The two-state model of
Indeed a full spectrum of
receptor activation
Paul Leff
Over the past few years, the concept that the activation
of G protein-coupled receptors and transmitter-gated
ion channels depends on a conformational change has
received increasingly widespread acceptance. As a
result, these two structurally distinct families of
receptors can now be considered to obey a similar two-
state mechanism. However, traditional receptor theory
has largely overlooked this concept. In this article,
Paul Leff explains and illustrates the predictions of the
two-state model of receptor activation and discusses its
impact on the analysis and interpretation of
agonist-receptor interactions.
The concept that the activation of a receptor by an agonist
involves a conformational change in the receptor has been
implicit in pharmacological thinking for some time. This
concept was originally made explicit by de1 Castillo and
Katz’ who proposed that binding of acetylcholine to
nicotinic acetylcholine receptors led to a conformational
change which resulted in channel opening. This model of
receptor activation invokes the existence of two receptor
states, a closed (or resting) and an open (or active) one,
and the agonist causes an increase in the ratio of active to
resting states. The application of this kind of model of
receptor activation is almost inevitable in the study of
transmitter-gated ion channels because the physical
nature of the measurements that can be made on them has
provided direct evidence for multiple states2. However,
this has not been the case for many other receptors, pre-
viously undefined in mechanistic terms but now known
to be G protein-coupled, where experimental analysis of
agonist action has, until recently, been necessarily in-
direct. Perhaps because of this, pharmacological receptor
theory (e.g. Refs 3-51, developed along non-mechanistic
lines and largely overlooked the two-state concept. An
unfortunate consequence of this is that the pharmaco-
logical methods to quantify agonist action that have
been devised from those theories are now recognized
as theoretically flawedh.
Over the past few years, the two-state model of recep
tor activation has been rejuvenated to explain some obser-
vations made on G protein-coupled receptors in cell lines
and in recombinant receptor-expression systems7-ll. A
key finding is the ability of such receptors to exist in a con-
stitutively activated state, that is, a state which is able to
initiate a biochemical response in the absence of the ago-
nist. A feature of these systems is their ability to dis-
close inverse agonism. Thus, certain ligands produce a
descending agonist concentration-effect curve rather than
0 1995,ElsevlerScience Ltd
the customary ascending one.
agonist activities is revealed, ranging from full and par-
tial agonism, through ‘silent’ activity (binding but no
effect; also true for antagonists), to partial and (appar-
ently) full inverse agonism (e.g. Ref. 10).Binding data cor-
relate with the functional measurements and support the
simple, two-state, interpretation that agonists have pref-
erential affinity for the active state, inverse agonists have
preferential affinity for the resting state, and ligands
which are silent have equal affinities for the two’.
It can now be advanced that both the two major super-
families of receptors, transmitter-gated ion channels and
G protein-coupled receptors, obey the same, two-state
mechanism of receptor activation. It is timely, therefore,
to address the pharmacological impact of the two-state
model, revisit what is already known about its predicted
behaviour from considerations of transmitter-gated ion
channel receptors, and to extend the applicability of these
predictions to G protein-coupled receptors. Accepting the
two-state model as ‘reality’has important consequences
with regard to the conceptual definition and quantifi-
cation of the pharmacological properties of agonists and
antagonists.
The two-state model
The simplest model that can be proposed for a two-
state mechanism of receptor activation (Box 1) is schemati-
cally identical to the model introduced by Monod,
Wyman and Changeux 12,the only difference being that
here it is applied to the interaction of agonists and recep
tors rather than to oxygen and haemoglobin.
In the scheme, R represents receptors in the resting
state, and R”represents receptors in the active state. L is
an equilibrium constant that determines the distribution
of receptors between the two states in the absence of lig-
and. Interaction of an agonist, A, with the receptor is
assumed to displace this equilibrium towards one or other
of the two states. If A has higher affinity for R*, it is an
agonist. If A has higher affinity for R, it is an inverse ago-
nist. This is determined by the values of KAand KA*,the
dissociation equilibrium constants for A at the two recep
tor states.
The properties of this model (and extended versions of
it) have been analysed in some depth by Colquhounls. A
number of theoretical observations were made concern-
ing its application to the pharmacological analysis of ago-
nist action. It was found that, in general, the operation of
the two-state model would closely resemble the predic-
tions of pharmacological receptor theory, but that it
would do so with a number of important distinctions.
First, the measured affinity of an agonist for the receptor
would be a complex quantity dependent on the two dis-
sociation constants, KAand KA*,and also on the value of
L. This contrasts with simple receptor theory which P. 1eff.
Head.
defines agonist affinity by a single constant. Second, the Depaitmentaf
efficacy of an agonist would also be dependent on K,4and Pharmacology.
Fisons
EakewelIRoad.
KA*, these being the quantities which determine the toughborough
degree to which an agonist displaces receptors towards UKLEll ORH
TiPS - March 1995 (Vol. 16) 8 9

the active state. Because affinity and efficacy depend on
the same underlying constants, they cannot be thought of
as being independent from one another, either conceptu-
ally or with regard to their measurement. This is a funda-
mental difference between the predictions of the two-state
model and those of pharmacological receptor theory.
Accepting the operation of the two-state mechanism in
practice means that these theoretical predictions are
unavoidable. Since this concept is now receiving wide-
spread acceptance, familiarization with Colquhoun’s
theoretical analysis is worthwhile. The present article will
be restricted to graphical illustration of the properties of
the two-state model which have most relevance to exper-
imental pharmacology, whether practised on native tis-
sues, cell lines or expressed receptors.
Predictions of the two-state model
Equations (1) or (2) (Box 1) were used to simulate the
two-state model and illustrate its pharmacological pre-
dictions. In each case, graphs of fR* versus log[Al are
plotted in accordance with pharmacological convention.
9 0 TiPS - March 1995 (Vol. 16)
K,‘(l + L)
(1 + LK,,*IK,J
Ligand (log,, M)
Fig.Simulation representing the fraction of receptors In the activated
state (fR*) against ligand concentration.
Curves which point in the upward direction represent
displacement of receptors towards R*. Curves which
point in the downward direction represent displacement
of receptors towards R. In each case, the plots represent
binding curves and so their mid-points are measure-
ments of the apparent binding affinities of ligands, KaPP.
These plots may be regarded as being representative of
agonist-concentration effect curves if the assumption is
made that the measured effect is directly proportional
to fR*.
Definitionof affinityandefficacy
The effect of varying the ratio between K, and K,* can
be determined (Fig. 1). The other parameters are kept con-
stant so this simulation depicts six different ligands act-
ing on the same receptor in the same cell or tissue. A value
of L was chosen such that, under basal conditions, the
ratio R : R* was 1: 1. KA was kept constant and K,* was
varied such that the relative affinity for the receptor states
ranged from 50-fold in favour of the active state to IO-fold
in favour of the resting state.
 PRINCIPLES

Efficacy
When KA:KA*~1, the ligand behaves as an agonist;
when KA:K,I’= 1, the ligand is ‘silent’, i.e. it produces no
agonist effect (this case represents competitive antagon-
ism as explained below); when KA:KA* 4, the ligand
behaves as an inverse agonist. The extent of agonist effect
in either direction depends on the ratio. Since this ratio is
a ligand-dependent quantity which determines the extent
and direction of the agonist effect it is a logical, and chemi-
cally meaningful, definition of intrinsic efficacy.

Affinity
When KA:KA* ~1, the apparent affinity constant, Kapp -10 -8 -6 -4
(the mid-point of the curve), approximates to the value of Ligand (log,, M)

K,*. Conversely, when K,: K,* < 1, K,, approximates to

Fig.1. Simulation representing six ligands with different K,:K,"
K,. This would be expected because an agonist prefers values acting in the same receptor system; I = 1.0; K, = 10-E;
R* and so its binding will be dominated by its affinity K,” = 2 X 1O-*;1O-';4 X 1O-? 104;3 X 1O-6; 1O-5.Affinity. log K,,,
constant for that state. For an inverse agonist, binding is values are indicated by 0. As &* increases, the agonist changes from
an agonist with high affinity to an inverse agonist with low affimty.
dominated by its affinity for R. Between the two extremes
Kappwill depend on both KA and KA*.
Two fundamental predictions of the model are illus- a
trated. First, there is an inescapable correlation between l.O- ,
efficacy and affinity; both measurements of agonist action
depend on the ratio KA: K,*. Second, a continuous spec-
0.8.
trum of agonist activities exists ranging from full inverse
to full agonism, for which the true intrinsic efficacy scale
0.6.
ranges from much less to much greater than unity.
C-F
Variation ofagonist affinity between cells or 0.4 -
tissues
The effect of varying 1, keeping KA and KA*constant
can be studied (Fig. 2a). This depicts the same agonist
acting on the same receptor but in six different cells or f
0.03
tissues amongst which the basal ratio of R : R* varies. L is -12 -10 -8 -6 -4
varied such that the basal ratio ranges from 100: 1 in Ligand (log,, M)

favour of R, to 25:1 in favour of R*.The simulationillustrates

how ligand binding, as measured by Kapp,varies with the
relative proportions of R and R*. In an R*-rich system,
binding is dominated by KA*, whereas in an R-rich sys-
tem, it is dominated by K,. Since the ligand is an agonist
in this example, it has higher affinity for R* than for R. As
a result, in the R*-richsystem it demonstrates higher affin- 0.6 -
ity than in the R-rich system. An inverse agonist will oper-
k
ate in the converse fashion, showing higher affinity in the
0.4-
R-rich system than in the R”-rich one.
r
Therefore, according to the two-state model, the 0 L= 100

measured affinity of a ligand is system-dependent. 0.2 -

Variation in relative efficacy between cells or o.o+

-12 -10 -8 -6 -4
tissues
Ligand (log,, M)
Three ligands acting in two different systems can be
simulated using this model (Fig. 2b). The three ligands Fig.2.a:System-dependence of affinity. Simulation representing the
same llgand in SIX separate systems which differ in the basal R:R* ratio
have different intrinsic efficacies (as defined above by IL): K, = 1O-6:K,"= 2 X 1 O-*; I= 100;20;4;1;0.2; 0.04. As 1 decreases,
their K,: K,* ratios) and L values are chosen to depict an agonist affinity increases as indicated by the log& values (01
R-rich (100: 1 in favour of R) and an R*-rich system (4 : 1 b: System-dependence of efficacy. Simulation representmg three ligands
acting in two systems which differ in the basal R:R* ratio (1); K, = lo!
in favour of R*).One of the ligands has a KA: KA* of 1: 1 so K,” = 1O-8;1O-7.10-e; I = 100; 0.3.With the decrease in I between the
it is ‘silent’in both systems. The other two are agonists two systems, the relative efficacy of the partial agonist IS increased
with K,: KA* values of 10 and 100. For these two ligands,

TiPS - March 1995 (Vol. 16) 9 1

 PRINCIPLES

a
1 illustrated here, the fraction of receptors in the R* form is
plotted directly, that is, no amplification step is included
1.0 in the modelling. Therefore, the two-state model accounts
for such variation at the receptor level. Accordingly, by
/
/ this model, there is a different conceptual basis for the
0.8
/ increase in agonist activity between systems. In a system
/ agonist which is enriched in R, an agonist may displace the ratio
I
0.6 / of receptor states towards R* but only to a finite extent,
/
and so the agonism is partial. In a highly R*-enriched sys-
tem the same alteration in the R :R*ratio can achieve near
complete displacement of the ratio towards R*,so the ago-
\ agonist nism appears full.
\ \
Therefore, according to the two-state model, system-
0.0 \ ‘\., dependent differences in agonist efficacy are predicted to
occur at the level of the receptor.
-12 -10 -6 -6 -4
Ligand (log,, M)
Competitive antagonism
b
As described above, ligands which have equal affinity
for the two receptor states are silent, that is, they are
devoid of agonism or inverse agonism. According to the
two-state model, such ligands behave as competitive
antagonists since they are still able to displace agonist
from the receptor. The antagonist is equally effective at
displacing the curves for the two types of agonist (Fig. 3a),
meaning that the same pK, estimate would be made in
& I
/ each case. This is one important property of the two-state
0.4 , model. Another is that the pK, estimate will be constant
- - - -
in different systems in which L varies (Fig. 3b). Intuitively,
0.2- these predictions are reasonable since the antagonist
competes at both receptor forms with the same affinity
and so its ability to displace an agonist is the same under
0.0 7
-12 -10 -8 -6 -4 all conditions.
Ligand (log,, M)

Fig. 3. a: Competitive antagonism of agonists and inverse agonists.

Simulation representing the interaction of a competitive antagonist
(dashed lines/ with an agonist and an inverse agonist in the same recep-
tor system; agonist: KA = IO”; KA* = 10-T; inverse agonist: K, = 10-8;
K,"= 10-6; competitive antagonist: KS= K,"= 10-6; [B]
= 0;1W; I = 1 .Ll
The antagonist produces the same degree of shift of both agonist curves.
b: System-independence of competitive antagonism. Simulation rep-
resenting the same agonist-antagonist interaction in three systems
which differ in I;agonist: K, = 10”; K’” = lo-‘; competitive antagonist
KS= KB*= IO";[B]= 0;IO+ L = 20;2;0.2. The competitive antagonist
shifts the agonist curve to the same extent in each system
in the R-rich system, clear differentiation between their
efficacies is visualized, whereas in the R*-rich system, the
differentiation is minimal. Also, both of these ligands
demonstrate higher affinities in the R*-richsystem than in
the R-rich system. This pattern of events is reminiscent
of the effects of receptor reserve differences according
to pharmacological receptor theory. For example, the
increase in intrinsic activity and potency of a partial ago-
nist between two systems would conventionally be
related to an increase in receptor density or coupling ef-
ficiency. According to pharmacological receptor theory,
such an interpretation requires there to be an amplifi-
cation step between the measured effect and agonist-occu-
pied receptors. In the simulations of the two-state model
Interaction between agonists and inverse agonists
The effects of an agonist in the presence of fixed con-
centrations of an inverse agonist can be determined. If the
basal ratio R:R* is set at 1: 1, the intrinsic effects of the
inverse agonist are manifested as a drop in the baseline of
the curves (Fig. 4a). If the basal ratio is set at 100: 1, the
inverse agonist cannot produce any measurable effect
(Fig. 4b). In both cases the inverse agonist displaces the
curves for the agonist to the right and the antagonism is
surmountable. However, the extent of displacement, as
measured at the midpoints of the curves, is less in the first
case than in the second. This is made clear when the cor-
responding Schild plots are plotted for the two cases (Fig.
4~1,which show that different apparent pK, estimates
would be made. These predictions for an inverse agonist
acting as an antagonist are in line with previous predic-
tions for its behaviour as an agonist. In a R-rich system,
an inverse agonist will demonstrate a higher affinity than
in a R*-rich system, hence its ability to compete with
another ligand will be higher in the first case than in the
second.
Therefore, according to the two-state model, when an
inverse agonist is used as an antagonist of an agonist (or
vice versa) the expressed affinity is a system-dependent
quantity.

9 2 TiPS - March 1995 (Vol. 16)

 PRINCIPLES

Irreversible antagonism
In principle, there are a number of ways in which irre- a increasing [B]
---------_-_*
versible antagonism can be modelled using two-state 1 .o
theory. An antagonist could bind and irreversibly inacti-
vate either R, or R*,or both. The example generated here 0.6
was chosen to best represent experience. To do this, it is
necessary to assume that receptors in the active state are
0.6
selectively reduced in concentration. This is simulated by
>
progressively increasing the value of L. 0.4
The effect of such an irreversible antagonist on an ago-
nist (KA*<< KA)can be studied (Fig. 5a). As L is increased,
0.2
the system becomes progressively enriched with recep-
tors in the resting state. KaPPis increasingly dominated
by K, and so the mid-points of the curves gradually
-12 -10 -8 -6 -4
approach this value. Also, since the basal ratio of recep-
Ligand (log,, M)
tors is increasingly towards R, the agonist becomes pro-
gressively less able to displace receptors towards R”.This
results in depression of the curves. This pattern of curve b
shifts agrees with conventional expectations for irre- 0.4
versible antagonism although the explanation for the
behaviour is different. According to pharmacological 1 increasing
------------_t
[Et]

receptor theory, rightward shift and depression of agonist

curves depends on the presence of amplification between
receptor occupancy and the measured effect, that is,
receptor reserve, and the progressive removal of this
amplification as receptor concentration is lowered. In con-
trast, the explanation provided by the two-state model is
independent of post-receptor coupling events.
The effect of the same irreversible antagonist on the
action of an inverse agonist can also be demonstrated (Fig.
5b). In this case, the agonist has preferential affinity for R
(KA*>> K,), so as R*is depleted and R is enriched, KaPPis
Ligand (log,, M)
increasingly influenced by KA. Therefore, the curves
undergo a leftward shift, their mid-points tending
towards this value.
c L=lOO
It is to be noted that, in both cases, the use of the irre 2.0,
versible antagonist is predicted to provide an estimate of
the affinity constant for the receptor form enriched by the
manouevre. Although only illustrated for the R-rich sys-
tem, the same would be true of an irreversible antagonist
on an R*-rich system.

Practicalconsequences of the two-state model

In principle, the predictions of the two-state
model have some important implications for applied
pharmacology.

The use of agonists in receptor classification

According to the model, the intrinsic quantities which
determine affinity and efficacy are the constants KAand Inverse agonist (log,, M)

K,*. However, the expressed affinity and efficacy, as

measured by KaPPand the maximal displacement of fa*,
respectively, are system-dependent quantities because
they each depend, in addition, on the basal R : R* ratio,
namely, L. Therefore, a possible concern is that variation
of L among tissues would alter agonist affinity, efficacy
patterns and potency orders in such a way as to indicate
false differences in receptor types. This would happen, for
Fig. 4. System-dependence of interactions of agonists and inverse ago-
nists. Simulations representing the interaction between an agonist and
an inverse agonist; agonist: KA = 10-s; K,” = 2 X lo-! inverse agonist:
Ks = 10”; Ks*= IO”; [B] = 0; 104; 3 x 10”; 10-S; 3 x 10-5;10-4. The
interaction is simulated in two systems, a: L = 1.O;b: L = 100.c:Schild
plot derived from (a) and(b), measuring concentration-ratios(r) from the
KaPpvalues (0) of the curves. The affinity of the inverse agonist varies
wrth the basal ratio, I.

TiPS - March 1995 (Vol. 16) 9 3

 PRINCIPLES
a important practical attribute. This property may be
Ligand (log,0 M)
b problem.
1 .o
0.6
0.0 4 -0 \ made so long as the quantitative relationships between
-12 -10 -8 -6 -4
Ligand (log,, M)
Fig.5. Irreversible antagonism of agomsts and Inverse agonists.
Simulations representing the action of an antagonist which Irreversibly
alters I in favour of R a: Effects of the alterations in I on an agonist
(K, = 10-6, KA* = lO~‘o, I = 20; 100: 500; 2500; 7500; 20000) and,
b: on an inverse agonrst (K, = 10mg; K,” = IO? I = 0.1; 0.5, 2 5, 12.5,
37.5; 100)acting In different systems The nghtward shift and depression
of curves for the agonist accords with pharmacological experience of
irreversible antagonism. The model predicts that the same irreversible
antagonist will left-shift and depress the curves for an inverse agonist.
example, if the two-state model allowed a change in
potency orders to occur between tissues despite the
values of K, and K,* being constant for each agonist.
The two-state model does not predict such behaviour.
Although absolute changes in affinity and efficacy values
are allowed, the relative values obey a relationship simi-
lar to the predictions of pharmacological receptor theory
(Fig. 2). Clearly the pattern of expressed affinities and effi-
cacies change between the tissues, but in a way which
would be expected on the basis of different receptor
reserves according to conventional thinking.
The use of antagonists in receptor classification
The expected behaviour of interactions between com-
petitive antagonists, agonists and inverse agonists can be
determined (Figs 3,4).
Since the affinity of competitive antagonists is ago-
nist- and system-independent, according to the two-state
model, they are reliable tools in receptor classification. In
particular, the fact that they are predicted to interact with
9 4 TiPS -March 1995 (Vol. 16)
agonists and inverse agonists with the same affinity is an
important, for example, when distinguishing between an
experimental system containing different receptors which
act in opposition from one in which a single kind of recep-
tor mediates agonist effects ‘in both directions’.
The use of inverse agonists as antagonists presents
something of a problem since their estimated affinities are
system-dependent. In systems with high basal R:R*,
inverse agonists will be difficult to distinguish from com-
petitive antagonists. However, amongst such systems,
where their affinity estimates are dominated by K,,
inverse agonists may be used reliably as antagonists. Only
when comparing among systems with widely varying
basal R : R”, does variable affinity estimation represent a
Quantitative analysis of agonist action
Implications for drug design
According to the model, affinity and efficacy estimates
for agonists will be necessarily correlated. This suggests
that there may be problems in the analysis of struc-
ture-activity data for agonists if, on the basis of pharma-
cological receptor theory, the assumption is wrongly
made that their expressed affinities and efficacies are
independent. However, useful interpretations can be
the two measurements are appreciated in terms of the
two-state model.
The key issue here is to access information about
changes in the underlying constants, K, and KA*,from the
changes in size and position of curves (in these examples
the same changes in K, or K,* in two systems with dif-
ferent basal RR” are studied). In practice, it is likely that
a single cell or tissue type is used at any one time to pre
vide structure-activity data but it is important to consider
the possible changes in interpretation that could be made
when different assays are used (in each example, a set of
agonists is studied).
When K, decreases at constant K,,\*,two things happen:
the relative affinity of the agonist for R* decreases and so
efficacy falls; the absolute affinity for R increases so K,,PI’
decreases, as indicated by a leftward shift of the curves
(Fig. 6). In an R-rich system, the Kc,PPfor agonists is
strongly influenced by K,, so reduction of K, causes a
pronounced leftward shift (Fig. 6a). In a system rich in R’,
the leftward shift is not so marked (Fig. 6b). In both cases,
the trend in the Kapp as the curves reduce in size is to-
wards the K,. Since, when the curve is completely flat, the
ligand is a competitive antagonist, this asymptotic value
of KaPPis also K,*.
Therefore, reduction of K, amongst a series of agonists
results in a decrease in efficacy but an increase in affinity.
When K,* increases at constant K,, the affinity of the
agonist for R” decreases, so efficacy falls; the influence of
K,\* on overall affinity is reduced so KaklPincreases, result-
ing in rightward shift of the curves. In the R-rich system
(Fig. 7a), the influence of K,” on KaFPis less than in the
 PRINCIPLES

-
a a
1.0 1.0 -
1
0.8 -

0.6 0.6 -
> C.F
0.4 0.4 -

0.2

0.0 0.0 1
-12 -10 -8 -6 -4 -12 -10 -8 -6 -4
Ligand (log,,, M) Ligand (log,,, M)

b b
1.0

0.8 0.8

0.6
>
0.4

0.2 0.2 -
1
0.0 L- 0.0 !
-12 -10 -6 -6 -4 -12 -10 -8 -6 -4
Ligand (fog,, M) Ligand (log,, M)

Fig. 6. Dependence of agonist activity on K,. Simulation
of agonists which differ only in their K, values, acting
which differ in I;K, = 1OF; 3 x 1lF; lo-‘; 5 X IV;
K,"= 1O-8; a: 1.= 20; b: L = 1.O With decreasing K,,the
increased affinity and reduced efficacy. These effects
nounced when L islarger
depicting a set Fig. 7. Dependence of agonist activity on K,".Simulations depictmg a
in two systems set of agonists which differ only in their KA*values, acting in two sys-
2 X lO@; 1tF; tems which differ in I;K, = 10-S; K,"= 3 X IF; 104; 3 x 10-7. 10-7,
agonists display 3 x 1CH; 111”; a: L = 20;b:1.O. With increasing K,",the agonists dls-
are more pro- play reduced affinity and efficacy.

R*-rich system (Fig. 7b), so the rightward shift ac- Estimation of KA and KA*fromaffinity and
companying its reduction is less marked in the former efficacy measurements
case than in the latter. As above, the trend in KaPPin both According to the two-state model (Box 1 Fig.) the loca-
cases is to the value KA= KA*. tion of the f,*/log[Al curve, KaPP,the basal value of ja*,
Therefore, increasing K,* among a series of agonists and the maximal value of fa*, are each defined in terms of
results in synchronous fall in efficacy and in affinity. 1, K,* and K,. The equations are:
When K, and K,* are varied equally, since the ratio
between the two constants remains the same, efficacy K app = K,*O + U/U + L K,*IK,) (1)
remains constant (Fig. 8). The only change is in Kapp.More-
over, the same relationship exists between the curves at basal = l/(1 + L) (2)
different basal ratios of R : R*.
These examples indicate that a certain degree of inter- max = l/(1 + LKA*/KA) (3)
petation of structure-activity relationships can be derived
from pattern recognition. Chemical changes that lead to From these relationships it can be shown that:
changes in K, or KA* alone are relatively straightforward
to recognise and distinguish from each other (Figs 6, 7). KaPP= KA* maxlbasal (4)
Chemical alterations that lead to absolute changes in both
constants but which don’t affect their ratio, can also be Since Kapp, max and basal are each measurable
detected (Fig. 8). quantities, KA* can be calculated from Eqn 4. L, in
However, in order to obtain more quantitative infor- principle, can be estimated from basal (Eqn 21,so KA can
mation on KA and KA*, the affinity and efficacy estimates then be calculated from max (Eqn 3) (see Fig. 9,
need more analysis. Table 1).

TiPS - March 1995 (Vol. 16) 9 5

 when it can be assumed that the measured agonist effects
are proportional tofa*, for example, when a set of agonists
displays overt partial agonist profiles, or when the effect
measurement is proximal to the receptor. If the measured
effect is indirect, experimental steps may need to be taken
to eliminate the complication of amplification. Third,
once these conditions are satisfied, the upper limit of the
effect ( fR*) scale must be known, and it must be possible
to measure HUZX values of individual ligand’s curves as
fractions of that value. In practice this requires a full ago-
nist to be available. Such an agonist, according to the two-
state model, is one which can produce virtually complete
conversion of receptors into R*. The analysis of full ago-
-10 -8 -6 -4
nists themselves, for which it is impossible to measure
accurate fractional max values, requires d manoeuvre
Ligand (log,,, M)
which can change L and so reduce the extent to which the
agonist can displace R to R*. An irreversible antagonist
Fig. 8. Effect of simultaneous variation of K, and KA* on
agonist activity. Simulation depicting three agomsts which have the acting in this way (Fig. 5a) affords for a full agonist an esti-
same ratio of KAand KA* but different absolute values, acting m two sys- mate of K,. Once K,& is known, KA*can be estimated using
tems which differ in L. K, = 3 X 104;10-E. IV, K,"= 3 X 10-8; 10-8, the equations above.
1O-9; I= 1;20.In both systems, the three agonists demonstrate the same
efficacies and the same relative affinities.
Summary
If the two-state concept of receptor activation is
Therefore, according to the model definitions, esti- accepted as a model for agonist action, then the predic-
mates of the agonist-dependent constants, K, and KA*, tions of this model have to be accepted. It follows that
are accessible from measurements of agonist affinity and interpretation and analysis of data obtained in systems
efficacy. In turn, this allows meaningful chemical inter- believed to operate by this mechanism must take into
pretation to be made from affinity and efficacy measure- account these predictions.
ments, even though these measurements do not them- Obviously, the simple model that has been used does
selves constitute the relevant information. not describe the whole process of agonism, either for
In practice, the analysis of agonists in this way is sub transmitter-gated ion channel or G protein-coupled recep-
ject to certain conditions. First, the measurement of basal tor systems. For example, it neglects the mechanistic com-
and, therefore, L, requires that an inverse agonist is avail- plexities associated with interaction between R* and AR*
able for the receptor under study in order to scale the and G proteins. Nevertheless, due to its simplicity, the
degree to which the baseline is elevated above the true model provides a valid starting point for assessing how
zero. Second, it must be recalled that, technically, the the two-state concept explains observations.
equations derived here represent fractional receptor Many of the phenomena which occur in pharmacology
changes ( fR*). They may be applied under conditions and which have been explained in terms of pharmaco-
logical receptor theory can be explained by the two-state
model. These include, for example, the tendency for a
ligand to behave as a partial agonist in one experimental
system and as a full agonist in another, and the effects of
irreversible antagonists on agonist-concentration effect
curves. An important property of the two-state model is
that such variations in the expression of agonist efficacy
are explained without the need to invoke the existence, or
variation, of receptor reserve. According to the model,
such phenomena are explained at the receptor level. This
is a fundamental difference between the two-state model
and traditional receptor theory, since, in the latter, a recep-
tor reserve is an obligatory assumption.
-10 -‘8 -6 -4 The two-state model also requires us to think differ-
Ligand (log,, M) ently about the meaning of agonist affinity and efficacy
and the relationship between them. The mechanistic def-
Fig.9. Estrmation of KAand K,"from affrmty and efficacy measurements. inition of both in terms of two underlying affinity con-
Simulation depicting two agonists acting in the same receptor system; stants means that they are correlated, and not indepen-
L = 4.0. Agonist 1. KA = 10-g M; K,"= IO-10 M Agonist 2: K,.= 10-EM;
&* = 3 X lo4 M.
dent properties as assumed in pharmacological receptor
L theory. This does not present problems in receptor

9 6 TiPS - March 1995 (Vol. 16)

 PRINCIPLES

redefinition of affinity and efficacy to be taken into

Table 1. Estimation of KAand K,* values
account. The affinities and efficacies expressed by ago-
Agonist 1 Agonist 2 nists need to be analysed into their component parts, the
two affinity constants, in order to make sense of the struc-
Measurements tural determinants of agonist activity. In this sense,
KaPP 3.63 x low 1.37 x IO-’ according to the model, affinity and efficacy must be con-
basal 0.2 0.2
sidered as measurements of agonist activity rather than
max 0.71 0.89
the intrinsic properties of agonists.
Calculations
K,"= K,,,.basaifmax(Eqn 4) 1.02X IOW 3.08X IO-8
Selected references
1 de1Castillo, J. and Katz, 8. (1957)Proc. R. SW. London Ser. R 146,
L = (1- basal)lbasal(Eqn 2) 4.0 4.0
369-381
2 Pallotta, B. S. (1991)FASEBJ. 5,203s2043
K,,
= KA*L max/(l
- max)(Eqn 3) 9.98 x IO-10 9.97 x 10-T 3 Stephenson, R. P. (1956)Br. I. Phmtuxo/. 11,379-393
4 Furchgott, R. F. (1966)in Advancesin Drug Research (Vol. 3) (Harper,
with the true values of K,and K,"used in the simulations
Comparison N. J. and Simmonds, A. B., eds), pp. 21-55, Academic Press
shows that these parameters can be estimated accurately from the 5 Black,J. W. and Leff, P. (1983)Proc.R. Sot. London Ser. B 220,141-162
curves. 6 Colquhoun, D. (1987)in Perspecfives on Recepfor CInssificatior~(Black,
J. W., Jenkinson, D. H. and Gerskowitch, V. P., eds), pp. 103-114,
Alan Liss
7 Costa, T. and Herz, A. (1989)Proc. NutI Acad.Sci. USA 86,7321-7325
Acknowled@ments
8 Costa, T. et al. (1992)Mol. Pharmcol. 41,549-560
classification, since the patterns of agonist activity 9 Samama, I? et al. (1993)1. Biol. Chem. 268,4625-4636 The author would like to
thank his colleagues Mark
predicted by the two-state model agree with traditional 10 Chidiac, P. et al. (1994)Mol. P/uzrmaco2.45,49&499 Robertson and lain
expectation and so do not throw up any interpretational 11 Samama, P. etal.(1994) Mol. Phmzacol. 45,390-394 Dougall for thw helpful
12 Monod, J. et al. (1965)J. Mol. Biol. 12,88-118 dlscusslon and
clashes. However, meaningful analysis of agonist ac- 13 Colquhoun, D. (1973) in Drug Receptors (Rang, H. I’., ed.), ~0nStr~tlve
critlclsm of
tivity requires the quantitative, as well as conceptual, pp. 149-181,Macmillan thn man”scr,Pt

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Tip.5 - March 1995 (Vol. 16) 9 7