Radiolabeling of the Allosteric Site of M2 Receptors
141
below) instead of a filtration assay were pelleted for 20 min at Hill equation. Because the observed Hill coefficients did not differ
20,900 ϫ g (15,300 rpm in a Beckman rotor model F241.5). Pellets significantly from unity (partial F test, p Ͼ 0.05, data not shown),
were resuspended in a buffer composed of 4 mM Na2HPO4 and 1 mM
IC50 values were determined from curve fits with nH fixed to 1. The
KH2PO4, pH 7.4 (Na,K,Pi buffer). Aliquots of 1 ml were frozen in binding parameters KD and Bmax were calculated according to De-
liquid nitrogen and stored at Ϫ80°. Protein content amounted to Blasi et al. (1989). [3H]Dimethyl-W84 inhibition curves were fitted
4.7–8.5 mg/ml membrane suspension.
by applying an equation for a two-site model. The binding parame-
Binding assays. Cardiac membranes at a protein concentration ters KD and Bmax were derived from the high and low affinity com-
ponents of this fit according DeBlasi et al. (1989). Ki values for the
inhibitory action of the test compounds on [3H]dimethyl-W84 binding
were obtained from IC50 values according to Cheng and Prusoff
(1973).
of 300–700 g/ml were incubated with the appropriate radioligand in
the Na,K,Pi buffer at a temperature of 23°. Assays for homologous
and heterologous competition measurements were carried out in
volumes of 1.5 ml with an incubation time of 2 hr. For kinetic
experiments, assays were prepared in larger volumes, and 1-ml
aliquots were removed at appropriate intervals over a total period of
120 min.
Analysis of the effect of dimethyl-W84 on the binding of [3H]NMS
was based on the ternary complex model of allosteric interactions of
Ehlert (1988) as described previously (eq. 3 in Tra¨nkle et al., 1998).
[3H]NMS dissociation data could be described by a monoexponen-
Effects of dimethyl-W84 on [3H]NMS binding. The binding
characteristics of [3H]NMS (0.2 nM) under control conditions were
investigated by homologous competition experiments. Nonspecific
[3H]NMS binding was determined in the presence of 1 M atropine
and did not exceed 5% of total binding when membranes were sep-
arated by filtration; in centrifugation experiments (see below), non-
specific [3H]NMS binding was Ͻ10% of the total. In filtration exper-
iments, the Ϫlog equilibrium dissociation constant of [3H]NMS
binding amounted to a pKD value of 9.80 Ϯ 0.11, and the density of
binding sites to Bmax was 100 Ϯ 18 fmol/mg protein (mean Ϯ stan-
dard error, three experiments); in centrifugation experiments, pKD
was 9.56 Ϯ 0.14, and Bmax was 79 Ϯ 21 fmol/mg protein (mean Ϯ
standard error, six experiments). The effect of dimethyl-W84 on the
equilibrium binding of [3H]NMS (0.2 nM) was determined in heter-
ologous inhibition experiments with membrane separation by filtra-
tion.
To determine the influence of dimethyl-W84 on the dissociation of
[3H]NMS, membranes were incubated for 60 min with dimethyl-W84
and the radioligand before radioligand dissociation was revealed by
the addition of 1 M atropine.
To measure the effect of dimethyl-W84 on the association of
[3H]NMS, dimethyl-W84 was incubated with the membranes for 30
min before [3H]NMS was added to start the formation of radioligand/
receptor complexes; 1-ml aliquots were drawn at appropriate inter-
vals.
tial decay function that yielded the rate constant of dissociation kϪ1
.
[3H]NMS association was monophasic. The rate constant of associa-
tion was obtained by fitting a straight line to the initial data points
of specific [3H]NMS binding (at 30 and 60 sec) as described, for
instance, by Bennett and Yamamura (1985).
The retarding action of dimethyl-W84 on the dissociation and
association of [3H]NMS was expressed in terms of a reduction of the
apparent rate constant of association kϩ1 (control, 1.131 Ϯ 0.070
Ϫ1
nM ⅐minϪ1, mean Ϯ standard error, four experiments) and dissoci-
ation kϪ1 (control, 0.165 Ϯ 0.002 minϪ1, mean Ϯ standard error, 16
experiments), respectively. Concentration-effect curves for the dim-
inution of the rate constants were obtained by curve fitting based on
a four-parameter logistic function.
The antagonistic action of obidoxime on the dimethyl-W84-in-
duced retardation of [3H]NMS dissociation was analyzed according
to Lazareno and Birdsall (1993) as described previously (eq. 4 in
Tra¨nkle et al., 1998).
Drugs. [3H]NMS was purchased from DuPont-New England Nu-
clear (Bad Homburg, Germany). Alcuronium dichloride was gener-
ously provided by Hoffmann-La Roche AG (Grenzach-Wyhlen, Ger-
many). Obidoxime chloride was a gift of Merck KG (Darmstadt,
Germany). Atropine sulfate, (Ϫ)-scopolamine N-methylbromide, and
gallamine triethiodide were from Sigma Chemical (Mu¨nchen, Ger-
many). W84 was synthesized by Dr. Joachim Pfeffer (University of
Kiel, Kiel, Germany).
Membranes were separated by rapid filtration (glass fiber filters
No. 6; Schleicher & Schuell, Dassel, Germany). Filters were washed
twice with 5 ml of ice-cold incubation buffer, dried, and placed into
vials containing 5 ml of Ready Protein (Beckman) for liquid scintil-
lation counting.
Results
Allosteric interaction of unlabeled dimethyl-W84
with [3H]NMS binding. Dimethyl-W84 allosterically inhib-
its the dissociation of [3H]NMS from porcine cardiac M2
receptors. The concentration-effect curve is illustrated in Fig.
2 using the apparent rate constant of [3H]NMS dissociation,
[3H]Dimethyl-W84 binding assay. The [3H]dimethyl-W84 ex-
periments were carried out in the presence of 1 M NMS to occupy
the orthosteric binding site of the M2 receptor. NMS was allowed to
equilibrate for 5–10 min with the receptors before [3H]dimethyl-W84
was added to the assay. Alkane-bis-ammonium compounds closely
related to dimethyl-W84 have been reported to interact with acetyl-
cholinesterase (Ohnesorge, 1969); to inhibit binding of [3H]dimethyl-
W84 to acetylcholinesterase, 1 M physostigmine was added to the
incubation mixture. Control experiments revealed that at the indi-
cated concentrations, neither NMS nor physostigmine had an allo-
steric effect on [3H]NMS dissociation and thus did not interact with
the allosteric site of the [3H]NMS-occupied M2 receptor (data not
k
Ϫ1, as a measure of the dissociation rate. The curve has a
slope not different from unity (partial F test, p Ͼ 0.05) and
levels off at kϪ1 ϭ 0, indicating that bound dimethyl-W84
may completely prevent the dissociation of [3H]NMS/receptor
complexes. The concentration of dimethyl-W84 causing a
50% decrease in the kϪ1 of [3H]NMS dissociation served as a
measure of potency and amounted to pEC50,diss ϭ 8.51 Ϯ 0.02
shown). Membranes were separated by centrifugation with 20,900 ϫ (mean Ϯ standard error, eight experiments; Table 1). This
g (15,300 rpm) in a Microfuge (Beckman) for 20 min at 23°. After
drawing off the supernatant, the tubes containing the tightly packed
pellet were carefully and quickly rinsed with 1.5 ml of cold Na,K,Pi
buffer to remove residual radioactivity from the tube wall. After
resuspension of the pellet in 1.5 ml of buffer, the suspension was
transferred into a scintillation vial filled with 10 ml of Ready Protein
(Beckman) for liquid scintillation counting.
value should reflect the affinity of dimethyl-W84 at NMS-
occupied M2 receptors (Lazareno and Birdsall, 1995).
Concentration-effect curves for the action of dimethyl-W84
on [3H]NMS association and on the equilibrium binding of
[3H]NMS also are included in Fig. 2. The inhibitive action of
dimethyl-W84 on [3H]NMS association results from occupa-
tion of free M2 receptors by the modulator. The concentra-
tion-effect curve approaches kϩ1 ϭ 0, suggesting that the
radioligand does not gain access to M2 receptors occupied by
Data analysis. Data from individual experiments were analyzed
by computer-aided, nonlinear regression analysis using Prism (ver.
2.01; GraphPAD Software, San Diego, CA). Analysis of homologous
competition data obtained with [3H]NMS was based on the general dimethyl-W84. The curve has a slope of unity and its inflec-