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

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

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Effects of 
-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 9–27-fold decrease intheir apparent affinity for the antagonists quinuclidinylbenzilate,
-methylscopolamine, and scopolamine whentreated with the thiol-selective reagent
-ethylmaleim-ide. Their apparent affinity for the agonists carbacholand oxotremorine-M was unchanged. Conversely, therate of alkylation by
-ethylmaleimide, as monitored bythe binding of [
3
H]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
-ethylmaleimide butslowed by quinuclidinyl benzilate and
-methylscopol-amine. The destabilizing effect of 
-ethylmaleimide wascounteracted fully or nearly so at saturating concentra-tions of each antagonist and the agonist carbachol. Sim-ilar effects occurred with human M
2
receptors differen-tially tagged with the c
-
Myc and FLAG epitopes,coexpressed in Sf9 cells, and extracted in digitonincholate. The degree of coimmunoprecipitation was un-changed by
-ethylmaleimide, which therefore waswithout discernible effect on oligomeric size. The dataare quantitatively consistent with a model in which thepurifiedreceptorfromporcineatriainterconvertsspon-taneously between two states (
i.e.
R
º
R*). Antagonistsfavor the R state; agonists and
-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
2
re-ceptorsthereforeappeartoexistpredominantlyintheiractive state.
Sulfhydryl-specific reagents have been useful probes of therelationship between structure and function in muscarinic andother G protein-coupled receptors (
 e.g.
Refs. 1–5). In recentstudies, such reagents have been tagged with environmentallysensitive fluorescent probes or spin labels and used to detect aconformational change linked to activation (1–3). That changeis thought to involve the rotation or tilting of the sixth trans-membrane helix (1, 3–5). The hydrophilic nature of some re-agents has been exploited to suggest that the conformationalchange 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 withmany others, it has been shown to affect receptor-G proteincoupling and to have a differential effect on the binding prop-erties of agonists and antagonists. With receptors in nativemembranes, the Hill coefficients for the binding of muscarinicagonists are increased from characteristically low values to values near or indistinguishable from 1; moreover, the shift inpotency brought about by guanyl nucleotides is reduced orabolished (
 e.g.
Refs. 6–9). The affinity of muscarinic antago-nists generally is not affected by
-ethylmaleimide (7–12),whereas the affinity of agonists can be either increased (8,10–14) or decreased (6, 9).It has been suggested that
-ethylmaleimide reacts prefer-entially with one of two spontaneously interconverting states of the receptor, thereby driving the equilibrium toward the reac-tive conformation (10–12). Further support for the existence of multiple interconverting states, only one or comparatively fewof which are functionally active, derives from the occurrence of constitutive activity as seen, for example, in mutants of the
2
adrenergic receptor (15) or the M
5
muscarinic receptor (16). Inthe latter case, it was demonstrated that the ligand-regulatedactivity of 13 constitutively active mutants, differing only atposition 465 in helix number 6, can be accounted for by shifts ina single equilibrium between an inactive and an active state(16). Also, the notion of spontaneously interconverting states isconsistent with the constitutive activity and related propertiesof M
1
–M
4
muscarinic receptors overexpressed in Chinese ham-ster ovary cells (17), and of M
5
muscarinic receptors coex-pressed with comparatively large amounts of G
q
(18).In the present study,
-ethylmaleimide has been used toprobe 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 thecountervailing effects of muscarinic ligands on the action of 
 N 
-ethylmaleimide, have been studied with the system underboth thermodynamic and kinetic control. The data are quantita-tivelyconsistentwithamodelinwhichthereceptorinterconvertsspontaneously between two states that are intrinsic to the recep-tor alone.
-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 Materials—N 
-[
3
H]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). (
)-[
3
H]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 of this 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: jwells@phm.utoronto.ca.
T
HE
J
OURNAL OF
B
IOLOGICAL
C
HEMISTRY
Vol. 277, No. 39, Issue of September 27, pp. 36188–36203, 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.org
36188
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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 
-[
3
H]methylscopolamine, obtained at the Molecular Medicine Re-search Centre of the University of Toronto. The absence of scopolaminefrom lot 3406009 of 
-[
3
H]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 highpurity 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 Receptors
 —
Purified M
2
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
KH
2
PO
4
, 20m
M
NaCl, 1 m
M
Na
2
EDTA, 0.1 m
M
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 of protein, as estimated from the maximal binding of [
3
H]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 withreceptor-specific antibodies. The purified receptor was devoid of the
subunits of G
o
, G
i1
, G
i2
, G
q/11
, and G
s
, 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 M
2
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
KH
2
PO
4
, 15.7 m
M
NaCl, 0.78 m
M
Na
2
EDTA, 0.078 m
M
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 humanM
2
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 M
2
receptor has been describedelsewhere (26).
 Reaction with N-Ethylmaleimide
 —
To prepare samples for use in thebinding assays, an aliquot of purified receptor from porcine atria or thesolubilized extract from Sf9 cells (28
65
l) was thawed and diluted toabout 450
l in buffer A (20 m
M
HEPES, 20 m
M
NaCl, 5 m
M
MgSO
4
, 1m
M
Na
2
EDTA, 0.1 m
M
PMSF, 0.1% digitonin, and 0.02% sodiumcholate, adjusted to pH 7.40 with NaOH) supplemented with
-ethyl-maleimide. A fresh solution of 
-ethylmaleimide was prepared for eachreaction. Controls were prepared in the same manner except that
-ethylmaleimide was omitted. The final concentration of 
-ethylmale-imideinthereactionmixturewas10m
M
exceptwherestatedotherwise;the concentration of receptor was 6.5
10 n
M
, as estimated from thebinding of [
3
H]quinuclidinyl benzilate to controls from which
-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.
-Ethylmaleimide was without effecton the binding of [
3
H]quinuclidinyl benzilate and therefore was notremoved in most experiments. All volumes were recorded, and theconcentration of receptor was corrected as required in subsequentcalculations.When the reaction mixture was to contain
-ethylmaleimide to-gether with either carbachol or
-methylscopolamine, the ligand waspreincubated with the undiluted thawed receptor in an ice bath for 30min.
 N 
-Ethylmaleimide then was added, dissolved in deionized water at10 times the final concentration of 10 m
M
. The total volume of thereaction mixture was 20
200
l. Controls were prepared in the samemanner except for the omission of 
-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 of 
-ethylmaleimide on the binding of [
3
H]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.
 Reconstitution
 —
Native and alkylated receptors were reconstitutedwith G proteins according to a procedure adapted
3
from that describedby Haga
et al.
(27). The G proteins were obtained as a mixture of isoforms purified from bovine brain (Calbiochem). Following incubationof the purified receptor for 24 h at 0
°
C with or without
-ethylmale-imide, as described above, the native or alkylated product (3.6
7.3 pmolin 36
80
l) was incubated with carbachol (10 m
M
) for about 15 min,also at 0
°
C. The receptor then was mixed (1:1) with a suspension of lipids (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 m
M
HEPES, 160 m
M
NaCl, 6 m
M
MgCl
2
, 0.8 m
M
Na
2
EDTA, and 1 m
M
dithiothreitol, 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 G
o
, 8
16 pmol G
i-1
, 8
16 pmolof G
i-2
,
8 pmol of G
i-3
, and 64 pmol of G
 
), which then was appliedto a column of Sephadex G-50 (0.8
5 cm) pre-equilibrated with bufferB. 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 n
M
.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 theSephadex column.
 Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blotting, and Immunodetection
 —
The co-immunoprecipitation of c-Myc-and FLAG-tagged M
2
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 Assays
 —
Binding was measured essentially as describedpreviously (21). Solutions of the radioligand and any unlabeled ligandswere prepared in buffer A or buffer C (25 m
M
KH
2
PO
4
, 4 m
M
HEPES,230 m
M
NaCl, 10 m
M
MgCl
2
, 0.8 m
M
Na
2
EDTA, and 0.1 m
M
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[
3
H]quinuclidinyl benzilate, the time of incubation was 2 h unlessindicated 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 independent
1
The abbreviations used are: QNB, quinuclidinyl benzilate; GMP-P(NH)P, 5
-guanyly-5
-yl imidodiphosphate; NEM,
-ethylmaleimide;NMS,
-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
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of time under the conditions of the experiments. In assays with
 N 
-[
3
H]methylscopolamine, the time of incubation was as stated in thetables or figures. To follow the time dependence of binding, an aliquot of the 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, andaliquots (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 m
M
unlabeled
-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 [
3
H]quinuclidinyl benzilate (
n
19)and 0.000018
0.000002 for
-[
3
H]methylscopolamine (
n
16).In assays with
-[
3
H]methylscopolamine, ethanol that accompaniedthe radioligand accelerated the inactivation of receptors pretreatedwith
-ethylmaleimide. Most or all of the ethanol therefore was evap-orated under argon before the radioligand was taken up in buffer. Insuchcases,theconcentrationofethanolinthereactionmixturewaslessthan 1% (v/v) at the highest concentration of 
-[
3
H]methylscopolamineand decreased proportionately at lower concentrations. Removal of eth-anol had no effect on the binding of 
-[
3
H]methylscopolamine to un-treated receptors or on the binding of [
3
H]quinuclidinyl benzilate toeither preparation. The radioligand therefore was used as supplied bythe manufacturer in those assays.
 Analysis of Data
 —
 All data were analyzed with total binding taken asthe dependent variable (
 B
obsd
) 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 (
 B
sp
,
B
max
) andtotal receptor ([R]
t
) denote the concentration in the binding assay (p
M
).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]
t
represent the total concentrations of the radiolabeled probe and anunlabeled ligand, respectively. In Equation 1,
B
sp
represents specificbinding at the corresponding value of [P]
t
, and
 B
max
represents maximalspecific binding; the parameter EC
50
denotes the concentration of un-bound radioligand that yields half-maximal occupancy, and
n
H
is theHill coefficient. The parameter
NS
represents the fraction of unboundradioligand 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
and
 B
[A]
represent the asymptotic levels of bindingwith respect to the concentration of unlabeled ligand; IC
50
is the con-centration of unlabeled ligand that reduces specific binding by one-half.In Equation 3,
  j
represents that fraction of specific binding defined bythe inhibitory potency IC
50(
  j
)
.
 B
obsd
B
max
([P]
t
 B
sp
)
n
H
EC
50
n
H
([P]
t
 B
sp
)
n
H
 NS

P
t
 B
sp
) (Eq. 1)
 B
obsd
 B
 A
 B
 A
0
 B
 A
IC
50
n
H
IC
50
n
H
 
 A
tn
H
(Eq. 2)
 B
obsd
B
 A
(
 B
 A
0
 B
 A
]
)
 j
1
n
 F 
 j
IC
50
 j
IC
50
 j
 A
t
(Eq. 3)Semi-empirical and mechanistic descriptions of the data were basedon Schemes I
III, 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 whichthe 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
.
 B
obsd
 B
sp
 NS
[P]
t
 B
sp

(Eq. 4)Scheme I comprises a mixture of distinct sites (R
  j
,
j
1, 2, . . . ,
n
)wherein those of type
j
bind P and A with the equilibrium dissociationconstants
P
  j
and
 A
  j
, respectively, and constitute the fraction
  j
of allsites (
i.e. F 
  j
[R
  j
]
t
 /[R]
t
, where [R
  j
]
t
[R
  j
]
[AR
  j
]
[PR
  j
], and [R]
t
  j
1
n
[R
  j
]
t
). Total specific binding of the probe was calculated according toEquation 5, and the required values of [PR
  j
] were obtained as describedbelow. Values of EC
50
were calculated from the fitted estimates of 
L
  j
(L
P or A) and
  j
(
n
1), as described previously (21).
 B
sp
 j
1
n
PR
 j
(Eq. 5)In Scheme II, each receptor of type
j
can exist in two spontaneouslyinterconverting states designated R
  j
and R
  j
*
. The ligands P and A bindto R
  j
with equilibrium dissociation constants
P
  j
and
 A
  j
(
 e.g.
[P][R
  j
]/ [PR
  j
]
P
  j
), and the relative affinity of the ligand for the two states of the receptor is designated
P
  j
and
 A
  j
, respectively (
i.e.
P
  j
P
  j
*/ 
 K 
P
  j
,
 K 
P
  j
*
[P][R
  j
*]/[PR
  j
*
];
 A
  j
 A
  j
*/ 
 K 
 A
 j
*
,
 A
  j
*
[A][R
  j
*
]/[AR
  j
*]). The pa-rameter
R
  j
represents the relative concentrations of R
  j
and R
  j
* atequilibrium in the absence of ligand (
i.e.
[R
  j
]/[R
  j
*]
R
  j
). Sites of type
 j
constitute the fraction
  j
of all sites, as in Scheme I (
i.e. F 
  j
[R
  j
]
t
 /[R]
t
,where[R
  j
]
t
[R
  j
]
[R
  j
*]
[AR
  j
]
[AR
  j
*]
[PR
  j
]
[PR
  j
*]).Thespecificbinding of the probe was calculated according to Equation 6, and therequired values of [PR
  j
] and [PR
  j
*] were obtained as described below.
 B
sp
 j
1
n
([PR
 j
]
[PR
  j
*
]) (Eq. 6)In Scheme III, the receptor can exist in two spontaneously intercon- verting states (R
1
and R
2
) that convert irreversibly to a third and fourthstate (R
3
and R
4
, respectively). The equilibrium between R
1
and R
2
isanalogous to that between R
  j
and R
  j
* for receptors of type
j
in SchemeII (
i.e.
[R
1
]/[R
2
]
 K 
R
, [PR
1
]/[PR
2
]
 K 
PR
). 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 thecalculations. The parameters
k
P(
  j
)
and
k
P(
  j
)
in Scheme III denote thefirst- and second-order rate constants for the binding of a radioligand(P) to receptors in state
j
, and
P
  j
is the corresponding equilibriumdissociation constant (
i.e. k
P(
  j
)
 / 
k
P(
  j
)
P
  j
). Similarly, the rate of interconversion between R
1
and R
2
is determined by
k
R(
)
and
k
R(
)
(
i.e.k
R(
)
 / 
k
R(
)
 K 
R
), and that between PR
1
and PR
2
is determined by
k
PR(
)
and
k
PR(
)
(
i.e. k
PR(
)
 / 
k
PR(
)
 K 
PR
). The rate of conversion of R
  j
and PR
  j
(
 j
1or2)toR
  j
2
andPR
  j
2
,respectively,isdeterminedby
k
R
  j
and
k
PR
  j
.The specific binding of the probe at any time
t
was calculated accordingto Equation 7, and the required values of [PR
  j
] were obtained as de-scribed below.
 B
sp
[PR
1
]
[PR
2
]
[PR
3
]
[PR
4
] (Eq. 7)The value of 
B
sp
in Equations 5 and 6 was calculated from theexpansions 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 equationsS
CHEME
1S
CHEME
2
 N-Ethylmaleimide and Purified Muscarinic Receptors
36190
  b  y  g u e s  t   , on O c  t   o b  e 8  , 0  0 www. j   b  c . o g ownl   o a d  e d f   om 

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