Introduction

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Apart from the field of muscarinic acetylcholine receptors, the investigation of allosterism among G protein-coupled receptors has been relatively little explored, despite the potential of allosteric sites as alternative targets for the development of subtype selective drugs (Birdsall et al., 1999). Thus, it is not known whether allosteric interactions are a general characteristic of this family of receptors. This is in contrast to the ligand-gated ion channel receptors, where multiple allosteric sites are known to exist (Galzi and Changeux, 1994). For example, it has been shown that benzodiazepines, barbiturates, and anesthetic steroids can act allosterically to enhance agonist responses at -aminobutyric acid_A receptors (for reviews, see Macdonald and Olsen, 1994; Smith and Olsen, 1995).

One therapeutically important group of G protein-coupled receptors is the adrenergic receptors. Within this group, allosterism has only been explored for the ₂-adrenergic receptor subtypes. It has been shown that amilorides act allosterically at the _2A-subtype (Nunnari et al., 1987; Leppik et al., 1998a), and possibly also at the _2B-subtype (Wilson et al., 1991). At the _2A-adrenergic receptor, binding of amilorides to the allosteric site increased the dissociation rate of antagonists such as yohimbine, from 2-fold for amiloride to ~150-fold for both 5-(N-ethyl-N-isopropyl)-amiloride (EPA) and 5-(N,N-hexamethylene)-amiloride (HMA) (Leppik et al., 1998a). Analysis of the data revealed that the amilorides exert strong negative cooperativities on the binding of the antagonists examined. Competition experiments indicated that the amilorides were acting via a common allosteric site, with no evidence for the presence of a second allosteric site (Leppik et al., 1998a).

It is not known whether an allosteric site exists on the ₁- or -adrenergic receptor subtypes, other members of the adrenergic receptor family. For the muscarinic receptor family, the relatively strong sequence identity, both within the putative transmembrane domains (containing the primary binding site) and in the extracellular loops (74 and 53%, respectively, between the human M₁ and M₂ receptors), means that it is not surprising that a comparable allosteric site exists on all five muscarinic receptor subtypes (Ellis et al., 1991). In contrast, there is a lower sequence identity between the human _1A- and _2A-adrenergic receptors in the comparable regions, being 44 and 33%, respectively, in the transmembrane domains and extracellular loops. Therefore, there can be no a priori assumption that ₁-adrenergic receptors have an allosteric site with a pharmacology similar to that described for the _2A-adrenergic receptor. In this study, we examine the effect of amilorides on the binding of the antagonist [³H]prazosin at one of the ₁-adrenergic receptor subtypes, the human _1A-adrenergic receptor.

	Experimental Procedures

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Materials. [³H]Prazosin (70-87 Ci/mmol) was from DuPont/NEN, Hounslow, Middlesex, UK. The amiloride analogs, phentolamine HCl, Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham, and polyethylenimine were from Sigma Chemical Co., Poole, Dorset, UK. Other tissue culture reagents were from Gibco BRL, Paisley, UK. Aqueous stock solutions (10 mM) of the amilorides in HEPES buffer were prepared fresh as required, as described previously (Leppik et al., 1998a).

Cell Culture and Membrane Preparation. The clonal Chinese hamster ovary (CHO)-K1 cell line stably expressing the human _1A-adrenergic receptor (Ford et al., 1997) was generously provided by Dr. Richard Eglen of Roche Bioscience (Palo Alto, CA). The cell line was grown in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 I.U./ml penicillin, and 50 µg/ml streptomycin, at 37° in 5% CO₂. The initial selection agent G418 was absent during routine cell culture. Membranes were prepared as described previously (Leppik et al., 1998a). Briefly, near-confluent cells were harvested in cold buffer 1 (20 mM Na-HEPES, pH 7.4, 10 mM EDTA), then homogenized and centrifuged. The pellet was resuspended in buffer 2 (20 mM Na-HEPES, pH 7.4, 0.1 mM EDTA), recentrifuged, again resuspended in buffer 2, then stored at 70°C. Protein concentrations were determined by the method of Bradford (1976), with BSA as the standard.

Radioligand-Binding Assays. For saturation experiments, membranes (2 µg of protein) were incubated with increasing concentrations (0.04-5 nM) of [³H]prazosin in duplicate, in a final volume of 1 ml of assay buffer (20 mM Na-HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂), at 20° for 120 min. Nonspecific binding was defined as the binding retained on the filter and membranes in the presence of 10 µM phentolamine. Where appropriate, amiloride analogs were added to both total and nonspecific binding-assay tubes to control for effects on binding. The use of organic solvents to dissolve the amilorides was avoided, due to the previously observed effects of the organic solvents tested on the equilibrium binding of antagonists to the _2A-adrenergic receptor (Leppik et al., 1998a). Bound and free ligand were separated by rapid filtration under vacuum through GF/B glass fiber filters (Whatman; Maidstone, Kent, UK), pretreated with 0.1% polyethylenimine, by using a Brandell cell harvester (Semat, St. Albans, Hertfordshire, UK). The filters were washed three times with cold 20 mM sodium phosphate buffer, pH 7.4, transferred to scintillation vials, scintillation cocktail (Beckman, Palo Alto, CA) added, the filters soaked overnight, and then counted. For competition experiments, membranes (5 µg of protein) were incubated with ~0.5 nM [³H]prazosin in duplicate, together with increasing concentrations of competing agent, in a final volume of 1 ml of assay buffer, at 20° for 60 min.

Data Analysis. Data were fitted by nonlinear regression analyses with the Grafit curve-fitting software (Erithacus Software, Staines, Middlesex, UK). This procedure allows the use of two or more independent variables (e.g., time and concentration), which was necessary for many of the analyses reported in this article. Logistic fits of the effects of amiloride, DMA, BZA and HMA on the k_obs of [³H]prazosin dissociation were fitted using the GraphPad Prism curve-fitting software (GraphPad Software, San Diego, CA).

	Results

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Characterization of Antagonist Binding at _1A-Adrenergic Receptor. Initially, the equilibrium-binding properties of [³H]prazosin at the human _1A-adrenergic receptor permanently expressed in a CHO-K1 cell line (Ford et al., 1997) were characterized. The nonspecific binding was defined as the residual binding measured in the presence of 10 µM phentolamine. The saturation curve for the specific binding of [³H]prazosin was compatible with the presence of a uniform population of binding sites. The log affinity value (log K_L) calculated for the [³H]prazosin binding (10.02 ± 0.04; five experiments) was in good agreement with that previously reported (9.92 ± 0.01) (Ford et al., 1997). However, the B_max estimate from these experiments was 16.0 ± 0.9 pmol/mg protein (five experiments), ~10-fold higher than that previously reported for this cell line (Ford et al., 1997). The higher B_max estimate found in the current study may be a reflection of the different growth and assay conditions used [D. Daniels, personal communication (Roche Bioscience, Palo Alto, CA)].

Effect of Amiloride Analogs on Equilibrium Binding of [³H]Prazosin. It has been reported previously that amiloride decreased [³H]prazosin affinity, but not the B_max, in equilibrium-binding studies with the ₁-adrenergic receptor in rat renal cortical membranes, and this effect was attributed to competition between the prazosin and the amiloride (Howard et al., 1987). However, this result is equally compatible with the ternary complex allosteric model (Fig. 1). It was important to also establish that, in CHO membranes containing the human _1A-adrenergic receptor, there was again no significant change in B_max with representative amilorides. Amiloride and HMA (Fig. 2) were chosen because they were found to have the smallest and largest effects, respectively, on the [³H]prazosin dissociation rate (see below). Both amiloride and HMA decreased the observed affinity constant for [³H]prazosin. At concentrations up to the highest practical concentration that either could be tested, no significant change in B_max was found (P > .1; Student's paired t test) (Table 1). These results are compatible with either a simple competitive or an allosteric model for the effect of the amilorides on [³H]prazosin binding.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the ternary complex allosteric model. In this scheme, radioligand L and allosteric agent X bind to two separate sites on the receptor R. KL and K1 are the affinity constants for L and X, respectively, binding to R; K2 is the affinity of X for RL and  (= K2/K1) the cooperativity factor between X and L. k-1 and k-2 are the rate constants for L dissociating from RL and XRL, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Structural formulae of amiloride and of the analogs examined in this study.

View this table: [in this window] [in a new window]   TABLE 1 Effect of amiloride or HMA on the binding of [3H]prazosin to the human 1A-adrenergic receptor Values are means ± S.E. from three saturation-binding assays, in which membranes containing the 1A-adrenergic receptor were incubated at 20°C for 120 min with increasing concentrations of [3H]prazosin (0.04-5 nM) in a final volume of 1 ml of assay buffer. LogKobs is the observed log affinity constant of the [3H]prazosin in the presence of amiloride analog at the 1A-adrenergic receptor. In the absence of amiloride analog, logKobs = logKL (Fig. 1).

View this table: [in this window] [in a new window]   TABLE 2 Affinity of the amiloride compounds at the 1A-adrenergic receptor The log affinities were determined in equilibrium inhibition experiments versus [3H]prazosin, performed at 20°C for 60 min. The data were fitted with a one-site equation (Leppik et al., 1998), with the slope factor set at 1, then the derived log apparent affinity constants were converted to the log affinity constants logK1 with the Cheng-Prusoff correction (Cheng and Prusoff, 1973). Kd = 1/K1. Values are means ± S.E. from three experiments.

Effect of Individual Amilorides on [³H]Prazosin Dissociation. The dissociation of [³H]prazosin alone from the human _1A-adrenergic receptor was found to be monoexponential, with a dissociation rate of 0.021 min¹ (t_1/2 = 33 min) at 20°C (Table 3). All of the amilorides were found to strongly increase the [³H]prazosin dissociation rate in a concentration-dependent manner (Figs. 3 and 4; Table 3). For all the amiloride analog concentrations investigated, the dissociation curves were monoexponential.

View this table: [in this window] [in a new window]   TABLE 3 Effect of amiloride analogs on the [3H]prazosin dissociation rate constant The experiments were performed at 20°C as described in the legend of Fig. 3, in the presence of the stated concentrations of the amiloride analogs. Values (min1) are means ± S.E. of three to six experiments. In the absence of amiloride analog, the [3H]prazosin dissociation rate was 0.0212 ± 0.0004 min1 (n = 19). The fold increase is the dissociation rate observed in the presence of the amiloride divided by the rate in its absence.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Dissociation of [3H]prazosin at 20°C in the absence or presence of various concentrations of amiloride. Membranes were first pre-equilibrated with [3H]prazosin, then the dissociation experiment initiated by mixing aliquots with phentolamine and various concentrations of amiloride. Individual data points from one experiment are shown. The lines represent the simultaneous fit of the one allosteric site equation (eq. 9), with time and amiloride concentration as independent variables. The results of five experiments are summarized in Table 3.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Dissociation of [3H]prazosin at 20°C in the absence or presence of various concentrations of DMA or HMA. The experiments were performed as described in the legend to Fig. 3. Individual data points from one experiment are shown. The data were fitted simultaneously to either the one-allosteric-site equation (A and C) or the two-allosteric-site equation (B and D) (eq. 9 and 8, respectively), with time and amiloride analog concentration as independent variables. The results of six (DMA) and three (HMA) experiments are summarized in Table 3.

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View this table: [in this window] [in a new window]   TABLE 4 Effect of amiloride on [3H]prazosin dissociation in the absence and presence of DMA The experiments were performed at 20°C, as described in the legend of Fig. 3. k1 and k2 are the dissociation constants for the dissociation of [3H]prazosin from either the unoccupied receptor or from the receptor occupied by amiloride, and logK2 is the log affinity of amiloride at the prazosin-occupied receptor (Fig. 1). The logobs is the difference between logK2 and the log affinity of the amiloride at the unoccupied receptor (logK1; Table 2). Values are means ± S.E. of n experiments.

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View larger version (19K): [in this window] [in a new window]   Fig. 5.   The concentration dependence of the amilorides on the observed dissociation rate (kobs) of [3H]prazosin. The experiments were performed as described in the legend to Fig. 3. For each amiloride analog concentration, the kobs was calculated with a single exponential decay equation. The derived kobs values, together with their calculated errors, were fitted to the polynomial equation kobs = a + b · [X] +c · [X]2, with b being the initial gradient, and X being the amiloride analog concentration. The straight lines portray the initial gradients for each analog. The initial gradients for EPA and MBA are superimposable, so are shown with the same line style. The results of two to six experiments are summarized in Table 5.

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View this table: [in this window] [in a new window]   TABLE 5 Comparison of the initial gradients for the concentration dependence of the effect of amilorides on the dissociation of [3H]prazosin or [3H]yohimbine from the 1A- or 2A-adrenergic receptors, respectively For the 1A-adrenergic receptor, the initial gradient estimates were derived as described in the legend of Fig. 5, and are expressed as means ± S.E. of n experiments. For the 2A-adrenergic receptor, the values were calculated with published data (Leppik et al., 1998). To enable comparison, each estimate was divided by the antagonist dissociation rate (k1) in the absence of amiloride analog (0.0212 ± 0.0004 min1 for [3H]prazosin/1A-adrenergic receptor, 0.034 ± 0.001 min1 for [3H]yohimbine/2A-adrenergic receptor). The k2 is the antagonist dissociation rate from the amiloride analog-occupied receptor, and K2 is the affinity of the amiloride analog at the antagonist-occupied receptor.

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View larger version (23K): [in this window] [in a new window]   Fig. 6.   Logistic fits of the effects of amiloride, DMA, BZA, and HMA on the kobs of [3H]prazosin dissociation at 20°C. Data from an individual experiment (DMA) or from combined experiments (four, two, or three experiments, respectively, for amiloride, BZA, or HMA) were fitted to eq. 13. The estimated slope factor, log affinity at the [3H]prazosin-occupied receptor (log K2), and maximum dissociation rate from the occupied receptor (kmax) for amiloride were 0.95 ± 0.17, 2.07 ± 0.47, and 0.35 ± 0.21 min1, respectively. The corresponding values for the analogs were DMA, 1.34 ± 0.14, 2.10 ± 0.22, and 1.85 ± 0.22; BZA, 1.20 ± 0.11, 1.94 ± 0.61, and 3.7 ± 4.9; and HMA, 2.06 ± 0.24, 3.29 ± 0.14, and 2.8 ± 1.1, respectively. The best-fit theoretical curves are shown on the main figure and the inset.

Competitive Interactions between Amiloride and DMA as Detected by Their Effects on [³H]Prazosin Dissociation. To further explore the allosteric interactions of amiloride, the effect of competition between it and DMA on the [³H]prazosin dissociation rate was examined. The concentrations of DMA were chosen such that its effect alone on the dissociation rate of [³H]prazosin could be reasonably well fitted to a one-site model. The dissociation data obtained were well fitted by the appropriate one-allosteric-site equation (eq. 9; Leppik et al., 1998a) (Fig. 7). From the fit, the parameter estimates which related to amiloride were defined, and were not significantly different (P > .05) from those obtained for the effect of amiloride alone on [³H]prazosin dissociation (Table 4). However, the DMA parameter estimates again were not defined, as found with DMA alone. The effects of DMA and amiloride on [³H]prazosin dissociation rate were additive, indicating that the concentrations of these ligands were insufficiently high to detect experimentally a significant modulation of the dissociation enhancing effects of one ligand by the other.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of DMA on the modulation by amiloride of [3H]prazosin dissociation at 20°C. The experiments were performed as described in the legend to Fig. 3. Individual data points from one experiment are shown. The lines represent the simultaneous fit of the data to the equation derived in a previous study (eq. 9; Leppik et al., 1998), with time, and amiloride and DMA concentrations as independent variables. The results of three experiments are summarized in Table 4.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

To further investigate the question as to whether -adrenergic receptors in general possess allosteric sites, we chose to examine the effect of amilorides on the binding of the antagonist [³H]prazosin at one of the ₁-adrenergic receptors, the _1A subtype. Equilibrium binding studies with the cloned human _1A-adrenergic receptor provided evidence that the amilorides were interacting competitively or with high negative cooperativity with [³H]prazosin (Table 2). Of the six analogs examined, amiloride had the lowest affinity (K_d = 11 µM), whereas the dissociation constants of the other amilorides clustered around 1 µM (Table 2). The dissociation constants calculated for amiloride, EPA, and BZA (Table 2) were 3- to 25-fold lower than those reported previously for ₁-adrenergic receptors on rat renal cortical membranes or on the Madin-Darby canine kidney cell line (Howard et al., 1987). These differences may be due to species variation or to different experimental conditions.

No correlation between affinity and size of the 5-N-alkyl side chain was found. This is in contrast to the _2A-adrenergic receptor, where the affinities increased progressively with increase in size of the 5-N-alkyl group (Leppik et al., 1998a). The structure-binding relationships of the amilorides at the unliganded _1A- and _2A-receptors are clearly different, suggesting that their effects on binding are not due to a simple membrane perturbation.

As found for the _2A-receptor, none of the inhibition curves obtained in the current study, even those carried out with a high concentration of [³H]prazosin, deviated from a simple binding isotherm. In addition [³H]prazosin binding in the presence of high concentrations of the amilorides did not differ from nonspecific binding. This is compatible with either competition of the ligands at the primary binding site of the _1A-receptor, or allosterism with high negative cooperativity (Ehlert, 1988). Hence, to explore potential allosteric interactions between the amilorides and prazosin, kinetic rather than equilibrium studies were required (Lee and El-Fakahany, 1991; Leppik et al., 1994; Lazareno and Birdsall, 1995).

All of the amilorides examined strongly increased the [³H]prazosin dissociation rate (Table 3), indicative of an allosteric modulation of the [³H]prazosin binding. For all ligands and concentrations the dissociation curves were monoexponential. At any given concentration of the amilorides, the magnitude of the increases in rate is dependent on the size of the 5-N-alkyl side chain (Table 3), in a manner similar but not identical with that found at the _2A-adrenergic receptor. However, such a rank order of dissociation rates is not necessarily a reflection of the rank order of the affinities of the allosteric ligands at the allosteric site of an occupied receptor, as has been demonstrated for the antagonist-occupied _2A-receptor (Leppik et al., 1998a). Thus, quantitative analyses of the effects of several concentrations of amiloride analogs on [³H]prazosin dissociation are required to derive the estimates and rank orders of allosteric ligand affinities at the antagonist-occupied _1A-receptor.

Such an analysis proved feasible with the parent amiloride, where the data were well fitted with the equation derived from the simple allosteric model (Fig. 3), indicating that amiloride indeed acts at an allosteric site to modulate [³H]prazosin dissociation. The observed cooperativity between amiloride and prazosin is strongly negative (Table 4), compatible with the competition data. For the radiolabeled antagonists examined, amiloride has a comparable affinity at both the antagonist-occupied human _1A- and _2A-adrenergic receptors (Leppik et al., 1998a). However, the maximum fold increase in the antagonist dissociation rate caused by the binding of amiloride is 10-fold higher for the _1A-adrenergic receptor than that observed at the _2A-adrenergic receptor (Leppik et al., 1998a).

The dissociation data for HMA (and the other amiloride analogs examined) are compatible with it modulating [³H]prazosin dissociation via two allosteric sites (Fig. 4D), but not one site (Fig. 4C). Despite the excellent fit to the two-site model, estimates of parameters describing the allosteric interaction were not obtained. The explanation was evident when the observed dissociation rates (k_obs) of [³H]prazosin in the presence of the amiloride analogs were plotted against analog concentration (Fig. 5). The k_obs values increased monotonically in an upwardly concave manner in the limited concentration range imposed either by the insolubility of the analogs or by our inability to measure high dissociation rates (>1 min¹). There was no apparent indication of a plateau being approached. This is in contrast to amiloride itself (Fig. 5), and to the situation found for the effect of the amilorides on [³H]yohimbine dissociation from the _2A-adrenergic receptor, where the approach to a k_obs plateau is evident (data not shown).

It was however possible to fit some of the k_obs data to a logistic equation (eq. 13) (Fig. 6). The calculated slope factors ranged from 1.2 to 2.1, reflecting the complex apparently positive cooperative interactions evident even in the tail of the dose response curves (Figs. 4 and 5). The slope factors were essentially independent of the uncertainty of the k_max values associated with the considerable extrapolation. Any interpretation of the k_max values obtained should be treated with caution, because eq. 13 is a logistic equation and not directly derived from a model.

Analyses of the effect of the amilorides on the k_obs of [³H]prazosin (Fig. 5) gave estimates of the initial gradients for all six amilorides (Table 5). This, from the one- or two-site models (see Appendix), is K₂ · (k₂  k₁) and is a measure of the sensitivity of the antagonist dissociation kinetics to low concentrations of the amilorides. With amiloride, where estimates of the individual parameters are obtainable (Table 4), the calculated product is in agreement with the initial gradient. The product K₂ · (k₂  k₁) also was calculated for the modulation of [³H]yohimbine dissociation by amilorides at the _2A-adrenergic receptor (Leppik et al., 1998a) (Table 5). To enable a more direct comparison of the values for the two receptor subtypes, the initial gradient values were normalized by dividing by k_-1. The patterns of the normalized values differ between the two adrenergic receptor subtypes, the estimates being ~20-fold larger at the _1A-adrenergic receptor for amiloride and BZA, ~5-fold larger for DMA, approximately the same for MBA and EPA, and ~2-fold lower at the _1A-adrenergic receptor for HMA. With amiloride, the ~20-fold difference reflects in large part the difference in antagonist dissociation rates from the amiloride-occupied receptors, given that the amiloride affinities at both antagonist-occupied receptor subtypes are comparable (Table 4; Leppik et al., 1998a).

Multiple allosteric sites for different ligands are well known with ion channel receptors (Galzi and Changeux, 1994), and with G protein-coupled receptors the obvious example of two distinct allosteric sites is the modulation of agonist binding by both an allosteric ligand and a G protein. For the _2A-adrenergic receptor, it also has been shown that Na⁺ and amilorides act via different sites to modulate [³H]yohimbine binding (Horstman et al., 1990). However, there are few reports suggesting two allosteric sites for the same ligand on G protein-coupled receptors. In one such report, the biphasic effect of gallamine on [³H]quinuclidinylbenzilate dissociation from muscarinic receptors at low, but not high, ionic strength was attributed to gallamine acting via two allosteric sites (Ellis and Seidenberg, 1989). However, none of the studies attempted to fit data to a model, to support and quantitate the postulated mechanism.

An alternative explanation of the results reported herein is that receptor dimerization (if it occurs for these receptors) may be modulated by the amilorides. However, as the data are compatible with the simpler model with two allosteric sites on a monomeric receptor, we have not considered a more complex dimerization model that involves 13 molecular species and 12 equilibrium constants.

Studies on the effect of competition between two allosteric ligands on antagonist dissociation is a useful technique for exploring further the interactions that are occurring (Ellis and Seidenberg, 1992; Waelbroeck, 1994; Proska and Tucek, 1995; Leppik et al., 1998a). When the effect of competition between amiloride and DMA on [³H]prazosin dissociation was examined, the data obtained were well fitted by the appropriate one-allosteric-site equation (Fig. 7). The parameter estimates from the fit that relate to amiloride were defined, and agreed with those obtained for the effect of amiloride alone on [³H]prazosin dissociation (Table 4). However the effects of amiloride and DMA were additive, suggesting that the concentrations of amiloride and DMA were both too low relative to their respective K₂ values for one ligand to affect the dose-effect curve of the other. This experiment did however indicate that DMA was not causing a nonspecific perturbation of either the receptor or the membrane and supports the conclusion that all the kinetic effects observed are receptor-specific.

In summary, the results from the current study are compatible with amiloride, in the concentration range tested, interacting with a single, defined, allosteric site on the human _1A-adrenergic receptor to modulate binding of the antagonist [³H]prazosin, with no evidence for interaction at a second allosteric site. In contrast the data for the amiloride analogs examined are compatible with interaction at two sites to modulate antagonist binding.