THE OF CHEMISTRY Vol. 255, No. 21, Issue November 10, pp ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 21, Issue of November 10, pp. 10144-10156, 1980 Printed m U.S.A.

The Relationship between Agonist Occupation and the Permeability Response of the Cholinergic Receptor Revealed by Bound Cobra a- Toxin*

(Received for publication, May 2, 1980, and in revised form, July 7, 1980)

Steven M. Sine and Palmer Taylor From the Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92093

The decrement in functional capacity of the nicotinic receptor on intact BC3H-1 cells has been simultaneously compared with the fractional occupation of the receptor by cobra a-toxin. A parabolic, concave inward relationship between the fractional occupation of receptors by a-toxin and the decrement in permeability response is observed when the latter is tested over a range of agonist concentrations. Since a-toxin binding appears equivalent at each site on the receptor, the observed relationship is accommodated by a model where activation of a permeability response requires agonist occupation of two toxin-binding sites per functional receptor. Furthermore, the binding of a-toxin and agonist appears to be mutually exclusive, but occupation of either of the two sites by a-toxin is sufficient to block the functional capacity of the receptor. Con- sistent with this model, when a major fraction of sites is occupied by a-toxin, the concentration dependence for either carbamylcholine-mediated activation or desensitization of the remaining functional receptors is not detectably altered and retains positive cooperativity. In contrast, progressive occupation of the available sites by a-toxin leads to a decrease in apparent affinity and a corresponding loss of positive cooperativity for agonist occupation functions generated upon instantaneous or following equilibrium exposure to the agonist. A t high degrees of fractional occupancy by a- toxin, where the dominant species capable of binding agonist would contain a single bound toxin molecule, the Hill coefficient for the equilibrium occupation function for full agonists falls from a value of 1.4 to 0.7. By contrast, the binding isotherms for antagonists which typically exhibit values less than 1.0 are not altered following fractional irreversible occupation by a-toxin. Thus, the two binding sites on the receptor oligomer are not intrinsically equivalent for the binding of agonists and reversible antagonists. A scheme for desensitization of the receptor is presented which incorpo- rates both nonequivalence in the two agonist binding- sites and the maintenance of symmetry in the receptor states undergoing transitions.

The action of acetylcholine at the motor endplate of verte- brate skeletal muscle produces a rapid activation of many individual ion channels in the cell membrane (1,2). When the

* This work was supported by Grant GM 24437 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

agonist concentration is maintained, the number of active receptor channels slowly declines; the latter phenomenon is termed desensitization (3). Activation of the receptor results from agonist association with a low affinity, resting state of the receptor while the comparatively slow development of desensitization parallels the appearance of a state of the receptor with an enhanced affinity for the agonist (4). Voltage clamp and tracer ion flux studies of the rapid activation process have provided clear evidence that binding of 2 or more agonist molecules to the receptor is required to open the ion channel (4-8). Similarly, recent studies of desensitization in cultured muscle cells reveal that occupation of the receptor by at least 2 agonist molecules must be considered to account for the concentration dependence of agonist binding and desensitization (4). Thus, through apparent cooperative interactions, both activation and desensitization of the receptor are regulated over a narrow range of agonist concentrations. Al- though several molecular mechanisms can account for cooperativity in activation and desensitization, it is not known whether cooperativity occurs in agonist binding, through interactions between equivalently occupied receptor subunits, or at both levels.

Because elapid a-toxins share a common binding domain with agonists, they are particularly valuable probes for determining the degree of occupation of the receptor by agonists (9, 10). Moreover, since fractional occupation of the receptor by a-toxin results in a random distribution of a-toxin bound to the available sites on the receptor (11,12), and is practically irreversible, the toxin provides a potential tool for determining the number of toxin-binding sites per functional receptor oligomer. Considered together with the stoichiometric ratio of toxin- and agonist-binding sites, this determination would also reveal the number of agonist molecules required to bind and to activate the functional receptor unit. Specifically, the relationship between the fractional occupancy by a-toxin and the corresponding reduction in functional capacity depends on the number of a-toxin-binding sites per functional receptor and whether hybrids containing bound agonist and a-toxin are formed and are functional. Biochemical studies of purified receptor from Torpedo reveal that the smallest macromolec- ular receptor entity is 250,000 daltons and contains two a- toxin-binding sites (13). If this species or multiples of it represented the receptor unit, several classes of models for the functional receptor arise which yield different predictions for the relationship between the fractional toxin occupancy and the loss of the permeability response.

A system in which occupation of the receptor by a-toxin may be compared with the permeability increase elicited by the agonist is necessary to approach this question. By coupling a determination of fractional a-toxin occupancy with tracer

10144

Ligand Occupation and the Response of the Cholinergic Receptor 10145

ion flux techniques, it is possible to measure simultaneously occupation by a-toxin and the permeability response from receptors on the same cell. However, the precise measurement of the fractional activation of receptors by radioisotopic meth- ods requires that the rate of tracer ion flux be proportional to both the number of receptors activated and the duration of exposure to the agonist. Thus, an ideal experimental system would have a low permeability to the test ion in the absence of agonist, a homogeneous population of enclosed compart- ments with a narrow size distribution, and large internal volumes enclosed by a membrane containing a comparatively low density of surface receptors. These factors would enable one to measure a unidirectional flux of ions into the cell prior to equilibration of the tracer ions with the internal volume. Furthermore, conditions which limit desensitization of the response ensure that a measurement of the permeability change is linear with time. These criteria can be largely met by employing the mammalian clonal muscle cell line, BC3H- l ( 1 4 ) .

In the present study, the fractional occupation of the receptor by a-toxin is compared with the capacity of the receptor to elicit a permeability increase to sodium ions in the presence of the agonist. In addition, following a fixed degree of irreversible occupation by a-toxin, parameters associated with activation and desensitization are compared with those for agonist occupancy of the low and high affinity states of the receptor. Several models relating toxin occupancy of the receptor with the functional capacity for activation of the permeability change are considered, and only one is consistent with the experimental findings.

EXPERIMENTAL PROCEDURES

Materials-The BC3H-1 cell line was kindly provided by Drs. Patrick and Boulter of the Salk Institute. Dulbecco's modified Eagle's medium (DME) and Ham's nutrient mixture, F-12, were obtained in powdered form from Gibco and mixed with fresh double-distilled H20. Fetal calf serum was from Reheis and horse serum was from Gibco. All drugs were obtained from Sigma Chemical Co. with the exception of suberyldicholine, which was a gift from Drs. R. B. Barlow and G. Ungar of the University of Bristol. Radionuclides, l r s I and "Na+, were purchased from New England Nuclear. Pure cobra a-toxin was prepared from Naja naju siamensis venom (15) obtained from Ross Allen Reptile Inst., Inc., and cobra a-monoiodo toxin was prepared and separated from noniodinated and diiodo species by isoelectric focusing as described previously (11).

Cell Cultures-Stock cultures of BC3H-1 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere of 12% Cor, 88% air, at 37"C, and maintained in the log phase of growth by subculture every 5 days. Experimental cultures were seeded from stock cultures as described previously (4). In the present experiments, optimal growth conditions were achieved with a medium containing a 3:1 mixture of Dulbecco's modified Eagle's and Ham's F12 media supplemented with 8% fetal calf serum and 2% horse serum. The resulting differentiated cells adhere tightly to the dish and produce highly uniform quantities of surface a-toxin-binding sites (1 to 1.5 pmol/dish).

Kinetics of '""Ilabeled a-Toxin Binding an,d Competition with Cholinergic Ligands-Experiments were performed a t either 21" or 35°C in a temperature-controlled room. A depolarizing buffer containing 140 mM KCI, 5.4 mM NaCI, 1.8 mM CaC12, 1.7 mM MgS04, 1 mM Na2HP04, 5.5 mM glucose, 25 mM (4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid, 0.06 mg/ml of bovine serum albumin, ad- justed to pH 7.4 with 11 mM NaOH was employed for both the a- toxin-binding and "Na+ flux determinations as described previously (4). In brief, identical cultures were rinsed free of growth medium and incubated in the presence or absence of a specified concentration of cholinergic ligand for 30 to 60 min prior to the competition assay, which was initiated by replacing this solution with an identical solution containing a stoichiometric excess of '"I-labeled a-toxin (typically 10 to 20 nM). After a prescribed interval (0.5 to 1.0 min), free radioligand was thoroughly removed by three 6-ml washes (or five 6- ml washes for experiments a t 3.5"C). The rate constant for toxin association, k ~ . was determined from the resulting specific binding (4)

and initial free toxin and receptor concentrations according to the bimolecular rate equation (4). The dependence of k~ on competing ligand concentration was analyzed empirically by the method of Hill (16) to determine the concentration of free ligand which reduces k~ by 50%, defined as the protection constant, K,. and the Hill coefficient, n. A linear regression of plots in the Hill formulation was derived from experimental points between 10 and 90% saturation.

Measurement of Permeability to "Na'-As described previously (4), 2zNa+ uptake was measured under conditions identical with those described above for a-toxin binding. However, when either binding or permeability measurements were performed at 3.5"C, adequate reten- tion of the cells on the culture dish was achieved by initially covering cells with assay buffer at 21°C and allowing them to cool slowly to 3.5"C for 30 min. Cells were then suitable for measurements of functional properties of the receptor following application of cholinergic ligands and 22Na+. The permeability increase to sodium ions, k t : ,

was determined according to Equation 1 from the specific uptake, x, measured during the interval, t, and the equilibrium uptake into the freely exchangeable intracellular volume, m (see Fig. 1 and Ref. 4):

m

w - x In - = h(;t (1)

Double Isotope Determination of Receptor Occupancy by '251- labeled a-toxin and Permeability to 22Na'-Progressive occupation of surface receptors by ""I-labeled a-toxin was achieved a t 21°C by incubation of cells with a-toxin for prescribed intervals. After thorough removal of unbound toxin (three 6-ml washes), cells were covered with 1 ml of buffer and cooled slowly at 3.5"C, and the amount of "YNa+ influx elicited by a fixed concentration of carbamylcholine was determined. The resulting cell-associated radioactivity was simultaneously determined for each isotope by using a dual channel y counter (Searle model 1197) with each channel optimized for either "'1 or T2Na+. The small overlap (-10%) of "'Na+ counts to the "'1 channel was corrected for in each lZ5I determination.

RESULTS

Since the subsequent experiments rely on measurements of receptor function at 3.5"C to minimize desensitization, we initially compared activation and occupation parameters at low temperature with those established at 21°C (4). A critical requirement for determining the relative number of receptors activated by the agonist is to establish conditions in which the permeability increase is detectable yet does not desensitize during the assay interval. Furthermore, calculation of the permeability increase from the rate of agonist-induced L'2Na+ uptake requires an understanding of the mechanism of the permeability change. When receptor channels open, sodium is free to diffuse from extracellular space into the intracellular compartment. Permeability, kc;, is then described by Equation 1 for a fist order exchange process. In this expression, calculation of the permeability change from the rate of tracer "Na+ uptake requires the correct assignment of the equilibrium uptake, 00, corresponding to the intracellular volume in which free exchange occurs.

Kinetics of Carbamylcholine-stimulated Uptake of "Nac at 3.5'C-Carbamylcholine stimulates a highly specific ac- cumulation of 22Na+ which may be resolved into two distinct kinetic components (Fig. 1). As documented previously for BC3H-1 cells a t 21°C (4), the dominant and rapid component seen here a t 35°C represents f i i n g of an intracellular compartment, freely exchangeable with the external pool of 2PNa+, while the slow component reflects exchange between this pool and a secondary, less accessible one. Elimination of the contribution of the slow component of uptake permits the kinetic analysis of the rapid phase shown in Fig. 1 (inset). When tracer 'jNa+ influx is monitored upon instantaneous or following equilibrium exposure to carbamylcholine, the kinetics can be described by a single rate constant for at least 45 s. Thus, at 3.5"C, the permeability increase determined in the presence of 100 ,UM carbamylcholine yields a reliable measure of the relative number of receptors in the active state, since desen-

10146 Ligand Occupation and the Response of the Cholinergic Receptor

O 1 2 3 TlME(min)

TIME (min)

FIG. 1. Time dependence of "Na+ influx elicited by 100 p~ carbamylcholine at 35°C. Cells were rinsed and cooled slowly to 3.5OC as described under "Experimental Procedures" and incubated for 30 min in the presence or absence of 100 p~ carbamylcholine. Influx of tracer 22Na+ elicited by 100 p~ carbamylcholine was monitored for the durations indicated U, influx in the presence of carbamylcholine when cells were previously incubated in buffer for 30 min; H, influx in the presence of carbamylcholine when cells were previously exposed to 100 p~ carbamylcholine for 30 min; A-A, influx in the presence of carbamylcholine monitored in cells initially incubated with a-toxin (0.5 p ~ ) for 20 min at 21°C prior to cooling to 3.5'C. Inset, first order plot of specific influx where the contribution of the slow phase of uptake has been eliminated providing kinetics of '*Na+ exchange between extracellular and freely exchangeable intracellular volumes. Analysis of the kinetics of rapid influx was according to Equation 1, where x is the influx determined in the interval, t , and m is the equilibrium influx value corresponding to the capacity of the freely exchangeable internal volume (61% of uptake achieved in 4 h). In this experiment, cultures contained 1.10 pmol of a-toxin-binding sites per 35-mm dish.

sitization is minimized and an initial rate for a unidirectional flux of 22Na+ can be monitored prior to equilibration of the tracer in the freely exchangeable volume of the cells. However, when saturating concentrations of agonist (i.e. 1 mM) are approached, the initial permeability increase is no longer described by a single rate constant even a t 3.5"C. As shown subsequently, this is largely a consequence of the enhanced rate of desensitization.

Comparison of the Kinetics of Carbamylcholine-induced Desensitization a t 21 ' a n d 3.5"C-While a temperature of 21°C is satisfactory for measurements of agonist occupation and receptor activation upon instantaneous addition of low and intermediate concentrations of agonist (4), in the concentration range approaching saturation, desensitization occurs at rates that exceed the temporal resolution of a manual assay. Hence, when it is necessary to measure a state function for activation or a binding function generated upon the instantaneous addition of agonist, deviations from ideal behavior are anticipated. Since low temperature is reported to decrease the rate of desensitization (17), the kinetics of desensitization onset promoted by several concentrations of agonist was compared at 21" and 3.5"C (Fig. 2). The relative number of active receptors present following various durations of exposure to the test agonist concentration was determined from the rate of ""a' uptake elicited during a short term (15-4 incubation with 100 PM carbamylcholine. The kinetics of desensitization for all three concentrations of carbamylcholine reveals a marked decrease in rate at 3.5"C. Furthermore, two components in the onset kinetics are apparent for all agonist concentrations at 3.5"C, with the amplitude of the more rapid phase in greater proportion with increasing agonist concentration. It is possible that two components are present at 21"C, but the temporal resolution of the measurements is not sufficient to detect them. In addition, the low temperature condition results in a smaller degree of equilibrium desensitization a t low agonist concentrations, but it becomes comparatively greater

in the concentration range near saturation. As considered in more detail subsequently, this trend appears due to both a higher dissociation constant for the desensitization process and a slower rate of desensitization a t 3.5"C. Thus, at agonist concentrations near the dissociation constant for activation, incubation a t 3.5"C reduces the development of desensitization during the assay interval by as much as 10-fold, permit- ting an accurate measure of the agonist-mediated permeability change (cf. Table I). In the following low temperature experiments where binding and state functions are determined following prolonged agonist exposure, time was sufficient for both the fast and slow processes to come to equilibrium.

Comparison of Functional and Occupation Parameters for Carbamylcholine at 21" a n d 3.5"C-In our initial study, simultaneous comparison of parameters associated with activation, desensitization, and occupation of the receptor led to the consideration of a two-state concerted scheme to describe the slow desensitization process (4):

KH KX L + RR -- RLR + L + RLRL

L i R R ' -- R'LR + L -- R L R L M 1 1 1

KW KH. SCHEME 1

In this scheme, the agonist ( L ) rapidly associates with the receptor in either the low ( R ) or high (R') affinity state in a noncooperative fashion. However, when transitions in state are perfectly concerted, a ligand with a preference for the R' state will exhibit homotropic cooperativity in both the equilibrium binding and state functions (18, 19). In this section,

D E F

TIME(mln)

FIG. 2. Concentration and temperature dependence of the kinetics of carbamylcholine-mediated desensitization. In each panel, the relative permeability increase elicited by 100 p~ carbamylcholine is plotted against the duration of prior exposure to the specified concentrations of agonist. Experiments were performed at 3.5"C ( A to C) or 21°C (D to F). Cells were incubated in buffer at 21" or 3.5"C for 30 min and further incubated in the presence of carbamylcholine at the concentrations indicated. After the interval specified on the abscissa, the conditioning solution was removed and a test solution containing 100 p~ carbamylcholine and tracer "Na+ was immediately applied to the cells for 15 s for the measurement of the initial rate of sodium influx. The resulting permeability change, hr;, was calculated as described in the legend to Fig. 1 and is expressed relative to h ~ , determined in the presence of 100 p~ carbamylcholine in cells exposed to buffer alone. In this experiment, the capacity of the freely exchangeable internal volume, m = 10,838 dpm and k w (21°C) = 2.12 X lO-'s-' and k ; ~ (3.5OC) = 2.07 X 10-'s-'. Cultures contained 1.20 pmol of a-toxin sites per 35-mm dish. The curues represent exponential fits to the rapid phase of desensitization: at 3.5OC, the apparent half-lives for the fast kinetic component of desensitization of the permeability response are: t1/2 = 51 s (100 p ~ ) , t l I2 = 116 s (60 p ~ ) , tIr2 = 86 s (30 p ~ ) . At 21"C, tlrL = 18 s (100 p ~ ) , i!l/? = 43 S (60 pM), t l , ~ = 55 S (30 pM).


TABLE I Comparison of parameters for carbamylcholine occupation,

activation, and desensitization of the receptor a t 21" and 3.5OC Temperature

21°C 3.5"C -

K (inst)" (M) 7.01 X 1.48 X 10.' (1) n' 1.23 f 0.14 (3) 1.43 f 0.16 K, (equil)" ( M ) 1.76 f 0.46 X 4.06 X lo-:' nh 1.39 f 0.07 (4) 1.42 f 0.11 (1)

KactCi ( M ) 7.14 X 1.18 f 0.10 X n 1.51 & 0.07 1.70 & 0.02 (2)

( M ) 1.78 X 3.31 X lo-:' ne 1.74 f 0.08 (') 1.66 f 0.09 (1)

R E G ' 0.19 f 0.04 (2) 0.11 f 0.03 (3) JmaJ (Na' ions/s. 6.08 f 1.7 X 10' (3) 1.14 f 0.26 X IO4 (3)

recentor) ~

" K, (inst) is the concentration of carbamylcholine which decreases the initial rate of '251-labeled a-toxin binding by 50% when carbamylcholine and a-toxin are added simultaneously and the rate of binding is determined in a 30-s interval. Values are expressed together with the standard deviation for the number of determinations given in parentheses.

Hill coefficients associated with competition or activation isotherms are determined as described under "Experimental Proce- dures.'' Values for single isotherms are expressed together with the standard error of the mean slope of the regression line for data formulated in the Hill equation. Where multiple isotherms were available, mean values are listed with the standard deviation.

'' KJequil) is the concentration of carbamylcholine which decreases the initial rate of '"1-labeled a-toxin binding by 50%' when cells are incubated for 30 (21°C) or 60 min (3.5OC) in the presence of carbamylcholine prior to measuring the rate of a-toxin binding.

"K,,, is the concentration of carbamylcholine which elicits a half- maximal increase in the permeability to sodium ions determined from the concentration dependence of the initial rate of "Na+ influx measured in a 15s interval.

KdeS is the concentration of carbamylcholine which produces a half-maximal reduction (170 - R,)/2 of the control permeability response following equilibrium exposure (30 min for 2loC, 60 min for 3.5"C) to agonist. This-valye is determined from the empirical Hill expression: log[(& - R)/(Ro - k ) ] = log L - nlog K d e s . I%, is the maximal permeability increase elicited by carbamylcholine (1 mM). R , is the relative permeability increase elicited following incubation with saturating concentrations of carbamylcholine; i? is the permeability increase elicited following incubation-with intermediate concentrations of agonist (L), again relative to Ro; and n is the Hill coefficient.

'Jmu is the rate of sodium ion influx per receptor unit elicited by a saturating concentration of carbamylcholine (1 mM). This value is calculated from the permeability increase, kc, which is determined from the initial rate of "Na+ influx elicited by carbamylcholine in a 15-s interval. The total number of receptors is calculated as one-half of the measured number of a-toxin sites on a paired culture dish (see Model 3). Note that JmaX corresponds to an external assay buffer solution containing a low concentration of sodium ions (18.4 mM) and the membrane potential approaches zero.

parameters of receptor function and occupation by carbamylcholine are presented for the 35°C incubation condition (Ta- ble I) and are evaluated in light of Scheme 1.

Receptor Actiuation-Upon short term exposure to the agonist, the concentration dependence of the change in permeability reflects a condition in which resting and active states of the receptor are in equilibrium. Since, at 2l0C, the rate of desensitization increases with agonist concentration and at saturation can exceed the temporal resolution of the assay, the measured dissociation constant (Kact) and Hill coefficient (n) likely provide lower limits for the true values. Thus, at 3.5"C, when desensitization is greatly reduced within the assay interval, both parameters are found to increase (Table I). This could also be a consequence of the temperature dependence of the occupation parameters in Scheme 1 or parameters associated with activation of the receptor channel.

Interestingly, the rate of "Na+ influx elicited by a saturating concentration of carbamylcholine, J,,,, is also enhanced at 35°C. In the present system, this parameter should depend on the temperature sensitivity of the mean channel lifetime ( T ) , the single channel conductance (y) , and the rate of desensitization. Since, in mammalian systems at low temperature, y decreases by at least as much as 7 increases ( Z O ) , a smaller rather than a larger rate of influx would be expected if only activation properties of the channel are considered. Therefore, the slower rate of desensitization should largely account for the observed enhancement in measured maximal flux rate.

State Function for Desensitization-The concentration dependence of agonist-induced desensitization reflects the equilibrium between the active and resting (low affinity) and the desensitized (high affinity) states of the receptor. As represented in Scheme 1, the state function for desensitization ( R ) is determined by the ratio of low and high affinity dissociation constants (KRIKK.), the allosteric constant ( M ) , and the number of interacting subunits (n = 2):

I: species in R state - 1

' + " ( l + L / K x )

R = I: total species

- 1 + L/Kx.

(2)

Experimentally, R is determined from by the maximal permeability increase elicited immediately following prior equilibrium exposure to a specified concentration of agonist. Since low concentrations of agonist produce only a slight degree of desensitization, and near saturation, desensitization a t equilibrium is maximal, a differential amount of desensitization can occur in the subsequent assay interval. If so, a t 21"C, the observed dissociation constant for desensitization ( Kdes) and the maximal fractional desensitization (E,) provide lower and upper limits, respectively. Consequently, the low temperature condition reveals an anticipated increase in Kd,. and a decrease in R, (Table I). However, the temperature dependencies of KR, KK., and M have not been analyzed independently.

Occupation of the Low Affinity State of the Receptor-The correlation between the dissociation constant for occupation of the receptor generated on short term exposure to the agonist (KJinst)) with K,,, for the permeability increase suggests that at 21"C, activation proceeds from the low affinity state of the receptor. In addition, a Hill coefficient for the agonist occupation function ( yiinst) greater than unity was observed. At 21"C, the experimental determinations again provide a minimum KJinst) and an overestimate of n because the rate of conversion of the receptor to the high affinity state increases with agonist concentration. At 3.5"C, K,>(inst) increases, and its close correspondence with KaCt (Table I) reinforces the previous interpretation that binding of agonist to the low affinity state and activation of the receptor are closely coupled. Positive cooperativity in yht persists when desensitization is minimized a t 3.5'C, suggestive that an appreciable fraction of the low affinity resting receptor can be drawn into the active state.

Occupation of the Receptor a t Equilibrium-In contrast to the instantaneous measure of agonist binding, exposure of the receptor to the agonist prior to the addition of '2sI-labeled a- toxin allows the low (resting and active) and high affinity (desensitized) receptor states to come to equilibrium. Thus, no systematic deviations in the occupation function ( yeqUil) should be evident. In addition, prior exposure to the agonist provides conditions identical with those prevailing just before the determination of the state function for desensitization ( R ) . Experimentally, yequd closely parallels R and both functions exhibit homotropic cooperativity. At 3.5"C, retains the original degree of cooperativity, and the corresponding dissociation constant, K,(equil), increases about 2-fold, again in


TABLE I1 Multisubunit models for a-toxin occupancy and functional

antagonism A similar analysis has been employed to examine the relationship

between a-toxin occupancy and covalent labeling by a sulfhydryl reactive antagonist (28).

Model Receptor species capable of activation

1 <' coco00

If the subunits in the dimer (Models 1 and 2) or tetramer (Model 5) were identical, then the species denoted as containing a single bound toxin molecule would be equivalent in each model.

Differs from Model 1 in that the hybrid species can elicit 0.5 times the conductance increase of the original species.

close accord with the value K d e s for the state function (Table I).

Antagonism of Carbamylcholine-induced Increase in Permeability to 22Na+ as a Function of the Fractional Oc- cupation of Receptors by a-Toxin-Several hypothetical models are considered which predict distinct relationships between the fractional occupancy of receptors by a-toxin ( y ) and the permeability response to carbamylcholine, k c (Table 11). In each model, the functional receptor is considered to be an oligomer of individual a-toxin-binding sites whose occupation by toxin is competitive with agonist binding. In addition, we assume that a-toxin binds to all available sites in a random distribution and its binding is irreversible for the duration of the experiment. In the following models, k p is the permeability change elicited by the agonist in the absence of prior a-toxin occupation.

Model 1-Functional receptor contains two binding sites and full activation results when one or two binding sites are occupied by agonist.' Occupation of both sites by toxin is required to block function.

.. ' kc; = kr:"(l - y2).

Model 2-Functional receptor contains two binding sites and activation results when one or two binding sites are occupied by agonist. Occupation of each site by a-toxin reduces the response capacity of that receptor by one-half.

:. hr: = k w ( 1 - y).

Model 3-Functional receptor contains two binding sites and activation requires occupation of both binding sites by agonist. Occupation of either site by a-toxin will completely block function.

:. kc = kou( 1 - y)?

Model 4-Functional receptor contains four binding sites and activation requires occupation of all four binding sites by agonist. Occupation of any one site by a-toxin will completely block function.

. . ' k c = hny( 1 - y)4.

Model 5-Functional receptor contains four binding sites and activation requires occupation of three or four binding sites by agonist. Occupation of more than one site by a-toxin will completely block function. Occupation of only one site

' In Models 1 to 5, the functional receptor is considered to possess an equal number of agonist- and toxin-binding sites (see under "Dis- cussion").

has no inhibitory effect.

:. kc = koo[(l - y).J(3y + l)].

To achieve progressive occupation of receptors by '2sI-labeled a-toxin, cells were incubated for various durations at 21°C with a-toxin and washed free of unbound radioligand. Upon incubation at 3.5"C, cells whose receptor sites were fraction- ally labeled with toxin were examined for their functional capacity by measuring the initial rate of z2Na+ uptake elicited by a fixed concentration of carbamylcholine. Consistent with our previous observations (4), the linearity in the bimolecular plot for '"I-labeled a-toxin binding shown in Fig. 3 (inset) reveals that a-toxin apparently combines with a kinetically homogeneous population of surface receptors on the cells. Since a-toxin binding kinetics exhibits neither positive nor negative cooperativity, toxin binds to available receptor sites on each oligomer with a random distribution. When cells with receptors partially labeled by a-toxin are examined for the permeability increase elicited by a range of subsaturating concentrations of carbamylcholine, a parabolic and concave inward relationship between the fractional occupation by a- toxin and the corresponding reduction in response is evident (Fig. 4).2 Moreover, close quantitative agreement with the predictions of Model 3 is observed. I t is noteworthy that a small but consistent deviation from Model 3 is observed at large degrees of toxin occupancy. The sigmoid curve for Model 5 approaches that for Model 3 near the maximal degree of toxin occupancy. However, by examining the entire range of fractional toxin occupancy, this model deviates significantly from the experimental data. Hence, a clear distinction between potential models requires a comparison of the functional capacity of receptors over the entire range of toxin occupancy.

This double isotope approach carries the advantage that the same population of cell-associated receptors are examined for a-toxin occupation and response, providing essential internal control. The monoiodotoxin has been purified from the noniodinated and diiodo species by isoelectrofocusing (10) and we estimate that these minor species constitute less than 5% of the total a-toxin. Still, it was necessary to establish that the potentially more rapid binding of trace amounts of unlabeled a-toxin, although not apparent from the kinetics of binding (Fig. 3 ) , was not distorting the profiies seen in Fig. 4. Therefore, an alternative method was employed where paired cultures were treated with unlabeled toxin for prescribed intervals to achieve progressive degrees of occupation of the receptors. Individual cultures were then subjected separately to measurements of agonist-mediated Z2Na+ influx or the initial rate of I2'I-labeled a-toxin binding. From the decrement of the initial rate of '"I-labeled a-toxin binding, kT, the degree of occupancy of receptor sites by unlabeled toxin can be determined for cultures incubated with a-toxin for a known duration and subsequently examined for the agonist-mediated

'When saturating concentrations of carbamylcholine (1 mM) are employed to elicit influx of sodium, the kinetics of the permeability change shows deviations from the linear relationship in Equation 1 even for short duration incubation intervals (10 to 20 s). This could be a consequence of the rapid rate of desensitization ( t l r ~ < 15 s) even a t 3.5"C, or result from appreciable equilibration of tracer "Na+

proaching 6091, of completion in 15 s. Under these conditions, the between external and freely exchangeable internal volumes, ap-

permeability versus a-toxin occupancy profiles where 1 mM carbamylcholine was employed to elicit the test response were variable in several experiments. In two experiments, Model 3 provided an accurate description of the data, while in three others, a parabolic, concave inward relationship was still observed but the data approached the description of Model 2 (linear). Thus, low agonist concentrations and short duration incubation intervals were routinely employed in the permeability measures to minimize the contribution of desensitization and the extent of equilibration of tracer "Na'.


TIME (min)

FIG. 3. Kinetics of association between '251-labeled a-toxin and surface receptors on BCIH-1 cells. Cells were rinsed and equilibrated in assay buffer at 21°C and incubated with 'z61-labeled a-toxin (17.5 n ~ ) for the specified intervals. Nonspecific binding determined in the presence of 3 mM carbamylcholine has been sub- tracted to determine specific binding. Inset, second order plot of specific binding to surface receptors according to the bimolecular rate equation (4). Linear regression of these data reveals a bimolecular rate constant, k ~ , of 1.04 X IO5 M" s". Ceus in this experiment were also examined for functional capacity and actually correspond to the data in Fig. 4C. Therefore, picomoles of a-toxin bound shown on the ordinate reflect '251-labeled a-toxin retained by the cells throughout the binding and subsequent permeability measurements. The kinetics of a-toxin association was routinely examined in all permeability versus a-toxin occupation experiments as shown here, providing a simultaneous verification that a-toxin association with surface receptors is bimolecular and fiactional a-toxin occupation results in a random distribution of labeled sites on each receptor oligomer.

permeability increase. Fig. 5 reveals a parabolic concave inward relationship between fractional occupancy of receptor by unlabeled a-toxin and the reduction in permeability response, which is again in reasonable agreement with Model 3. Since fewer experimental determinations can be generated in a single experiment by this method, somewhat greater varia- bility is apparent. Nonetheless, occupation of receptor sites by either labeled or unlabeled a-toxin yields comparable degrees of functional antagonism. These relationships strongly support the interpretation that the functional receptor contains two a-toxin-binding sites, and occupation of a single site is sufficient to block function.

The finding that occupation of receptors by labeled or unlabeled a-toxin yields the same reduction in the capacity for receptor activation is also dependent on establishing that a decrement of the initial rate of '?-labeled a-toxin binding, k ~ , actually reflects a diminished number of available sites. For example, application of labeled or unlabeled a-toxin to the cells for brief periods could conceivably label immediately accessible surface receptors and not those less accessible he- cause of diffusion barriers. Conversely, application of agonist to determine functional capacity may preferentially activate the hypothetical population of accessible receptors. Such a situation is not evident in the cell culture system, since the reduction of the initial rate of '251-labeled a-toxin binding, kT, produced by prior occupation of receptors by unlabeled toxin, is linearly related to the total number of receptor sites available to '251-labeled a-toxin (Fig. 6). Thus, both labeled and unlabeled a-toxin uniformly occupy the available surface receptors on the cells and the labeling rate is proportional to the number of unoccupied receptor sites.

Functional Properties of the Receptor after Partial Occu- pation by a-Toxin-In Model 3, the nonlinear reduction in the capacity of receptors to activate a change in permeability following progressive degrees of occupation by a-toxin appears due to the inability of receptor oligomers occupied by a single a-toxin molecule to activate the ion channel when the remaining vacant site combines with agonist. Therefore, since the

only receptor oligomers capable of activation would not be modified by a-toxin, the concentration dependency for agonist-mediated activation of the receptor should not be altered following partial occupation of receptor sites by a-toxin. A fixed degree of toxin occupancy was achieved by incubation of cultures for a prescribed interval with unlabeled a-toxin. Following thorough removal of free toxin, the permeability increase elicited by various concentrations of carbamylcholine was examined. Occupation of the majority of the available sites by a-toxin results in no detectable change in the activation isotherm for the residual receptors (Fig. 7a). In one of these experiments where 60% occupancy by a-toxin was achieved, Model 3 predicts that 16% of the receptors would be totally unoccupied by a-toxin and 48% would exist as hybrid toxin-receptor species. Thus, the predominant hybrid species do not elicit a detectable permeability change in the presence of agonist.

FRACTIONAL OCCUPANCY BY a-TOXIN

.40

.20

1 ,005 c Sop M

30p M

100pM

FRACTIONAL OCCUPANCY BY i2*I-a.TOXIN FIG. 4. Reduction of the permeability increase, ko. elicited

by carbamylcholine resulting from progressive occupancy of receptors by "%labeled a-toxin. A, theoretical relationships for the fractional permeability response uersus fractional occupancy of receptors by a-toxin generated from Models 1 to 5 (Table 11): a-.,

Model 1; . . , Model 2; -, Model 3; -----, Model 4; - - -, Model 5. B to D, experimentally determined fractional kc following progressive degrees of occupancy by '251-labeled a-toxin. B, permeability increase, determined from the rate of sodium influx elicited by 30 p~ carbamylcholine in a 75-s interval. C, permeability increase resulting from activation by 60 PM carbamylcholine measured in a 30-s interval. D, permeability increase resulting from activation by 100 p~ carbamylcholine measured in a 15-s interval. In each panel, the solid line corresponds to the function h~ = k ~ o ( 1 - y ) 2 resulting from Model 3, where y is the fractional occupancy of receptors by a-toxin. In each experiment, fractional occupancy of receptors by 'Z51-labeled a-toxin was achieved as detailed in the legend to Fig. 3, and the cells were cooled to 3.5"C for 30 min prior to measuring the initial rate of sodium influx upon addition of the specified concentration of carbamylcholine and tracer '*Na+. The permeability increase, k ~ , was determined as described in the legend to Fig. 1 and is expressed relative to kGn

measured in cells incubated in the absence of a-toxin. In each experiment, a measured equilibrium influx value, m, is employed corresponding to the capacity of the freely exchangeable internal volume as shown in Fig. 1. The fractional occupancy by lZ5I-labeled a-toxin corresponding to each permeability measure isdeterminedias in Figure 3, and a-toxin association examined in each experiment exhibited bimolecularity as shown in Fig. 3 (inset).


TIMEImml

1.0Oh c

FRACTIONAL OCCUPANCY BY a.TOXIN

.40

.20

.20 .40 .60 .80 1.00 FRACTIONAL OCCUPANCY FRACTIONALOCCUPANCY

BY a.TOXIN BY a.TOXIN

FIG. 5. Reduction of the permeability increase, ko, resulting from progressive occupancy of receptors by unlabeled a-toxin. A, time dependency of the reduction in the initial rate, k ~ , of lZ5I-

labeled a-toxin binding following incubation of cells with unlabeled a-toxin for the specified intervals. B to D, permeability increase, kc, elicited by carbamylcholine following progressive degrees of occupancy by unlabeled a-toxin relative to ks measured in cells without prior toxin occupancy. B, permeability increase determined from the rate of sodium influx elicited by 30 p~ carbamylcholine in a 75-s interval. C, permeability increase elicited by 60 PM carbamylcholine measured in a 30-s interval. D, permeability increase elicited by 100 p~ carbamylcholine measured in a 15-s interval. Fractional occupancy of receptors, y , by unlabeled toxin was determined from the measured time of incubation of cells with unlabeled toxin and the reduction in initial rate of '251-labeled a-toxin binding measured in one of the set of paired culture dishes. In A, the curve is an exponential fit to the experimental points with an apparent half-life of 153 s. In B to D, the curves correspond to the function kc = kco(1 - y)' resulting from Model 3. In A, progressive occupancy of receptors was achieved by incubation of cells with unlabeled a-toxin (50 nM) for the specified intervals at 21"C, and tollowing three 6-ml washes, cells were covered with 1 ml of buffer. The initial rate of '251-labeled a-toxin binding, h ~ , was then measured in a 60-s interval as described under "Experimen- tal Procedures." In B to D, occupation of receptors by unlabeled toxin was achieved as in A, and one dish from each paired set was cooled to 3.5"C for the determination of the permeability change, kc, resulting from agonist action as described in the legend to Fig. 4.

A second measure characteristic of functional receptors is the concentration dependence for desensitization, termed the state function, R . It is evident upon inspection of Scheme 1 that the associated state function (Equation 2) is determined by the parameters KR, K E , and M , as well as the inherent requirement of subunit symmetry in the transitions in state. Hence, if partial toxin occupancy yields activatable receptors altered in these parameters or if a receptor occupied by 1 toxin and 1 agonist molecule could open the channel but displayed different activation and desensitization properties, a change in the state function, R , should be evident. As shown in Fig. 7b, occupation of 62% of the sites by a-toxin produces no change in the experimental state function, R. Thus, in accord with Model 3, functional receptors that remain following partial occupation by a-toxin appear unaltered in either their capacity for activation or desensitization.

Occupation of Receptors by Carbamylcholine following

100 kA,

80 -

60 -

40 -

20 -

0-0 FRACTION OFSITES

AVAILABLE TO '*% a.TOXIN

FIG. 6. Decrease of the initial rate, k~ of '261-labeled a-toxin binding as a function of the corresponding loss of total available receptors resulting from progressive occupancy of receptors by unlabeled a-toxin. Paired culture dishes were treated with unlabeled toxin (50 nM) for the specified intervals (inset ( B ) ) . Follow- ing removal of unbound toxin (three 6-ml washes), cells were covered with 1 ml of buffer and one dish of the pair was subjected to an initial rate determination of k~ (60 s), while the other dish was incubated with '*'I-labeled a-toxin (14 nM) for 90 min to determine the total available receptor sites. Inset ( B ) , time dependence of the reduction of kT resulting from unlabeled toxin association for this experiment. The curue is an exponential with an apparent half-life of 115 s.

[CarbamylchollnelM [CarbamylcholinelM

FIG. 7. Effect of prior toxin occupation on agonist-mediated activation and desensitization of the receptor. A, influence of prior fractional toxin association on the concentration dependence of carbamylcholine-mediated permeability increase to "Na+. Cells were washed once with 3 ml of buffer and incubated in the presence ( E " - B ) or absence (H) of unlabeled toxin (100 nM) for 60 s. After removal of unbound toxin (three 6-ml washes), cells were cooled to 3.5"C for 30 min and the initial rate of sodium influx was determined in a 15-s interval in the presence of the specified concentrations of carbamylcholine and tracer "Na+. The resulting permeability change, kc, was determined as described in the legend to Fig. 1 and is expressed relative to the maximal kc elicited by a saturating concentration of agonist. To determine the fractional occupancy of receptors by a-toxin, cultures treated in parallel were subjected to an initial rate measurement, k ~ , of '251-labeled a-toxin binding as described in the legend to Fig. 5. In the experiment shown here, 42% of the available sites were occupied by unlabeled toxin and the maximal permeability increase elicited in these cells was found to be 36% of that measured in control cells. For untreated cells, K,,, = 111 p~ and n = 1.68 f 0.06, and for cells partially occupied by a-toxin, Kact = 106 p~ and n = 1.77 f 0.15. In three separate experiments, 42, 47, and 59% of the available sites were occupied by a-toxin, resulting in a mean K,,, = 102.6 f 11.5 p~ and n = 1.71 f 0.07 (mean f S.D.). B, influence of prior fractional a-toxin association on the concentration dependence of carbamylcholine mediated desensitization. Surface receptors on cells were incubated in the presence (M) or absence ( O " - O ) of a-toxin as described for A and incubated at 3.5"C in the presence of the specified concentrations of carbamylcholine for 50 min. The resulting state function for desensitization was determined by replacing the incubation medium with buffer containing 1 mM carbamylcholine and tracer "Na+ for a 15-s interval. The resulting permeability change, kc, and the fractional occupancy by unlabeled toxin were determined as described for A. In the experiment shown, 62% of the total available sites were occupied by a-toxin and the maximal permeability increase, kc, elicited in these cells was 27% of that measured for control cells. For control cells incubated in buffer alone, K d e s = 33.1 p~ and n = 1.66 f 0.09, and for cells with receptors partially occupied by a-toxin, K d e . = 27.9 p~ and n = 1.76 f 0.23.


Partial Occupation by a-Toxin-Although receptors capable of activation following partial occupation by a-toxin exhibit their original functional properties, a substantial portion of unoccupied receptor sites should retain the ability to bind both a-toxin and carbamylcholine, yet should not elicit activation of the ion channel. From inspection of Scheme 1, which can account for the cooperativity in agonist occupation of receptors at equilibrium ( ye,,,il) and the concentration dependence of equilibrium desensitization (E), it seemed likely that parameters for agonist occupation would be altered if a major fraction of receptor sites that bind carbamylcholine have neighboring sites on the same receptor unit occupied by a-toxin. For example, a vacant binding site paired with a site occupied by a-toxin might be expected to bind agonist with an initial low affinity. However, while the slow conversion to the high affinity state may still occur, the extent of conversion would lack the enhancement provided by subunit symmetry in the state transitions. As a result, the occupation function for carbamylcholine, yeequil, would reveal a gradual increase in the apparent dissociation constant, KJequil), and a loss of cooperativity as receptors are progressively occupied by a- toxin. Thus, in contrast to the native receptor, where the equilibrium state and occupation functions for agonist nearly coincide, following partial occupation of receptor sites by a- toxin, R and yequa should diverge. A fixed degree of occupation by a-toxin was achieved by incubation of cells for an appro- priate interval with unlabeled toxin. After thorough removal of unbound toxin, cells were covered with buffer containing various concentrations of carbamylcholine. Following a period sufficient to reach equilibrium, the initial rate of '251-labeled a-toxin binding was monitored to determine agonist occupancy of the residual receptor sites. In Fig. 8, the family of equilibrium competition isotherms reveals that progressive degrees of occupation of receptors by a-toxin produce a decrease in the apparent affinity and a simultaneous loss of the cooperativity typical of agonist binding. Moreover, occupation of 85% of the available sites by a-toxin causes a significant flattening of the experimental binding function yielding a Hill coefficient, n = 0.70. In the case of Model 3, the fraction of original oligomers containing a single bound toxin molecule is 2y(l - y) and the fraction unoccupied by toxin is (1 - y)'. In this experiment, when y = 0.85, 25.5% of the original dimers of binding sites are half-occupied by toxin and only 2.25% remain totally unoccupied. If both toxin-binding sites on the individual receptor unit were identical with respect to carbamylcholine affinity, the Hill coefficient for the equilibrium binding function should approach unity as the fraction of receptor species with both sites unoccupied by a-toxin approaches zero. Thus, the observed deviation from a unitary Hill coefficient suggests that the two agonist-binding sites on the functional receptor have different intrinsic affinities for carbamylcholine. It should be emphasized that since agonist and receptor are allowed to come to equilibrium prior to the competition assay, the resulting series of binding isotherms cannot be distorted by transitions in state.

Exposure of the receptor to agonist only during the short term interval required to measure the initial rate of '251-labeled a-toxin binding permits an estimate of occupation of the low affinity state of the receptor by the agonist (4). To examine whether the change in the equilibrium occupancy of receptor by carbamylcholine seen after fractional occupation by a-toxin is partially due to a loss in the ability of receptors to exhibit a slow increase in affinity, the competition isotherm generated upon instantaneous addition of carbamylcholine and 1251-labeled a-toxin is compared with the equilibrium exposure condition (Fig. 8). When 50% of the receptors sites are occupied by unlabeled a-toxin, binding of agonist upon short

\ 401 t "1 40 \

LLL 20 O 10-6 1 0 ~ 5 10-4 10-1 iLi?L O 10-6 10-5 10' 1 0 ~ 1

[CarbamylcholinelM

FIG. 8. Influence of progressive degrees of prior fractional a-toxin association on the equilibrium binding function for carbamylcholine ( Yequil) at 21°C. Cells were washed once with 3 ml of buffer and incubated in buffer alone (A) or in buffer containing unlabeled a-toxin to achieve: 38% (65 nM, 1 min), 64% (80 KIM, 1 min), and 85% (80 nM, 2 min) occupation. After removal of unbound toxin (three 6-ml washes), cells were incubated in buffer containing the specified concentrations of carbamylcholine for 45 min prior to the determination of the initial rate of '251-labeled a-toxin binding in the presence of identical concentrations of carbamylcholine. Sufficient quantities of '2sI-labeled a-toxin. receptor complexes were generated by employing 60-, 90-, 120-, and 240-s incubation intervals in the presence of '251-labeled a-toxin for the profiles presented in A, B, C, and D, respectively. Fractional occupation by unlabeled toxin in each panel was determined from the initial rate of '2511-labeled a-toxin binding in the absence of carbamylcholine for cells with a given degree of prior a-toxin occupancy. Thus, the initial rate of toxin binding in the presence of a given concentration of carbamylcholine is expressed relative to this value. A (control), KJequil) = 18.1 PM, n = 1.39 & 0.07; B (38% occupancy), KJequil) = 25.1 p ~ , n = 0.94 f 0.05; c (64% occupancy), &(equil) = 36.0 pM, n = 0.85 & 0.02; D (85% occupancy), KJequil) = 31.0 p ~ , n = 0.71 k 0.03.

exposure is also shifted to a lower apparent affinity, again accompanied by a marked broadening of the isotherm (Fig. 9). Comparison of the instantaneous and equilibrium exposure isotherms (Figs. 8 and 9) shows that receptor oligomers with one of their two sites occupied by a-toxin, despite their inability to activate, are still capable of exhibiting a slow increase in affinity following complexation with agonist. Furthermore, since the condition of short duration of exposure to agonist retains a receptor population largely in the low a f f i t y state, the observed Hill coefficient of 0.85 suggests that the individual sites on the receptor dimer in this state, R, are also not equivalent with respect to carbamylcholine binding.

Occupation of Receptors by d- Tubocurarine following Par- tial Occupation by a-Toxin-The less than unitary Hill coefficients characteristic of antagonist binding (4, 21) could be described by a mechanism where antagonist binds to sites on the receptor oligomer possessing intrinsically different affinities (i.e site nonequivalence pre-exists ligand binding) or one in which antagonist binds to a receptor composed of multiple sites that are initially similar, but exhibit weaker binding when individual sites on the oligomer are progressively occupied (negative cooperativity). To distinguish between these alternatives, occupation of a major portion of receptor sites by a-toxin was accomplished as detailed above and the competition between d-tubocurarine and '251-labeled a-toxin was determined for the remaining sites. As shown in Fig. 10, no alteration in the binding function for d-tubocurarine is evident when 64% of the available receptor sites are occupied by a-


toxin. The negative cooperativity mechanism requires a convergence of the binding function towards a unitary Hill slope with prior occupation of the sites by a-toxin. Thus, as in the case of agonists, antagonists appear to associate with nonequivalent binding sites on the receptor that pre-exist the binding event.

Ligand Specificity of the Alteration in the Binding Func- tion Revealed by a-Toxin Occupancy-The influence of partial occupation of the receptor by a-toxin on the equilibrium

e

[Carbamylcholine]M FIG. 9. Influence of prior fractional a-toxin association on

the binding function for carbamylcholine geperated upon instantaneous exposure to agonist at 21OC ( Yht). M, ''% labeled a-toxin and carbamylcholine added simultaneously to control cells for a 40-s interval; H, a-toxin and carbamylcholine added simultaneously for a 40-s interval to cells with 50% of the surface sites previously occupied by a-toxin. Surface receptors were partially occupied by a-toxin as described in the legend to Fig. 8 and cells were incubated in buffer for 20 min prior to the determination of the initial rate of '*'I-labeled a-toxin binding in the presence of the specified concentrations of carbamylcholine. For control cells, KJinst) = 62.7 ELM and n = 1.29 f 0.06, and for cells with receptors partially occupied by a-toxin, KJinst) = 128 and n = 0.85 f 0.05.

binding functions for several cholinergic ligands of differing efficacy is summarized in Table 111. The binding functions for two full agonists, carbamylcholine and suberyldicholine, are modified to the same degree by prior a-toxin occupation. Decamethonium, a ligand with functional properties of a partial agonist in BC3H-1 cells ( 4 ) shows a similar increase in KJequil) and a reduction in Hill coefficient following a-toxin occupation. Hence, the alteration of the ligand-binding parameters by prior occupation of the sites by a-toxin, most notably the dramatic loss of positive cooperativity, appears to be specific for agents which can initiate opening of the receptor channel.

2 o l " 2 1 ? 5 0 10-7 10-8 10-5 10-4

[d.tubocurarine]M FIG. 10. Influence of prior fractional a-toxin association on

the equilibrium binding function for d-tubocurarine at 21°C. O " 4 , competition between 1251-labeled a-toxin and d-tubocurarine for receptors on control cells. H, competition between "'I- labeled a-toxin and d-tubocurarine for receptor on cells with 64% of the sites previously occupied by a-toxin. Surface receptors were partially occupied by a-toxin, cells were incubated in the presence of the specified concentrations of d-tubocurarine for 45 min, and the initial rate of '*'I-labeled a-toxin binding was determined in the presence of d-tubocurarine as described in the legend to Fig. 8. For control cells, K,(equil) = 4.85 X M and n = 0.54 f 0.02, and for cells with 64% occupation by a-toxin, KJequil) = 3.27 f M and n = 0.50 f 0.07.

TABLE I11 Specificity of the alteration in parameters of ligand occupancy revealed by prior a-toxin occupancy

Cholinergic ligands are grouped according to their pharmacological efficacy as described previously (4). For each experiment, progressive degrees of a-toxin occupancy, y, were achieved as described in the legend to Fig. 9, and the competition with the initial rate of '*'I-labeled a- toxin binding was determined for the specified ligand and for the condition where the ligand was added with the initiation of (instantaneous) or 30 min prior to (equilibrium) the measurement of the initial rate of a-toxin binding. Kp is the protection constant and n is the Hill coefficient (see under "Experimental Procedures") corresponding with the specified degree of prior a-toxin occupation and the prior ligand exposure condition.

Percentage of a- toxin occupancy K P

n

Ligand (exposure condition) Experiment Experiment Experiment

1 2 3 1 2 3 1 2 3

M M M

Full agonists Carbamylcholine (equilibrium) 0 0 0 1.81 X 1.39 X 2.28 X 1.39 f 0.07 1.41 f 0.18 1.48 f 0.15

36 37 33 2.51 X lo-' 1.54 X 4.12 X 0.94 f 0.05 0.78 f 0.12 1.08 * 0.07

85 83 62 62 73 3.60 X 4.24 X 5.01 X lo-' 0.85 f 0.02 0.76 f 0.04 0.86 f 0.09

3.10 X 4.26 X 0.71 f 0.03 0.70 rt 0.04

Carbarnylcholine (instantaneous) 0 0 50 50

6.27 x lo-' 7.36 x lo-' 1.29 f 0.06 1.07 f 0.08 0.83 f 0.05 0.70 f 0.05 1.28 X 10-~ 1.21 X

Suberyldicholine (equilibrium) 0 1.71 X lo-' 55 2.56 X 10"

Partial agonist Decamethonium (equilibrium) 0 1.57 X

55 2.23 X

Antagonists d-Tubocurarine (equilibrium) 0 4.85 X IO-'

64 3.27 X

Gallamine (equilibrium) 0 1.76 X 67 1.52 X lo-'

1.46 f 0.07 0.79 f 0.02

1.04 f 0.03 0.80 f 0.03

0.54 f 0.02 0.50 f 0.07

0.83 f 0.05 0.75 f 0.03


DISCUSSION

Progressive occupation of the cholinergic receptor of intact BC3H-1 cells by a-toxin apparently results in a random distribution of occupied toxin sites among the surface receptors. Simultaneous examination of the receptor response reveals a parabolic, concave inward relationship between the loss of the capacity of the agonist to elicit a change in permeability to sodium ions and the fractional occupancy of receptors by a- toxin. Over the entire range of cu-toxin occupancy, this finding is closely described by a model where the functional receptor has two toxin-binding sites and requires occupation of both sites by the agonist to produce significant activation of the ion channel (Table 11). Accordingly, since the concentration dependencies for activation and desensitization of the remaining active receptors are not altered following occupation of the majority of the receptor sites by a-toxin, hybrid species occupied by a single toxin molecule appear virtually silent in the presence of agonist. Finally, partial occupation of receptors by a-toxin to form the hybrid species not only eliminates the positive cooperativity of agonist binding to the residual sites but also reveals an intrinsic nonequivalence in the two agonist- binding sites of the receptor molecule. The nonequivalence in the sites for binding of cholinergic antagonists as revealed by Hill coefficients less than unity persists when the receptor sites are partially occupied by a-toxin.

Employing the intact BC3H-1 clonal muscle cells to monitor simultaneously occupation of the receptor by a-toxin and the agonist-mediated permeability increase confers several key experimental advantages. First, experiments performed on the intact cell maintain the natural disposition of the receptor in the plasma membrane interposed between many of the in situ ionic and metabolic environments. In this situation, both occupation by agonists and function of the receptor are con- veniently measured under identical conditions. Second, a feature inherent to a nonfusing clonal cell line is the uniformity of experimental cultures (ie. a narrow distribution of cell volumes and uniformity of receptor number within individual batches of culture dishes resulting from an original stock culture of identical precursors). Furthermore, nearly ideal conditions are achieved for measurements of the relative number of receptor channels activated by the agonist. As developed previously (4), dissipation of the transmembrane sodium and potassium gradients provides a sustained depo- larization in spite of permeability changes resulting from agonist action. Hence, the rate of sodium influx should not be influenced by changes in membrane potential. Moreover, the comparatively low density of surface receptors and the large internal cell volume are desirable, since the rate of unidirectional flux may be accurately determined prior to equilibration of the exchangeable internal volume with radioactive tracer. Finally, since this method measures primarily influx of sodium ions, the resulting permeability change reflects the direction- ality and current-carrying ions that prevail in the in situ condition. These latter conditions are presently difficult to achieve with in vitro membrane vesicles from Torpedo. When monitored by conventional techniques (22), the exceedingly high density of surface receptors and small internal volumes preclude an accurate initial rate determination of the permeability change elicited by the agonist. Such studies where permeability is not ascertained from initial flux rates do not distinguish alternative mechanisms for receptor activation (23, 24).

Reduction of the incubation temperature from 21" to 3.5"C minimizes the development of desensitization of the agonist- mediated permeability increase during the assay interval. Under these conditions, the permeability measure is linear with the time of agonist exposure. The reduced rate of desen-

sitization is also apparent when parameters associated with activation, desensitization, and occupation of the receptor by agonist are compared at the two temperatures. Accordingly, the maximal rate of sodium influx, the extent of desensitization, R , , the dissociation constant, IC,,,, and the Hill coefficient for activation (Table I) all change in a manner anticipated from a decrease in the rate of desensitization, hence minimiz- ing the influence of this process in the assay interval. Of particular importance is the refinement in the determination of the concentration dependencies for activation and desensitization achieved at low temperature. By spanning a greater range of agonist concentration, these results further support the conclusion that both activation and desensitization of the receptor are cooperative processes in the BC3H-1 culture system.

The kinetics of a-toxin association shows that the available binding sites behave as a kinetically homogeneous population towards this ligand. The lack of either positive or negative cooperativity in the occupation of individual sites within a receptor oligomer by toxin means that species fully or partially occupied are generated purely on the basis of stochastic probability. With this provision, five models for a functional receptor oligomer composed of either two or four toxin-binding sites were examined which place specific constraints on the functional capacity of the species generated by partial toxin occupancy. When examined over the entire range of fractional toxin occupancy, the reduction in the functional capacity of receptors is most closely described by the model where the functional receptor oligomer contains two toxin-binding sites and activation requires the association of at least 2 agonist molecules. The occupation of either binding site by a-toxin is sufficient to block the permeability change in the presence of agonist (Model 3). Small deviations from this theory are observed in the region of large fractional occupancy of the receptors by a-toxin, where the agonist-mediated permeability change is somewhat greater than expected. A modification of Model 3 which allows the hybrid species possessing a single bound toxin molecule to elicit a small but significant change in sodium permeability on combination with agonist (i.e. channel opening would occur but with a very low probability) may account for this slight departure from theory. Finally, in further support of Model 3, parameters associated with the state functions for either receptor activation or desensitization are not altered when a majority of receptors are occupied by a-toxin. Hence, as revealed by these two properties of functional receptors, the only receptors capable of activation and desensitization of the permeability response following partial occupation by a-toxin are those free to bind 2 agonist molecules, as specified by Model 3.

Although there is disagreement on the stoichiometric ratio of agonist- to a-toxin-binding sites in receptor from Torpedo (9, 25, 26), more recent reports strongly favor a 1:l ratio, determined from equilibrium binding of acetylcholine (9, 25) and d-tubocurarine (25), the binding of a spin-labeled partial agonist (lo), and from affinity labeling by the irreversible antagonist, p-(trimethy1ammonium)benezenediazonium fluo- roborate (27). Only one of two toxin sites on the receptor monomer will react with the irreversible affinity labels, 4-(N- maleimide)benzyltrimethylammonium (28) and bromoacetyl- choline (29). This absolute difference in reactivity requires prior reduction of existing disulfide bonds in the receptor and the 0.5:l.O ratio of MBTA3 to toxin sites is likely due to the absence of a reactive sulfkydryl in the vicinity of one of the toxin-binding sites following reduction. Thus, the available biochemical data support an equivalent number of agonist-

The abbreviation used is: MBTA, 4-(N-rnaleirnide)benzyltri- rnethylarnrnoniurn.


and toxin-binding sites on the receptor, consonant with the 1:l stoichiometry intrinsic to Model 3.

Although implicit in Model 3, a 1:1 stoichiometry is not required. A four-toxin-site, two-agonist-site, one-channel model, while inconsistent with the 1:l stoichiometry determined in some laboratories, could accommodate the present experimental findings (24). In this situation, a specified pair of toxin sites would be constrained to share a common binding domain with two agonist sites, while the remaining two would not. However, occupation of the latter sites by a-toxin would be silent in blocking function. Thus, the number of agonist molecules required to activate the receptor and the number of toxin sites related to functional antagonism would be two per functional receptor unit.

We have assumed in Model 3 that the receptor monomer containing two binding sites and a single channel is the primary functional unit. In the case where the functional receptor is a dimer of this basic set of subunits, certain models may be generated which are equivalent to Model 3: (a) a four-binding- site, two-channel model where there is strict pairing between two binding sites and a given channel, or (b ) a four-binding- site, one-channel model where activation requires that a specified pair of sites become occupied by agonist.

Scheme 1, which can describe desensitization of the receptor in the present cell culture system, is readily accommodated by Model 3, where the functional receptor oligomer contains two sites for which the binding of a-toxin and agonist is mutually exclusive. Within this framework, following occupation of a majority of the available sites by a-toxin, the predominant population of receptor oligomers to which agonists can still bind would be associated with a single toxin molecule. Under these conditions, the major receptor population available for agonist binding can be formally considered to obey a single-site, two-state scheme (cf. Ref. 10) lacking the previous enhancement in binding provided by subunit symmetry. Consistent with this interpretation, prior occupation by a-toxin reveals an increase in the apparent dissociation constant for equilibrium binding of agonist. However, the Hill coefficient associated with both the equilibrium and instantaneous binding functions falls markedly below unity, at var- iance with the prediction for agonist saturation of a single class of binding sites. Since the Hill coefficient associated with the equilibrium binding function decreases steadily with progressively greater degrees of occupancy by toxin, reaching an extreme value of 0.7, the agonist-binding sites on an individual receptor oligomer are likely intrinsically nonequivalent. Thus, the resulting agonist-binding function results from the super- imposition of two different isotherms that are individually devoid of homotropic cooperativity. An alternative model where two populations of receptor oligomers exist, each of which contains intrinsically equivalent binding sites, could account for the apparent nonequivalence observed following partial occupation by a-toxin. Nonequivalence from the loss of homotropic cooperativity in the binding of agonist within each receptor population. However, the latter interpretation seems less likely since the two populations must be present in near equal proportions to give rise to the symmetry in the isotherm of Fig. 8. True negative cooperativity where the binding sites on an individual receptor unit are initially similar but exhibit weaker binding when one of the pair in the oligomer is occupied by agonist (30) cannot account for the prevailing low Hill coefficient observed following occupation of most of the available sites by a-toxin. In the limit of maximal degrees of a-toxin occupancy, Model 3 dictates that only one site for agonist binding remains on each receptor oligomer and, hence, it cannot be influenced by binding of agonist to a neighboring site. Finally, it is conceivable that

occupation of one site on a receptor oligomer by a-toxin might modify the intrinsic affinity of the remaining site for the agonist. While this possibility cannot be excluded, the sites for agonist binding still show a pre-existing nonequivalence; otherwise, a unitary Hill coefficient associated with agonist binding to a single class of altered sites would be observed as one approaches complete toxin occupancy.

Damle and Karlin (28) have also provided evidence for the nonidentical nature of the two ligand-binding sites on the Torpedo receptor. Only one of the two sites will react with the irreversible affinity label, MBTA, and this labeling is blocked only when toxin is bound to the labeling site. The possibility that two nonequivalent populations of receptors prevail which differ in their reactivity to MBTA was considered unlikely, since they are not separable during purification and the ratio of a-toxin to MBTA sites is virtually constant at all stages of the purification. This possibility could be further tested by following the loss of functional capacity with progressive degrees of MBTA labeling (23).

In the present cell culture system (4) and in the receptor from Torpedo (21,25), the occupation function for antagonist binding to receptor is typified by Hill coefficients markedly less than 1.0. As considered above for agonists, the latter distinction could result from negative cooperativity in antagonist binding or from a pre-existent nonequivalence in the binding sites for antagonists. Since the isotherm for antagonist binding to receptor is not altered following partial occupancy of receptor sites by a-toxin, the binding sites for antagonists are clearly nonequivalent prior to antagonist binding. This finding provides a means for distinguishing agonists from antagonists based on binding properties alone and reveals an interesting correlation between the binding properties of agonists and antagonists in the absence of homotropic cooperative interactions.

Recent studies have established that the purified Torpedo receptor contains four polypeptide chains in a stoichiometric ratio of 2:l:l:l; the individual subunits have been designated aePlylGl (31). The molecular weights of the subunits sum nominally to 250,000 daltons, corresponding to the monomeric unit which has been found to contain two agonist- or a-toxin- binding sites (13). Since the a subunit is the primary locus for toxin association (32) as well as for the binding of irreversible agonists (28) and antagonists (27, 33), it seems likely that cooperativity arises from an initial interaction with this subunit pair. Therefore, is the nonequivalence between the two binding sites a consequence of different primary structure for the two a subunits, or are they identical but differ in their intersubunit contacts within the receptor oligomer? With Torpedo receptor, electrophoretic migration behavior and partial sequences do not distinguish two different types of a subunits. The inclusion of a 2-fold axis of symmetry in the receptor unit perpendicular to the membrane seems improb- able for the disposition of within the membrane. Moreover, even with a symmetric arrangement of subunits, the individual peptide chains are expected to be asymmetric, and dissimilar intersubunit contacts between the two a subunits and their neighbors would prevail. Asymmetry of other membrane-associated oligomeric proteins has been well documented (34).

The receptor from BC3H-1 cells has been purified to specific activities approaching those of the Torpedo receptor and a distribution of subunits similar to that of Torpedo was evident (36). Although multiple a species were not immediately apparent in the gel electrophoresis profiles, no information is available on labeling stoichiometry of individual subunits with irreversible agonists and antagonists. In contrast, the receptor from denervated rat skeletal muscle contains two a subunits


with different molecular weights as revealed by MBTA labeling, and an overall stoichiometry is 1 MBTA/a-toxin site (35). Whether the differing a subunits are contained within the same receptor oligomer or two populations of receptor oligomer is presently not known.

Scheme 1 is the simplest two-state scheme which c y accommodate homotropic cooperativity in the binding ( Y ) and state ( E ) functions for desensitization for receptors in BC3H- 1 cells. An inherent feature of this two-state scheme is that the binding of agonist to individual protomers on the receptor is equivalent and binding to receptor confined to a single state occurs in a noncooperative fashion. Moreover, when transitions in state are perfectly concerted, an agonist possessing an energetic preference for the receptor in the R' state will exhibit positive homotropic cooperativity in the binding and state functions (18, 19). The assumption of subunit equivalence in Scheme 1 can be relaxed to accommodate the nonequivalence observed in the binding of agonist to the two protomers within the functional receptor, while preserving the principle of subunit symmetry in the state transitions:

+ TBhRARBL .RALRB + L

L + R'AR'B 1 + \ L + R'aR'sL =":"" R'a LR'B + L R'a LR'BL

Scheme 2

As developed for hemoglobin (37) and subsequently hypoth- esized for the cholinergic receptor (38), the two protomers of the receptor, designated A and B, have intrinsically different affinities for the agonist, L. By analogy to Scheme 1, in Scheme 2, the receptor can exist in two principal states, R and R', whose ratio in the absence of agonist is determined by the allosteric constant, M. In addition, the bimolecular binding steps are rapid while the transitions among states (top plane to bottom plane) are relatively slow and occur in a perfectly concerted fashion. Thus, in spite of the nonequivalent binding sites present in either the low or high affiiity state, at equilibrium the binding and state functions exhibit positive homotropic cooperativity. The degree of cooperativity and the relationship between the binding and state functions depend on the average difference in affinity of the agonist for the receptor in the R and R' states, the allosteric constant M, and on the number of interacting protomers within the oligomer (n = 2). In the case of the hemoglobin dimer, it has been possible to assign many of the kinetic constants in a two-state model containing nonequivalent subunits (39).

While the complexity of this scheme would preclude a unique assignment of each parameter based on a fit to the experimental equilibrium binding and state functions, it never- theless represents the simplest two-state scheme consonant with the experimental demonstration of nonequivalence in the two sites in terms of agonist affinity. When the majority of the available sites are occupied by a-toxin, two hybrid populations of receptor predominate in equal amounts: one capable of binding agonist to the vacant A site, and the other to the vacant B site. Therefore, the receptor which was originally capable of traversing all six planes in Scheme 2 on combination with agonist is restricted to one of the two vertical planes extending from the equilibrium defined by M. This results in a partial convergence of the instantaneous and equilibrium binding functions (y-t, yquJ which are associated with Hill coefficients markedly less than 1.0. The less than unitary Hill slopes are a consequence of two separate populations of receptor subunits binding the agonist with different affinities, while

the convergence of yequa and yiinst results from the loss in the enhancement of conversion in state originally provided by the symmetry maintained between the interacting subunits.

In addition to a resting and desensitized state, an overall description of receptor function must include the active or conducting state. The present experiments demonstrate that only the receptor species free of bound toxin is able to open channels with a measurable conductance increase or with a high probability. If the hybrid receptor species with 1 toxin and only 1 agonist molecule bound has the same low probability for receptor activation as does the receptor with 1 agonist bound, in the simplest case, only a single equilibrium needs to be incorporated into Scheme 2:

RALRBL -" R**LRs*L. Alternatively, a second cubic

scheme analogous to Scheme 2 could represent the activation process where species RaRn, RaLRn, and RARBL have low but finite probabilities for achieving the active R* state. In both cases, a ratio of a to P <1 could give rise to cooperativity in the binding function generated upon short exposure to agonist

Electrophysiological measurements of the concentration dependence of the agonist-mediated conductance increase have sought a mechanistic description of the process of receptor activation. Within the precision of the measurements, several molecular schemes are capable of describing the available data, and a lower limit of 2 molecules of agonist required to activate the receptor has been firmly established (5,6,40,41). As considered above, a two-state (open and closed channel) scheme analogous to Scheme 2 might describe receptor activation and is compatible with a functional receptor possessing two nonequivalent binding sites. Unfortunately, such a scheme contains many unknown (and to date unmeasurable) parameters, and neither the present data nor the existing electrophysiological data are sufficient to yield a unique fit to such a mechanism. In principle, if one could resolve the occupation and state functions associated with either activation or desensitization of the receptor, schemes such as Scheme 2 could be rigorously tested. The present experiments provide evidence that the functional receptor unit contains two mutually exclusive a-toxin- and agonist-binding sites, activation and desensitization involve the cooperative interaction of at least 2 agonist molecules, the binding of 1 toxin molecule blocks function of the receptor unit, and the binding of both agonists and antagonists to the two sites is nonequivalent.

P a

( Y i m t ) .

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