Allosteric activation of plasma membrane receptors--physiological implications and structural origins

Allosteric modulation of receptors has physiological not just pharmacological significance. Thus, the chemical context in which an agonist signal is received can have a major impact on the nature of the physiological response by modifying receptor sensitivity and/or maximal activity-even the nature of the signalling response. In addition, recognising that an endogenous activator is the allosteric modulator of a known receptor, rather than the agonist of a novel receptor, has the potential to solve, in dramatic fashion, key physiological questions. What is an allosteric modulator and why are allosteric effects on receptors so diverse and frequently complex? What is the scope of allosteric effects? Can the existence of endogenous modulators be predicted from a receptor's amino acid sequence? How should screening for endogenous allosteric modulators be undertaken? These questions form the framework of this mini-review on physiological and structural aspects of receptor allostery.     

Ago-allosteric modulation and other types of allostery in dimeric 7TM receptors

Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own and as enhancers for the endogenous agonists in both increasing agonist potency and providing additive efficacy-superagonism. The additive efficacy can also be observed with agonists, which are neutral or even negative modulators of the potency of the endogenous ligand. Based on the prevailing dimeric concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but-importantly-in the "other" or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric modulators can act through binding in the "other" protomer. It is suggested that for the in vivo, clinical setting a positive ago-allosteric modulator should be the preferred agonist drug.     

Ago-Allosteric Modulation and Other Types of Allostery in Dimeric 7TM Receptors

Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own and as enhancers for the endogenous agonists in both increasing agonist potency and providing additive efficacy—superagonism. The additive efficacy can also be observed with agonists, which are neutral or even negative modulators of the potency of the endogenous ligand. Based on the prevailing dimeric concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but–importantly–in the “other” or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric modulators can act through binding in the “other” protomer. It is suggested that for the in vivo, clinical setting a positive ago-allosteric modulator should be the preferred agonist drug.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

A Monod-Wyman-Changeux mechanism can explain G protein-coupled receptor (GPCR) allosteric modulation

The Monod-Wyman-Changeux (MWC) model was initially proposed to describe the allosteric properties of regulatory enzymes and subsequently extended to receptors. Yet despite GPCRs representing the largest family of receptors and drug targets, no study has systematically evaluated the MWC mechanism as it applies to GPCR allosteric ligands. We reveal how the recently described allosteric modulator, benzyl quinolone carboxylic acid (BQCA), behaves according to a strict, two-state MWC mechanism at the M1 muscarinic acetylcholine receptor (mAChR). Despite having a low affinity for the M1 mAChR, BQCA demonstrated state dependence, exhibiting high positive cooperativity with orthosteric agonists in a manner that correlated with efficacy but negative cooperativity with inverse agonists. The activity of BQCA was significantly increased at a constitutively active M1 mAChR but abolished at an inactive mutant. Interestingly, BQCA possessed intrinsic signaling efficacy, ranging from near-quiescence to full agonism depending on the coupling efficiency of the chosen intracellular pathway. This latter cellular property also determined the difference in magnitude of positive cooperativity between BQCA and the orthosteric agonist, carbachol, across pathways. The lack of additional, pathway-biased, allosteric modulation by BQCA was confirmed in genetically engineered yeast strains expressing different chimeras between the endogenous yeast G(pa1) protein and human Gα subunits. These findings define a chemical biological framework that can be applied to the study and classification of allosteric modulators across different GPCR families.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor

Ligand binding to extracellular domains of G protein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg¹⁰-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca²⁺ or phosphoinositide turnover by a leftward shift of the dose-response curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primary human endothelial cells inhibited the allosteric enhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling.     

Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y₂R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca²⁺). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca²⁺. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.     

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

Allosteric modulator ORG27569 induces CB1 cannabinoid receptor high affinity agonist binding state, receptor internalization, and Gi protein-independent ERK1/2 kinase activation

The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and Δ(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.     

Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M₁ muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M₁ mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85^2.64 in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397^7.32 and Trp-400^7.35 in TMVII as residues that contribute to the BQCA binding pocket at the M₁ mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described “common” allosteric site in the extracellular vestibule of the M₁ mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102^3.29 and Asp-105^3.32) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.     

Allosteric effects of four stereoisomers of a fused indole ring system with 3H-N-methylscopolamine and acetylcholine at M1-M4 muscarinic receptors

We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.     

A missense mutation in the seven-transmembrane domain of the human Ca2+ receptor converts a negative allosteric modulator into a positive allosteric modulator

G protein-coupled receptors (GPCRs) are the most common targets of drug action. Allosteric modulators bind to the seven-transmembrane domain of family 3 GPCRs and offer enhanced selectivity over orthosteric ligands that bind to the large extracellular N terminus. We characterize a novel negative allosteric modulator of the human Ca(2+) receptor, Compound 1, that retains activity against the E837A mutant that lacks a response to previously described positive and negative modulators. A related compound, JKJ05, acts as a negative allosteric modulator on the wild type receptor but as a positive modulator on the E837A mutant receptor. This positive modulation critically depends on the primary amine in JKJ05, which appears to interact with acidic residue Glu(767) in our model of the seven-transmembrane domain of the receptor. Our results suggest the need for identification of possible genetic variation in the allosteric site of therapeutically targeted GPCRs.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

The pharmacologic characterization of allosteric molecules: Gq protein activation

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo.

Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales.

Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1–(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration–response curves were fit to the functional allosteric model to yield indices of agonism (τ_B), effects on affinity (α cooperativity), and efficacy (β cooperativity).

Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model.

Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.     

Calcium-dependent block of P2X7 receptor channel function is allosteric

Among purinergic P2X receptor (P2XR) channels, the P2X7R exhibits the most complex gating kinetics; the binding of orthosteric agonists at the ectodomain induces a conformational change in the receptor complex that favors a gating transition from closed to open and dilated states. Bath Ca(2+) affects P2X7R gating through a still uncharacterized mechanism: it could act by reducing the adenosine triphosphate(4-) (ATP(4-)) concentration (a form proposed to be the P2X7R orthosteric agonist), as an allosteric modulator, and/or by directly altering the selectivity of pore to cations. In this study, we combined biophysical and mathematical approaches to clarify the role of calcium in P2X7R gating. In naive receptors, bath calcium affected the activation permeability dynamics indirectly by decreasing the potency of orthosteric agonists in a concentration-dependent manner and independently of the concentrations of the free acid form of agonists and status of pannexin-1 (Panx1) channels. Bath calcium also facilitated the rates of receptor deactivation in a concentration-dependent manner but did not affect a progressive delay in receptor deactivation caused by repetitive agonist application. The effects of calcium on the kinetics of receptor deactivation were rapid and reversible. A438079, a potent orthosteric competitive antagonist, protected the rebinding effect of 2'(3')-O-4-benzoylbenzoyl)ATP on the kinetics of current decay during the washout period, but in the presence of A438079, calcium also increased the rate of receptor deactivation. The corresponding kinetic (Markov state) model indicated that the decrease in binding affinity leads to a decrease in current amplitudes and facilitation of receptor deactivation, both in an extracellular calcium concentration-dependent manner expressed as a Hill function. The results indicate that calcium in physiological concentrations acts as a negative allosteric modulator of P2X7R by decreasing the affinity of receptors for orthosteric ligand agonists, but not antagonists, and not by affecting the permeability dynamics directly or indirectly through Panx1 channels. We expect these results to generalize to other P2XRs.     

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.     

Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.