Alpha-adrenergic receptors on human platelets.

[³H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E₁ (PGE₁) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [³H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE₁-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE₁-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE₁-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE₁-receptor activating cAMP production. The inhibition of PGE₁-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.     

Effects of reserpine and adenosine agonist on α2-adrenergic receptor of rat vas deferens examined with [3H]yohimbine and [3H]clonidine

The changes of [³H]yohimbine and [³H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [³H]clonidine binding sites but had no influence on affinity and number of binding sites of α₂-antagonist, [³H]yohimbine. Amount of [³H]yohimbine binding sites was found to be higher than that of [³H]clonidine with or without the treatment. Inhibition curves of α₂-agonists, clonidine and norepinephrine, on [³H]yohimbine binding were less than unity though α₂-antagonist inhibited with about 1.0 of n_H. The treatment with adenosine agonist reduced the IC₅₀ value of agonists on the [³H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in α₂-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist.On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for α₂-adrenergic receptors and also increased the amount of the α₂-receptors.     

Agonist-induced desensitization of an octopamine receptor

The octopamine-sensitive adenylate cyclase associated with haemocytes of the American cockroach, Periplaneta americana, has been used as a model system with which to study desensitization of the octopamine receptor. Preincubation of the haemocytes with octopamine results in a large decrease in subsequent maximal stimulation of cyclic AMP production by octopamine with little change in affinity of the receptor for the agonist. This effect of preincubation is dependent upon the concentration of octopamine in the preincubation media and on the duration of exposure. The attenuation appears to be a receptor-mediated event rather than an artifact of the preincubation. Octopamine receptor agonists (octopamine, synephrine, N-demethylchlordimeform) induce desensitization while biogenic amines with poor octopamine receptor affinity (dopamine, serotonin, norepinephrine) are without affect. In contrast, the octopamine receptor antagonist, phentolamine, appears to enhance subsequent stimulation by octopamine. The attenuation of octopamine stimulation of adenylate cyclase is conserved in broken-cell preparations with no alteration of responses to NaF or forskolin. Incubation of the cells with dibutyryl cyclic AMP or forskolin does not induce desensitization. The data indicate that the OA receptors coupled to AC in cockroach haemocytes undergo an homologous desensitization in response to exposure to agonists.     

Author index

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as $\text{p-}\text{azido}\text{-m-[}{}^{\mathrm{<mtext>125</mtext><mtext>I</mtext><mtext>]</mtext><mtext>iodobenzylcarazolol</mtext>}}\text{. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su?}$ variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Cardiomegaly produced by chronic beta-adrenergic stimulation in the rat: comparison with alpha-adrenergic effects.

Chronic administration of d, l isoproterenol, 0.2 – 5 mg/kg/day, for 14–21 days in the male rat produced marked increases in dry ventricle weight (21.1 – 43.6%; p < 0.001). In comparison, an α-adrenergic agonist, phenylephrine (7.5 mg/kg/day) decreased ventricle weight (?15.3%; p < 0.025). Also, isoproterenol injection at 5 mg/kg/day decreased cardiac actomyosin ATPase activity by 23.3% (p < 0.0025) while phenylephrine, administered as above, did not influence ATPase activity. The effect of isoproterenol on heart weight was completely blocked by the β₁-adrenergic antagonist practolol (5 mg/kg/day). Albuterol, a relatively specific β₂-adrenergic agonist was less potent than isoproterenol in producing cardiac hypertrophy. l-Epinephrine injection, 0.8 mg/kg/day for 14 days, had no effect on heart weight. However, l-epinephrine produced cardiac hypertrophy (22.4% p < 0.001) when the animals were preinjected with the α-adrenergic antagonist, phenoxybenzamine (5 mg/kg/day). The data indicate that cardiac hypertrophy can be produced by stimulation of the β₁-adrenergic receptors of the heart; apparently, stimulation of α-adrenergic receptors opposes β-adrenergic hypertrophic effects.     

Binding of agonists and antagonists to the porcine adipose tissue β-adrenergic receptor(s)

1. Affinities of agonists for porcine adipose tissue β-adrenergic receptors, determined by competitive ligand binding with ³H-dihydroalprenolol to crude adipose tissue membranes in vitro, varied from 50 times > to 25 times < than isoproterenol. Affinities for antagonists varied from 8 times > to 1000 times < propranolol.2. Receptor affinity was not related to the ability to stimulate or inhibit lipolysis, or to the agonist or antagonist purported receptor subtype specificity.3. Modeling of ligand-binding data indicated more than one binding site for several ligands. The assignment of β-adrenergic subtypes to the individual binding sites was unclear because this would depend on the individual ligands used to establish binding sites.     

Cannabinoid CB2 Receptors Regulate Central Sensitization and Pain Responses Associated with Osteoarthritis of the Knee Joint

Osteoarthritis (OA) of the joint is a prevalent disease accompanied by chronic, debilitating pain. Recent clinical evidence has demonstrated that central sensitization contributes to OA pain. An improved understanding of how OA joint pathology impacts upon the central processing of pain is crucial for the identification of novel analgesic targets/new therapeutic strategies.Inhibitory cannabinoid 2 (CB₂) receptors attenuate peripheral immune cell function and modulate central neuro-immune responses in models of neurodegeneration. Systemic administration of the CB₂ receptor agonist JWH133 attenuated OA-induced pain behaviour, and the changes in circulating pro- and anti-inflammatory cytokines exhibited in this model. Electrophysiological studies revealed that spinal administration of JWH133 inhibited noxious-evoked responses of spinal neurones in the model of OA pain, but not in control rats, indicating a novel spinal role of this target. We further demonstrate dynamic changes in spinal CB₂ receptor mRNA and protein expression in an OA pain model. The expression of CB₂ receptor protein by both neurones and microglia in the spinal cord was significantly increased in the model of OA. Hallmarks of central sensitization, significant spinal astrogliosis and increases in activity of metalloproteases MMP-2 and MMP-9 in the spinal cord were evident in the model of OA pain. Systemic administration of JWH133 attenuated these markers of central sensitization, providing a neurobiological basis for analgesic effects of the CB₂ receptor in this model of OA pain. Analysis of human spinal cord revealed a negative correlation between spinal cord CB₂ receptor mRNA and macroscopic knee chondropathy.These data provide new clinically relevant evidence that joint damage and spinal CB₂ receptor expression are correlated combined with converging pre-clinical evidence that activation of CB₂ receptors inhibits central sensitization and its contribution to the manifestation of chronic OA pain. These findings suggest that targeting CB₂ receptors may have therapeutic potential for treating OA pain.     

Beta-adrenoceptors of human lymphocytes: decrease in affinity of agonist for (-)3h-dihydroalprenolol binding sites by cytoplasmic fractions

The beta-adrenoceptors of intact human lymphocytes were investigated by binding assays with a radiolabeled β-adrenergic antagonists, (?)³H-dihydroalprenolol (DHA). Results shown are 1.48 ± 0.57 × 10⁴ binding sites for the ligand per cell. Binding of (?)³H-DHA to the membrane fractions of human lymphocytes was studied for comparison. The affinity of (?)³H-DHA for membrane was similar to that for whole lymphocytes, but the binding to the membranes was inhibited more strongly by β-adrenergic agonist. Moreover, the cytoplasmic fraction of human lymphocytes decreased the inhibition by (?)-isoproterenol of (?)³H-DHA binding to the membrane fraction. The results imply that the cytoplasmic fraction of human lymphocytes lowered the affinity of (?)-isoproterenol to (?)³H-DHA binding sites and left no effect on the binding of the β-adrenergic antagonist to the sites.     

Affinity of different α1-agonists and antagonists for the α1-adrenoceptors of rabbit and rat liver membranes

In membranes prepared from rabbit liver, competition with [³H] prazosin by different α₁-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned α_1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned α_1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these α₁-adrenergic subtypes.     

Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

Background

Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta ₂-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta₂-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.

Results

The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta₂-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta₂-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β₂-adrenergic receptor for those of Ste2 creates a Ste2/beta₂-adrenergic receptor chimera displaying insulin-stimulated internalization.

Conclusion

Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

M2-Muscarinic Receptors: How Does Ligand Binding Affinity Relate to Intrinsic Activity?

Abstract

In this study we looked for evidence regarding a correlation between M₂-muscarinic receptor binding affinity and ligand intrinsic activity. Guanine nucleotide-binding protein-coupled receptors have been shown to exist in both a high affinity and a low affinity, agonist state. The agonist [³H]Oxotremorine-M, was used to determine the affinity of compounds for the high affinity state and the antagonist, [³H]N-methylscopolamine, plus GppNHp, was used to determine the affinity for the low agonist state. The magnitude of the difference in the affinity a compound has for the high versus the low agonist state of the receptor has been related to the intrinsic activity of the compound. NMS/Oxo-M ratios were established for muscarinic agonists, partial agonists and antagonists. NMS/Oxo-M ratios varied from 1695 for the agonist carbachol to 1.9 for the antagonist AFDX-116 with intermediate values for the partial agonists oxotremorine-M, pilocarpine and RS86 (233, 36 and 17 respectively). Intrinsic activity was assessed by receptor-mediated G_i-protein GTPase activity. Indeed, a close correlation (r=0.92) was found between the NMS/Oxo-M ratios of the ligands on the one hand, and their ability to activate the M₂-receptor coupled G_i-protein on the other.     

Fused tricyclic pyrrolizinones that exhibit pseudo-irreversible blockade of the NK1 receptor

Previously, we had disclosed a novel class of hNK₁ antagonists based on the 5,5-fused pyrrolidine core. These compounds displayed subnanomolar hNK₁ affinity along with good efficacy in a gerbil foot-tapping (GFT) model, but unfortunately they had low to moderate functional antagonist (IP-1) activity. To elaborate on the SAR of this class of hNK₁ compounds and to improve functional activity, we have designed and synthesized a new class of hNK₁ antagonist with a third fused ring. Compared to the 5,5-fused pyrrolidine class, these 5,5,5-fused tricyclic hNK₁ antagonists maintain subnanomolar hNK₁ binding affinity with highly improved functional IP-1 activity (<10% SP remaining). A fused tricyclic methyl, hydroxyl geminally substituted pyrrolizinone (compound 20) had excellent functional IP (<2% SP remaining), hNK₁ binding affinity, off-target selectivity, pharmacokinetic profile and in vivo activity. Complete inhibition of agonist activity was observed at both 0 and 24 h in the gerbil foot-tapping model with an ID₅₀ of 0.02 mpk at both 0 and 24 h, respectively.     

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [³H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca²⁺ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological properties.     

Spinal β-adrenergic receptors’ activation increases the blood glucose level in mice

We examined the role of spinally located β-adrenergic receptors in the regulation of the blood glucose level. The intrathecal (i.t.) injections with dobutamine (β₁-adrenergic receptor agonist) or terbutaline (β₂-adrenergic receptor agonist) caused an elevation of the blood glucose level, whereas metoprolol (β₁-adrenergic receptor antagonist) or butoxamine (β₂-adrenergic receptor antagonist) did not. In addition, i.t. pretreatment with pertussis toxin (PTX) attenuated the hyperglycemic effect induced by dobutamine or terbutaline. Moreover, plasma insulin level was increased by dobutamine but not by terbutaline, and PTX reduced dobutamine-induced up-regulation of the plasma insulin level. Terbutaline significantly increased plasma corticosterone level, and PTX further enhanced terbutaline-induced corticosterone level. Furthermore, intraperitoneal (i.p.) pretreatment with hexamethonium- (a preganglionic blocker) attenuated dobutamine- and terbutaline-induced hyperglycemic effects. Our results suggest that activation of spinal β₁- and β₂-adrenergic receptors produces hyperglycemic effects in a different manner. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by terbutaline. Furthermore, dobutamine- or terbutaline-induced hyperglycemia appears to be mediated through the spinal nerves.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Characteristics of β-adrenergic receptors in bovine corneal epithelium: Comparison of fresh tissue and cultured cells

The characteristics of the β-adrenergic receptors in homogenates of fresh tissue and cultured bovine corneal epithelium were compared using [³H]dihydroalprenolol. High affinity, specific binding sites were observed in both preparations. Fresh tissue exhibited a higher binding site density (165 fmol/mg protein) than did cells in culture (57 fmol/mg protein). Studies with various β-adrenergic agonists and antagonists indicated that binding characteristics were typical of β-adrenergic receptors, predominantly of the β₂ subtype. These results demonstrate that β-adrenergic receptors exist in both fresh and cultured bovine corneal epithelium and that these receptors are qualitatively and quantitatively similar.     

Evaluation of N-substituent structural variations in opioid receptor profile of LP1

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9–15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (K_i^MOR = 38 ± 4 nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA₂) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD₅₀ = 2.0 mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function.     

Effects of thiol modification and Ca++ on agonist-specific state transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax.

The agonist binding affinity of nicotinic acetylcholine receptor (nAChR) from $\text{Torpedo}$$\text{californica}$ electroplax, as inferred from ability of agonist to inhibit specific curaremimetic neurotoxin binding to nAChR, is sensitive to the duration of exposure to agonist. The concentration of carbachol necessary to prevent one-half of toxin binding over a 30 min incubation with nAChR (K₃₀) is 10 μM when toxin and carbachol are simultaneously added to membrane-bound nAChR, and 3 μM when nAChR are pretreated with carbachol for 30 min prior to the addition of toxin. These alterations in agonist affinity may be mimicked by modification of nAChR thiol groups. Affinity of nAChR for carbachol is decreased following treatment with dithiothreitol (DTT). Dithio-bis-nitrobenzoic acid treatment of DTT-reduced membranes yields K₃₀ values of 5 μM for carbachol, while N-ethylmaleimide treatment of DTT-reduced nAChR produces nAChR with reduced affinity for carbachol, reflected in K₃₀ values of about 400 μM. In the absence of Ca⁺⁺, K₃₀ values for carbachol binding to native and DTT-reduced nAChR are diminished 3–6 fold. These affinity alterations are not observed with d-tubocurarine (antagonist) binding to nAChR. Thus, Ca⁺⁺ and the oxidation state of nAChR thiols appear to affect the affinity of nAChR for agonists (but not antagonists), and may therefore be related to agonist-mediated events in receptor activation and/or desensitization.