LKL) for [3H]NMS in terms of Scheme II (Table IV)); similarly, the value of log KRj was fixed at 1.28 and 3.28 for native and

alkylated receptors, respectively (Table V). The values of log KP3 and log KP4 were fixed arbitrarily at 3 to preclude appreciable binding at thehighest concentration of [3H]NMS. The fit is independent of the values of the rate constants for the interconversion between R 1 and R2, or betweenPR1 and PR2, provided that each step is rapid on the time scale of the binding assay. The fitted estimates of the rate constants for inactivation areas follows (min1): kR1 (native and alkylated receptor) 0.00008 0.0025, kPR1 (native and alkylated receptor) 0.00014 0.00045, kR2 (nativereceptor) 0.0024 0.0010, kR2 (alkylated receptor) 0.013 0.001, kPR2 (native and alkylated receptor) 0.00053 0.00033. The fittedestimates of other parameters are as follows ( M1 min1): kP(1) 5.0 10

8, kP(2) 5.6 107, kP(3) 2.9 10

3, and kP(4) 3.0 104. Because

each value of kP(j) was defined by only a shallow minimum in the sum of squares, no estimate of the error is shown. Values plotted on the y axiswere normalized to the mean value of [R]t for [

3H]NMS (native receptor, 678 76 pM, n 3; alkylated receptor, 327 27 pM, n 4). Values atthe lowest concentration of [3H]NMS in panel B (E) are the means of two experiments. Further details are described in the legend Fig. 3.



8/16

As illustrated in Fig. 9A, N-methylscopolamine slowed the

rightward shift induced by N-ethylmaleimide in the binding

curve for [3H]quinuclidinyl benzilate. When the data were an-

alyzed collectively in terms of Scheme I (Equation 5, n 2), the

shift can be described as a time-dependent interconversion of

sites from a state of higher affinity (log KP1 9.31 0.02) to

a state of lower affinity (log KP2 8.29 0.03). Native

receptors were wholly in the state of higher affinity. Aftertreatment for 30 min in the absence of antagonist, the low

affinity sites represented about 60% of the total binding (F2

0.62 0.02); after 30 min in the presence of N-methylscopol-

amine, they represented less than 40% of the total binding

(F2 0.36 0.03). After more prolonged treatment in the

absence and presence ofN-methylscopolamine, the low affinity

sites represented more than 80% (4 h, F2 0.84 0.02; 24 h,

F2 0.83 0.02) and about 60% (4 h, F2 0.61 0.02; 24 h,

F2 0.59 0.02) of the total binding, respectively.

The rate of the reaction with N-ethylmaleimide was unaf-

fected by carbachol. As illustrated in Fig. 9B, the binding

profiles obtained after alkylation for 30 min with and without

the agonist are virtually superimposable. In terms of Scheme I,

about 70% of the sites were in the state of lower affinity for

[3H]quinuclidinyl benzilate in either case (no carbachol, F2

0.67 0.01; with carbachol, F2 0.71 0.01).

Oligomeric Status of M2 Receptor Extracted from Sf9

CellsG protein-coupled receptors are known to occur as oli-

gomers (22, 26, 33). The relationship between oligomeric status

and binding therefore was examined in extracts from Sf9 cells

coexpressing the c-Myc- and FLAG-tagged M2 receptors. Be-

cause the FLAG epitope coimmunoprecipitated with the c-Myc

epitope, as identified on Western blots (Fig. 10, A and B), at

least some of the receptors were present as dimers or larger

oligomers in vivo. No signal is obtained if membranes from cells

infected separately with the two baculoviruses are mixed prior

to solubilization (26). The coprecipitated receptor migrated pri-marily as two immunoreactive bands with molecular masses

indicative of monomers (59.5 1.6 kDa, n 6) and dimers

(93.4 2.6 kDa, n 6), based on the calculated value of 51,673

Da for a monomer (24, 25). Some blots also contained a minor

band exhibiting a molecular mass of 165 19 kDa (n 3),

perhaps indicating a trimer.

Tagged receptors extracted from Sf9 cells were stable at

30 C, because preincubation for 20 min affected neither the

capacity for [3H]quinuclidinyl benzilate nor the degree of coim-

munoprecipitation (Fig. 10). N-Ethylmaleimide was without

effect on the degree of coimmunoprecipitation, which was un-

changed after preincubation at 30 C in the absence of ligand or

in the presence of either quinuclidinyl benzilate or N-methyl-

scopolamine (Fig. 10). The alkylated receptor was functionally

FIG. 5. Stability of M2

receptor purified from porcine atria.

Native (E, ) and alkylated (, ) receptor was obtained by pretreat-ment of the purified product with or without NEM. Aliquots were mixedwith buffer A (E, ) or with buffer A containing a small amount of[3H]QNB at final concentrations of 5.0 (, native receptor) or 3.9 nM (,alkylated receptor). Each aliquot contained sufficient material for onemeasurement, and each measurement was performed in triplicate.Upon the addition of buffer A with or without [3H]QNB, the replicateswere transferred from 0 to 30 C and maintained at that temperaturefor the length of time shown on the abscissa. The capacity of eachsample then was estimated by incubation for 2 h at a saturatingconcentration of [3H]QNB (316 nM), and the three values were aver-aged. Each time course was measured 37 times. The lines representthe best fit of the biexponential expression Bobsd fRBt0 (Bt0

fRBt0){(1 F2) exp(k1t) F2 exp(k2t)} to the data from allcurves acquired under the same conditions. For the alkylated receptor(, ) and the native receptor preincubated with [3H]QNB (), single

values of kj, F2, and fR were common to all of the data, whereas aseparate value of Bt0 was assigned to each curve. Native receptor

preincubated without [3H]QNB (E) exhibited considerable variabilityamong the seven curves, and the values at each time therefore wereaveraged prior to the analysis. Values plotted on the y axis were nor-malized to the mean value of Bt0 for the native (n 10) or alkylated(n 7) receptors, as appropriate. To determine the level of bindingachieved at 5.0 and 3.9 nM [3H]QNB, samples were incubated at 30 Cfor 2 h and assayed without additional radioligand (f, native receptor;, alkylated receptor).

FIG. 6. Stabilization of the alkylated receptor by carbachol.Purified receptor from porcine atria was reacted with NEM, and ali-

quots of the alkylated product were preincubated for 15 min as follows:on ice (E, , ), at 30 C (R, diamond with , v), and at 30 C in thepresence of 10 mM carbachol (Q, diamond with , ). All samples thenwere desalted on Sephadex G-50 (0.8 5 cm) and assayed for thebinding of [3H]QNB. Each experiment included samples processed inparallel under each set of conditions, and the data from three separateexperiments are shown (E, R, Q;, diamond with , diamond with ;, v, ). The lines represent the best fit of Equations 4 and 5 (SchemeI, n 2) to all of the data taken together. Single values of KPj and Fjwere common to all data acquired under the same conditions. Thecapacities of samples preincubated on ice were estimated in absoluteunits (i.e. [R]t). The capacities of samples preincubated at 30 C wereestimated relative to [R]t, with a single value of the ratio fR for the datafrom all three experiments. The fitted parametric values are as follows:preincubated on ice, log EC50 8.10, [R]t 510 19 pM (n 3);preincubated at 30 C without carbachol, log EC50 8.01, fR 0.19 0.02; preincubated at 30 C with carbachol, log EC50 8.11, fR 1.03 0.02. Values plotted on the y axis were normalized to the mean

value of [R]t.



9/16

unstable, however, in that about 60% of the original sites were

lost before or during the binding assay (Table I, Fig. 10 C). The

overall loss increased to 86% after preincubation for 20 min and

to 97% after preincubation for 1 h (Fig. 10C).

Alkylation by N-ethylmaleimide was similar in its effect on

the binding properties of M2 receptors from Sf9 cells and por-

cine atria. Both preparations underwent a reduction in their

affinity for [3H]quinuclidinyl benzilate (Table I) and an in-crease in the rate of inactivation at 30 C (cf. Figs. 5 and 10C),

although the latter effect was much greater with the recombi-

nant receptor. BecauseN-ethylmaleimide was without effect on

the degree of coimmunoprecipitation from Sf9 extracts, the loss

of function apparently was not accompanied by any change in

the oligomeric status of the receptor.

DISCUSSION

Effects of N-Ethylmaleimide on the M2 Muscarinic Recep-

torMuscarinic ligands and the sulfydryl-specific reagent

N-ethylmaleimide exhibit a pattern of complementary effects

at M2 receptors purified from porcine atria. Alkylation caused

a 9 27-fold reduction in the affinities of three muscarinic an-

tagonists, as estimated in terms of Scheme I, whereas the

affinities of two agonists were unchanged. Conversely, the re-

action with N-ethylmaleimide was slowed by the antagonist

N-methylscopolamine but not by the agonist carbachol. The

receptor also underwent a time-dependent, irreversible loss of

capacity that was faster after alkylation and slower in the

presence of quinuclidinyl benzilate or N-methylscopolamine.

The destabilizing effect of N-ethylmaleimide was counteracted

fully or nearly so at saturating concentrations of either antag-

onist or the agonist carbachol. The protective effect of

[3H]quinuclidinyl benzilate was greater than that of N-[3H]

methylscopolamine, as indicated by the time dependence of

binding. Because [3H]quinuclidinyl benzilate dissociates more

slowly than N-[3H]methylscopolamine, it follows that the de-

gree of stabilization appears to depend upon the residency time

of the ligand on the receptor.

The change effected by N-ethylmaleimide in the affinity of

antagonists was not a consequence of its destabilizing effect on

the receptor, notwithstanding the protection afforded by

N-[3H]methylscopolamine and [3H]quinuclidinyl benzilate. Al-

though instability can account for the progressively weaker

binding of N-[3

H]methylscopolamine to alkylated receptors

FIG. 7. Binding of antagonists and agonists to M2

receptor purified from porcine atria. Native receptor (AC) and receptor treated withNEM (DF) were prepared as described under Materials and Methods. Panels A and D, binding was measured at graded concentrations of[3H]QNB (E,,) and [3H]NMS (, , ). Different symbols denote data from different experiments (n 3). The time of incubation with [3H]NMSwas 45 (native receptor) or 15 min (alkylated receptor). Panels B and E, the binding of [3H]QNB was measured at two concentrations of theradioligand (B, 1.0 1.2 and 1113 nM; E, 8.712 and 49 94 nM) and graded concentrations of unlabeled NMS (E, ), scopolamine (, ), or QNB(, ). Panels C and F, the binding of [3H]QNB (C, 1.11.4 nM; F, 1114 nM) was measured at graded concentrations of carbachol (E) oroxotremorine-M (). The data represented in all panels were combined and analyzed in terms of Scheme I (Equations 4 and 5, n 2) to obtainthe parametric values listed in Table III. The same data also were combined with the data represented in Fig. 9A and analyzed in terms of SchemeII (Equations 4 and 6). The lines in the figure represent the best fit of Scheme II, and the parametric values are listed in Tables IV and V. Valuesplotted on the y axis were normalized to the mean value of [R]t obtained for [

3H]QNB (native receptor, 610 18 pM, n 15; alkylated receptor,572 26 pM, n 23). The capacity for [3H]NMS in panels A and D was scaled relative to that for [3H]QNB according to the ratio listed in TableII (native receptor, 0.95; alkylated receptor, 0.77). There were fewer constraints with Scheme I ( n 2) than with Scheme II, and the sum of squaresis 15% less for the data represented in Fig. 7, but the fitted curves from Scheme I are almost superimposable with those shown in the figure.



10/16

(Fig. 3B), as described below, the major change occurred prior

to the binding assays. Moreover, the decrease in affinity was

retained under conditions when the alkylated receptor was

comparatively stable. [3H]Quinuclidinyl benzilate afforded

more protection than did N-[3

H]methylscopolamine (Figs. 2

and 4), yet the change in affinity was similar or the same for

both radioligands (Table III). Also, there was a comparable

change in the affinities of three unlabeled antagonists, as in-ferred from their inhibitory effect under conditions that are

expected to preclude inactivation. In binding assays conducted

overnight at 0 C, treatment with N-ethylmaleimide was found

to reduce the affinity but not the capacity of purified receptors for

N-[3H]methylscopolamine. The independent nature of the

changes in affinity and capacity is illustrated further by a com-

parison of receptors from Sf9 cells and porcine atria. Whereas the

destabilizing effect of alkylation was markedly greater with the

Sf9 extract, the decrease in affinity for [3H]quinuclidinyl benzi-

late was similar in the two preparations.

It has been reported that N-ethylmaleimide can react with G

proteins, thereby precluding their interaction with the receptor

(34) and inhibiting GTPase activity (35). Because the purified

receptor was devoid of o, i1, i2, q/11, and s, the effects ofalkylation found in the present investigation were independent

of G proteins; rather, the observed changes in binding and

stability apparently derived from an effect intrinsic to the

receptor alone. When the purified receptor was reconstituted

with Go/i in phospholipid vesicles, the binding of agonists was

affected by N-ethylmaleimide in the manner that is character-

istic of muscarinic receptors in native membranes (e.g. Refs. 6,

8, and 9). In particular, the reagent decreased the magnitude of

the effect of GMP-P(NH)P. The purified receptor therefore re-

tained its native functionality with respect to the interaction

with Go/i and the allosteric interaction between the agonist and

GMP-P(NH)P; also, the related effects of N-ethylmaleimide are

largely independent of whether alkylation occurs in the native

membrane or after purification.N-Ethylmaleimide did not affect the nature or quantity of

oligomers formed by differently tagged human M2 receptors, at

least as monitored by the coimmunoprecipitation of c-Myc and

FLAG epitopes from extracts of coinfected Sf9 cells. Cloned

receptors and receptors from porcine atria underwent qualita-

tively similar changes in affinity and stability, with the two

preparations differing only in the magnitude of the change. It

therefore appears that neither the instability nor the decreased

affinity for [3H]quinuclidinyl benzilate arose from any effect of

alkylation on the oligomeric status of the receptor.

Evaluation in Terms of Scheme IIWhereas the changes in

affinity and stability appear to be mutually independent, the

reciprocal nature of the various effects argues for a common

cause. Many studies have shown that muscarinic and other G

TABLE IIIAffinities of antagonists and agonists for native and alkylated receptor, estimated semi-empirically in terms of Scheme I

The data represented in Fig. 7 were analyzed simultancously in terms of Scheme I (Equations 4 and 5, n 2) to obtain the parametric valuesfor all ligands (radiolabeled, L P; unlabeled, L A). All experiments were performed at least three times except for the inhibition of [3H]QNBby unlabeled QNB in panel E, which was done once. Assays at two concentrations of [3H]QNB generally were performed in parallel (panels B and

E). Single values of KLj were common to all of the relevant data acquired with the same preparation of receptor ( i.e. native or alkylated). In thecase of QNB, the radioligand and the unlabeled analogue could be assigned single values of KLj without affecting the sum of squares (i.e., KPj

KAj) (p 0.37); in the case of NMS, the same constraint compromised the fit (p 0.00001). The sum of squares was significantly lower with twoclasses of sites rather than one (p 0.00001), and a single value of F2 was common to all of the data (F2 0.58 0.06). The mean values of [R]tobtained for [3H]QNB are as follows: native receptor, 633 19 pM (n 15); alkylated receptor, 632 28 pM (n 23).

Ligand L NEM Log KL1 Log KL2 Log EC50 Log EC50

([3H])Quinuclidinyl benzilate P 9.78 0.12 8.70 0.09 9.13A, P 8.58 0.08 7.70 0.11 8.06 1.07

N-[3H]Methylscopolamine P 9.01 0.08 8.25 0.08 8.56P 8.11 0.08 7.23 0.17 7.59 0.97

N-Methylscopolamine A 9.05 0.17 7.91 0.14 8.36A 7.27 0.15 6.68 0.16 6.92 1.44

Scopolamine A 7.95 0.15 6.98 0.12 7.37A 6.39 0.14 5.84 0.16 6.07 1.30

Carbachol A 3.93 0.16 2.50 0.17 3.05A 3.94 0.18 2.99 0.21 3.37 0.33

Oxotremorine-M A 4.54 0.18 3.62 0.25 3.99A 4.49 0.15 3.47 0.18 3.88 0.11

FIG. 8. Effect of N-ethylmaleimide and GMP-P(NH)P on thebinding of oxotremorine-M to M

2receptor purified from porcine

atria and reconstituted with Go/G

i. Native receptor (E, ) and

receptor treated with NEM (q, f) was prepared and reconstituted withG proteins as described under Materials and Methods. The binding of[3H]QNB was measured at graded concentrations of oxotremorine-Malone (E, q) and together with 0.1 mM GMP-P(NH)P (, f). Theconcentration of [3H]QNB was selected to achieve 50% occupancy inthe absence of oxotremorine-M (native receptor, 1.0 1.1 nM; alkylatedreceptor, 1215 nM). Each curve represents two experiments performedon different batches of purified receptor. The lines represent the best fitof Equation 3 (n 2). Single values of IC50(j) and Fj were common to

both sets of data acquired under the same conditions, and separatevalues ofB[A]0 and B[A]3 were assigned to the data from each exper-iment. Values plotted on the y axis were normalized to the fittedasymptotes taken as 100 and 0. The parametric values are as follows:native receptor (no GMP-P(NH)P), log IC50(1) 5.43 0.17, logIC50(2) 3.89 0.13, F2 0.56 0.07; native receptor (with GMP-P(NH)P), log IC50(1) 4.16 0.19, log IC50(2) 3.11 0.53, F2 0.31 0.22; alkylated receptor (no GMP-P(NH)P), log IC50(1) 5.37 0.24, log IC50(2) 3.52 0.08, F2 0.75 0.05; alkylated receptor(with GMP-P(NH)P), log IC50(1) 4.48 0.43, log IC50(2) 3.18 0.17, F2 0.74 0.14.



11/16

protein-coupled receptors can interconvert spontaneously be-

tween two or more states of different intrinsic activity. Ago-

nists bind with higher affinity to the more active state and shift

the equilibrium accordingly, whereas antagonists favor the less

active state (e.g. Refs. 15, 16, 36, and 37). The molecular dis-

tinction between the two states remains unclear, because the

effects of mutation and other perturbations typically are mon-

itored by means of a receptor-elicited response. According to

one view, however, the inactive and active states correspond to

the free receptor and the receptor-G protein complex, respec-

tively (38). An extension of that view posits that the two states

of the receptor differ in their affinity for the ligand on the one

hand and the G protein on the other (15, 39, 40). It also has

been suggested that N-ethylmaleimide reacts with muscarinic

receptors only in the agonist-specific state, thereby locking the

receptor in that conformation and causing an observed increase

in the affinity for agonists (10, 11).

A model that incorporates the notion of two interconverting

states offers a plausible explanation for the effects of N-ethyl-

maleimide on purified receptors in the present investigation.

The lines in Figs. 7 and 9A represent the best fit of Scheme II

to the data, taken together, and the parametric values arelisted in Tables IV and V. It was assumed that there were two

noninterconverting forms of the receptor overall (n 2). Native

receptors were wholly of one form (e.g. Figs. 7, AC, j 1, F2

0), and receptors treated with N-ethylmaleimide for 24 h were

wholly of the other (e.g. Fig. 7, DF, j 2, F2 1).5 This

restriction presupposes that alkylation was complete after 24 h

in the absence ofN-methylscopolamine, and it follows from the

observation that the potency of [3H]quinuclidinyl benzilate was

essentially the same after treatment with N-ethylmaleimide

for 24 h and 4 days (Table I). The reaction otherwise was

assumed to be incomplete: that is, after treatment for less than

24 h in the absence of N-methylscopolamine or for any time in

the presence of N-methylscopolamine (Fig. 9A). Accordingly,

those data were analyzed as a mixture comprising both forms

of the receptor (i.e. 0 F2 1).

It also was assumed that N-ethylmaleimide was without

effect on the affinity of the ligand for either R or R*. All of the

data therefore shared single values of KLj and Lj for each

ligand (i.e. KL1 KL2 and L1 L2). With this constraint

and the assumption of homogeneity after alkylation for 24 h,

the effects of N-ethylmaleimide are attributed exclusively to

a change from KR1 to KR2; that is, alkylation is assumed to

cause a shift in the distribution of receptors between the two

states.

Preliminary analyses with Scheme II indicated that the con-

straints described above were without effect on the sum of

squares, but neither were they sufficient to yield a unique set of

parametric values. The ambiguity arises in part from uncer-tainty over the absolute value of KRj, particularly after alkyla-

tion. The relative value of KRj is better defined, however, and a

lower bound of about 100 was determined by mapping the sum

of squares with respect to the ratio of KR1/KR2. The value ofKR1is about 0.05 0.1 at all acceptable values of KR1/KR2, and the

results obtained with KR1/KR2 fixed at 100 are listed in Tables

IV and V.

The good agreement between Scheme II and the data indi-

cates that the model can account for all of the effects illustrated

in Figs. 7 and 9A. Based on the fitted value of KR1, purified M2receptors were predominantly but not exclusively in the R*

state in the absence of muscarinic ligands (log KR1 1.28,

Table V). In the presence of an antagonist, however, the net

interconversion associated with comparatively high values of

5 With this restriction, the native receptor and the product aftertreatment for 24 h with N-ethylmaleimide are described as distinct butsingle classes of sites in terms of Scheme II. In each case, the sum ofsquares is the same as that obtained from Scheme I with a single classof sites. The sum of squares from Scheme II is lower with two classes ofsites rather than one, as found with Scheme I (Table III), but theparameters are poorly defined, and the fitted curves are almost indis-

tinguishable from those shown in Fig. 7.

FIG. 9. Effect ofN-methylscopolamine and carbachol on the rate of alkylation by N-ethylmaleimide, as monitored by [3H]quinu-clidinyl benzilate. Purified receptor from porcine atria was reacted with NEM at 0 C for different times in the absence of ligand (dashed lines)and in the presence of either 1 M NMS (A) or 10 mM carbachol (B) (solid lines). Samples that were reacted for the same time with and withoutthe ligand were processed in parallel. The receptor then was desalted on Sephadex G-50 and assayed for the binding of [ 3H]QNB. Panel A, bindingwas measured after treatment in the absence (E, , ) and presence (, , ) of NMS as follows: no NEM (E, ) (n 1), NEM for 0.5 h (, )

(n 3), and NEM for 4 h (, ) (n 2). Data acquired after treatment with NEM for 24 h are not shown but were included in all analyses ( n 2). Panel B, binding was measured after treatment with NEM for 0.5 h in the absence () and presence () of carbachol (n 2). The data shownin both panels were analyzed in terms of Scheme I (Equations 4 and 5, n 2), as described in the text. The data shown in panel A also werecombined with those represented in all panels of Fig. 7 and analyzed in terms of Scheme II (Equations 4 and 6). The parametric values are listedin Tables IV and V. The lines in panel A represent the best fit of Scheme II (n 2), and they are almost superimposable with those from the bestfit of Scheme I. Values plotted on the y axis were normalized to the mean value of [R] t for the experiments described above (534 8 pM, n 16).The lines in panel B represent the best fit of Scheme I (n 2). Values plotted on the y axis were normalized to the mean value of [R] t for theexperiments shown in the panel (553 14 pM, n 4).



12/16

L caused the R state to predominate at saturating concentra-

tions of the ligand. Because N-ethylmaleimide decreased KR by

100-fold or more, alkylation caused the R* state to predominate

not only in the absence of ligands, precluding even the minor

amount of R found with the native preparation, but also at

saturating concentrations of antagonist. This difference in the

ability of a ligand to promote a redistribution of sites from R*

to R accounts for the decrease of at least 10-fold effected by

N-ethylmaleimide in the apparent affinity of muscarinic antag-

onists (Table III).

Because the alkylated receptor was predominantly in the R*

state with or without antagonist (Table V), the values ofLKL

calculated for antagonists in terms of Scheme II (Table V) agree

closely with the corresponding values of EC50 obtained for

alkylated receptors in terms of Scheme I (Table III). In con-

trast, the values of KL and LKL from Scheme II (Table V)

bracket the corresponding values of EC50 obtained for the na-

tive receptor from Scheme I (Table III) (i.e. KL EC50 LKL).

The intermediate values of EC50 are a consequence of the

redistribution of sites from R* to R that occurs upon the bind-

ing of the antagonist to the native receptor (Table V).

The lower limit on KR1/KR2 is related to the marked prefer-

ence of alkylated receptors for the R* state under all conditions.

The extent of the redistribution effected by ligands is governed

by Lj. Antagonists bind more tightly to R (Lj 1, Table IV)

and therefore promote R over R* (KRj LjKRj, Table V).

Because R* predominates with native receptors in the absence

of ligand and with alkylated receptors irrespective of ligand

(Table V), the value of KR1 must approximate or exceed that of

L2KR2. The ratio KR1/KR2 therefore is limited by L2 (i.e. L2

KR1/KR2), or by the single value of L if N-ethylmaleimide is

without effect on the relative affinity of the ligand for R and R*

(i.e. L1 L2 L). The fit therefore is compromised at values

of KR1/KR2 that are substantially less than L; the value ofLis governed in turn by the difference in EC50 before and after

alkylation.

Because N-ethylmaleimide was without effect on the binding

of carbachol and oxotremorine-M, the relative affinity of ago-

nists for R and R* is unclear. This is illustrated in Fig. 11,

where the global sum of squares is shown to be independent of

L at lower values and to increase at higher values. The map

indicates that the data are consistent with any value of Lsmaller than about 100.2 for carbachol and 100.9 for oxotremo-

rine-M. This ambiguity arises from the preponderance of R*

under all conditions with respect to the agonist (Table V).

Because the system is predominantly in the state potentially of

higher affinity for agonists, the distribution of sites between R

and R* undergoes little or no change upon addition of the

ligand. The state of lower affinity for agonists therefore is

unobservable.Scheme II also can account for the opposing effects of N-

methylscopolamine and N-ethylmaleimide on the affinity of

purified receptors for [3H]quinuclidinyl benzilate (Fig. 9A). Na-

tive receptor was assumed to be exclusively R1 or R1*, as

described above, and the progress of the reaction was modeled

as the increase in the fraction of sites identified as R2 or R2*

(i.e. F2 [R2]t/([R1]t [R2]t). Upon treatment with N-ethylma-

leimide in the absence ofN-methylscopolamine, the fraction F2increased from zero initially to 0.72 0.03 after 30 min and to

0.92 0.02 after 4 h; the value after 24 h was set at 1. In the

presence of N-[3H]methylscopolamine, the value of F2 in-

creased to only 0.46 0.03 after 30 min and to 0.70 0.03 after

4 or 24 h. In contrast to N-methylscopolamine, carbachol was

without effect on the rate of interconverstion from R1 to R2 (Fig.9B). This difference between N-methylscopolamine and carba-

chol parallels the different preference of antagonists and

agonists for the two states of the receptor. It follows that

the antagonist slowed alkylation by favoring a state, in this

case the R state, that is comparatively unreactive to

N-ethylmaleimide.

Evaluation in Terms of Scheme III A kinetically deter-

mined variant of Scheme II can account for the opposing effects

of antagonists andN-ethylmaleimide on the rate of inactivation

(Figs. 4 and 5) and, in the case of the alkylated receptor, for the

time-dependent, rightward shift in the binding of N-[3H]meth-

ylscopolamine (Fig. 3B). Scheme III was formulated with time

as an independent variable and incorporates the possibility

that the receptor can decay to a state or states that do not bind

FIG. 10. Oligomeric and functional stability of M2 receptorextracted from Sf9 cells. FLAG-tagged and c-Myc-tagged receptorswere coexpressed in Sf9 cells and extracted in digitonin/cholate. Solu-bilized receptor was maintained at 0 C for 24 h in the absence ofreagent and in the presence of 10 mM NEM. The sample then waspreincubated at 30 C in the absence of ligand and in the presence ofeither QNB (0.3 M) or NMS (0.3 M), and at the designated time it wasdesalted on Sephadex G-50. Aliquots of the eluate were assayed for thebinding of [3H]QNB and for coimmunoprecipitation of the FLAG andc-Myc epitopes. Panels A and B, the receptor was immunoprecipitatedby the agarose-conjugated anti-c-Myc antibody and detected by theanti-FLAG antibody. The film was exposed for about 2 min. The arrowsindicate bands corresponding to molecular weights of 59,500 and93,400. Panel C, binding was measured at a saturating concentration of[3H]QNB (300 nM) (open bars), and the intensities of the monomeric andoligomeric bands were estimated by densitometry (solid bars). The

values plotted inpanel C are shown relative to the corresponding values

obtained with alkylated receptor that was not preincubated at 30 Cprior to being loaded on the Sephadex column. Each value is the meanof 2 6 experiments.



13/16

the radioligand, at least at the concentrations used in the

assays. The lines in Figs. 3B and 4 represent the best fit to the

pooled data, and the rate constants are listed in the legend to

Fig. 4.

Receptors in the state designated as R1 in Scheme III were

stable under the conditions of the assays, with a half-life meas-

ured in days either with or without N-[3H]methylscopolamine(i.e. kPR1 and kR1). The R2 state was almost as stable in the

presence of antagonist (kPR2). In the absence of ligand (kR2),

however, the decay was 5-fold more rapid with the native

receptor and 25-fold more rapid after treatment with N-ethyl-

maleimide. Alkylation therefore hastened inactivation by fur-

ther destabilizing the labile R2 state and by promoting R2 over

the stable R1 state. The protective effect of N-[3H]methylsco-

polamine derived in part from its preference for R1 over R2 and

the attendant redistribution of sites away from the labile state

(i.e. L 100, Table IV); also, either state of the receptor was

more stable in the presence of a ligand. The latter effect pre-

dominated after alkylation, because the value of LKRj was

such that most of the sites were in the R2 state even at satu-

rating concentrations of antagonist (Table V). Similarly, occu-

pancy per se also accounted for the stabilizing effect of carba-

chol (Fig. 6).

In the context of Scheme III, the rightward shift shown for

N-[3H]methylscopolamine in Fig. 3B derived from the opposing

effects of the antagonist and N-ethylmaleimide on the inacti-

vation of alkylated receptors. Because of the decay illustrated

in Fig. 4B, longer incubation times increased the concentrationofN-[3H]methylscopolamine required to achieve a given level of

binding. That led in turn to an increase in the value of EC50over time. Scheme III also can account for a decrease in max-

imal binding, as described below, but the predicted effects

differ from those illustrated in Fig. 3B. In terms of the model,

the capacity for N-[3H]methylscopolamine was 73 89% of that

for [3H]quinuclidinyl benzilate at each time of incubation. The

shortfall does not derive from the nonbinding species R3 and

R4, because the parameter [R]t comprises all forms of the re-

ceptor. Also, the amounts of R3 and R4 were assumed to be zero

at the outset, and the apparent loss of sites is expected to

emerge over time.

The predicted effect of time on the binding of N-[3H]methyl-

scopolamine to the alkylated receptor is illustrated in Fig. 12,

TABLE IVEffect of N-ethylmaleimide on the affinities of muscarinic ligands for purified M2 receptor, evaluated in terms of Scheme II

The data illustrated in Figs. 7 and 9A were analyzed in concert according to Equations 4 and 6. There were two classes of sites overall ( n 2),representing native receptor (j 1) and alkylated receptor (j 2). The reaction with NEM was complete after 24 h, and the data in the lower panelsof Fig. 7 therefore were treated as a homogeneous population of sites (j 2). The data in Fig. 9A were treated as a mixture of sites. Single valuesofKL, and L were common to all of the data acquired with both native and alkylated receptor, as described in the text. QNB and NMS were presentboth as the radioligand (L P) and as the unlabeled analogue (L A). The two forms of QNB yielded consistent estimates of KL and L, whichtherefore were optimized as a single value (i.e. P A L, KP KA KL). The same constraint with NMS led to a significant increase in thesum of squares. Capacities were estimated as the total concentration of sites ([R] t) and that fraction corresponding to alkylated receptor (i.e. F2 [R2]t/([R1]t [R2]t)). The number of curves represented in Fig. 7 is shown in parentheses (native receptor, alkylated receptor). Further details aredescribed in the legend to Table V.

Ligand Log KL Log L Log (LKL)

[3H]Quinuclidinyl benzilate (3, 3) 10.41 0.07b 2.29 0.08 8.12and quinuclidinyl benzilate (2)a

N-[3H]Methylscopolamine (3, 3) 9.87 0.08 2.18 0.09 7.69N-Methylscopolamine (5, 6) 9.68 0.08 2.86 0.08 6.82Scopolamine (6, 6) 8.65 0.08 2.64 0.08 6.01Carbachol (3, 3)c 3.11 0.2 3.31Oxotremorine-M (3, 3)c 4.85 0.9 4.00

a Unlabeled QNB was used only in assays with the alkylated receptor.b The value listed in the table is for the data in Fig. 7. A separate value was required for the data in Fig. 9A (log KL 10.68 0.08), perhaps

because a different batch of receptor was used in those experiments.c The parametric values listed for carbachol and oxotremorine-M correspond in each case to the upper bound on log L (Fig. 11). Lower values

were without effect on the sum of squares or on the values of log KL and log L obtained for other ligands. The latter were determined with thevalue of log L for each agonist fixed at 2.

TABLE

VEffect of N-ethylmaleimide and muscarinic ligands on the distribution of purified M2 receptor between interconverting states,evaluated in terms of Scheme II

The data illustrated in Fig. 7 and 9A were analyzed in concert according to Equations 4 and 6. The distribution of sites between R and R* in theabsence of ligand was evaluated as the single value ofKR2 common to all data acquired with the alkylated receptor and the single value of the ratio

KR1/KR2 common to all data acquired with the native receptor. The value of log (KR1/KR2) was fixed at 2 to obtain the optimized value of log KR2(3.28 0.08) and the corresponding value of log KR1 (1.28) listed in the table. The values of log (LKRj) were calculated from the value of log

KRj and the appropriate value of log L from Table IV. The fraction of sites in the R state was calculated from the value of KRj for the unligandedreceptor and from LKRj for the receptor at saturatin

Documents

Chi Shing Sum et al- Effects of N-Ethylmaleimide on Conformational Equilibria in Purified Cardiac Muscarinic Receptors