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At least 235 records · Page 13Linked to original sources
PubMed2001

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue.

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.

Adenosine Triphosphate↗
PubMed1998

Use of a spreadsheet to quantitate the equilibrium binding of an allosteric modulator.

Using the program Microsoft EXCEL, a spreadsheet was developed for constrained, simultaneous analysis of multiple datasets obtained from equilibrium binding experiments, according to an allosteric model of interaction. This approach was used to quantitate the interaction between the modulator (heptane-1,7-bis (dimethyl 3'-phthalimidopropyl) ammonium bromide) (C7/3-phth) and the radioligands [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate at cortical and atrial muscarinic receptors. The interaction between various concentrations of the radioligands and C7/3-phth, in the guinea pig atrium and in the rat cerebral cortex, could be well described by the allosteric model. The affinity of C7/3-phth for unoccupied atrial receptors was significantly higher than for cortical receptors. The negative cooperativity between [3H]quinuclidinyl benzilate and the modulator was higher in cortex than that between the modulator and [3H]N-methylscopolamine. It is suggested that the described method has wide applicability because of the extensive availability of spreadsheet programs, the analytical advantages offered by constrained, simultaneous nonlinear regression and the ability to adapt the spreadsheet to almost any model of ligand-receptor interaction.

Allosteric Regulation↗
PubMed1988

Potential-dependent allosteric modulation of 1,4-dihydropyridine binding by d-(cis)-diltiazem and (+/-)-verapamil in living cardiac cells.

We have studied allosteric effects of the Ca channel blockers d-(cis)-diltiazem, (+/-)-verapamil, and (S)- and (R)-devapamil on the specific binding of the 1,4-dihydropyridine derivative (+)-[3H]PN 200-110 to intact tissue cultured rat heart cells. In polarized cells (membrane potential, -38 +/- 4 mV) d-(cis)-diltiazem (5 microM) increased the affinity of the radiolabel 2-4-fold causing 100-187% enhancement of binding at (+)-PN 200-110 concentrations below 0.5 nM. (+/-)-Verapamil (0.1-3 microM) had a similar, although smaller, effect on (+)-PN 200-110 binding. The two enantiomers of devapamil were without effect. In depolarized cells (membrane potential, 0 mV) d-(cis)-diltiazem had a small and the phenylalkylamines a strong inhibitory effect on (+)-PN 200-110 binding, mainly due to a reduction of binding affinity. At 50% receptor occupation by the radioligand, (R)-devapamil, (+/-)-verapamil, and (S)-devapamil displaced 40, 55, and 75%, respectively, of specifically bound radiolabel. Half-maximal effects were reached with 50, 20, and 4.5 nM, respectively, of the three compounds. Compared with nominally Ca-free medium (containing 3-5 microM Ca), addition of 1.25 mM CaCl2 caused an increase in the maximal binding capacity for (+)-PN 200-110 in both polarized and depolarized cells. However, Ca had only marginal effects on the allosteric interactions between (+)-PN 200-110, d-(cis)-diltiazem, and verapamil. We conclude from our results that positive cooperative interactions between Ca channel blockers prevail under conditions in which the voltage-dependent Ca channel can fluctuate between closed, open, and inactivated states. Negative cooperativity is usually observed under conditions in which all channels are inactivated (depolarized cells, fragmented membranes). Therefore, it is impossible to predict the type and the extent of allosteric interactions in vivo from studies in cell homogenates.

Allosteric Regulation↗
PubMed2001

Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane.

Staphylococcal alpha-toxin forms heptameric pores on eukaryotic cells. After binding to the cell membrane in its monomeric form, the toxin first assembles into a heptameric pre-pore. Subsequently, the pre-pore transforms into the final pore by membrane insertion of an amphipathic beta-barrel, which comprises the "central loop" domains of all heptamer subunits. The process of membrane insertion was analyzed here using a set of functionally altered toxin mutants. The results show that insertion may be initiated within an individual protomer when its NH2 terminus activates its central loop. The activated state is then shared with the central loops of the residual heptamer subunits, which results in cooperative membrane penetration. This cooperation of the central loops commences while these are still remote from the lipid bilayer. Nevertheless, it is subject to modulation by the target membrane, which therefore acts across a distance much like an allosteric effector. However, while allosteric transitions usually are reversible, membrane insertion of alpha-toxin is an irreversible event, and we show here that it can proceed to completion in a domino-like fashion when triggered by as little as a single foreign atom within the entire heptamer.

Allosteric Regulation↗
PubMed1999

Dual allosteric modulation of pacemaker (f) channels by cAMP and voltage in rabbit SA node.

1. A Monod-Whyman-Changeux (MWC) allosteric reaction model was used in the attempt to describe the dual activation of 'pacemaker' f-channel gating subunits by voltage hyperpolarization and cyclic nucleotides. Whole-channel kinetics were described by assuming that channels are composed of two identical subunits gated independently according to the Hodgkin-Huxley (HH) equations. 2. The simple assumption that cAMP binding favours open channels was found to readily explain induction of depolarizing voltage shifts of open probability with a sigmoidal dependence on agonist concentration. 3. Voltage shifts of open probability were measured against cAMP concentration in macropatches of sino-atrial (SA) node cells; model fitting of dose-response relations yielded dissociation constants of 0.0732 and 0.4192 microM for cAMP binding to open and closed channels, respectively. The allosteric model correctly predicted the modification of the pacemaker current (If) time constant curve induced by 10 microM cAMP (13.7 mV depolarizing shift). 4. cAMP shifted deactivation more than activation rate constant curves, according to sigmoidal dose-response relations (maximal shifts of +22.3 and +13.4 mV at 10 microM cAMP, respectively); this feature was fully accounted for by allosteric interactions, and indicated that cAMP acts primarily by 'locking' f-channels in the open configuration. 5. These results provide an interpretation of the dual voltage- and cyclic nucleotide- dependence of f-channel activation.

Allosteric Regulation↗
PubMed1996

A behavioural and neurochemical study in rats of the pharmacology of loreclezole, a novel allosteric modulator of the GABAA receptor.

Loreclezole is an anticonvulsant and anxiolytic compound which has been reported to potentiate GABA via a novel allosteric site on the beta-subunit of the receptor. We have now studied in rats both the in vivo and in vitro pharmacology of the compound. The dose of loreclezole required to increase by 50% the dose of intravenous pentylenetetrazol eliciting a seizure was comparable to that of barbiturates and chlormethiazole (in mg/kg): diazepam, 1.3; pentobarbitone, 16; chlormethiazole, 22; loreclezole, 25; pentobarbitone, 36. Loreclezole dose-dependently decreased locomotion (dose to decrease locomotion by 50% (in mg/kg): chlormethiazole, 9; pentobarbitone, 16; loreclezole, 25). Loreclezole, chlormethiazole and pentobarbitone all failed to displace [3H]muscimol and [3H]flunitrazepam binding from a rat cortical membrane preparation. All three compounds fully displaced [35S]TBPS binding (IC50 values: loreclezole, 4.34 +/- 0.68 microM; pentobarbitone, 37.39 +/- 3.24 microM; chlormethiazole, 82.10 +/- 8.52 microM). Addition of bicuculline (10 microM) produced a major rightward shift in the loreclezole and pentobarbitone displacement curves, increasing IC50 values for [35S]TBPS binding by 25 times (loreclezole), 6 times (pentobarbitone) and 2.7 times (chlormethiazole), suggesting a greater involvement of GABA in the interaction of loreclezole with the chloride channel than in the case of chlormethiazole. Anticonvulsant activity of the compounds did not appear to relate to [35S]TBPS binding activity. Other binding data suggested that although the evidence of others indicates that loreclezole interacts with a specific allosteric site on the beta-subunit, it nevertheless also alters the binding characteristics of other modulatory sites.

Allosteric Regulation↗
PubMed1997

Allosteric modulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin.

We investigated the functional and allosteric effects of the 4-amino analogue of tetrahydrobiopterin, (6R)-2,4-diamino- 5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxypropyl) pteridine (4-amino-H4biopterin) on pteridine-free rat neuronal nitric oxide synthase. In the presence of added (6R)-5,6,7,8-tetrahydro-L-erythrobiopterin (H4biopterin; 10 microM), 4-amino-H4biopterin completely inhibited the conversion of both L-arginine and NG-hydroxy-L-arginine with half-maximally effective concentrations of 1.1+/-0.09 and 1.3+/-0.09 microM, respectively. Inhibition was reversible, as shown by a time-dependent restoration of citrulline formation upon dilution of the inhibitor-treated enzyme (t1/2=3.0 min). Binding of 4-amino-H4biopterin led to a complete conversion of the haem from low-spin to high-spin state, and to the formation of stable homodimers which partially survived electrophoresis under denaturating conditions. These results show that oxidation of both L-arginine and NG-hydroxy-L-arginine is pteridine-dependent, and that the allosteric effects of H4biopterin do not fully explain the essential role of the pteridine cofactor in nitric oxide biosynthesis.

Allosteric Regulation↗
PubMed1999

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels.

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.

Algorithms↗
PubMed1998

Depolarization differentially affects allosteric modulation by neurotoxins of scorpion alpha-toxin binding on voltage-gated sodium channels.

Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K+ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion alpha-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 mM K+). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 mM K+). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.

Allosteric Regulation↗
PubMed1998

Allosteric modulation of neuronal nicotinic acetylcholine receptors.

The structure-function relationship of the neuronal nicotinic acetylcholine receptor is examined in the light of the allosteric concepts. Effects of site-directed mutagenesis as well as those caused by allosteric effector of the physiological and pharmacological receptor properties are discussed.

Allosteric Regulation↗
PubMed2004

Development of a new type of allosteric modulator of muscarinic receptors: hybrids of the antagonist AF-DX 384 and the hexamethonio derivative W84.

Various fragments of the hexamethonio-type allosteric agent W84 were linked to the secondary amino group of the muscarinic M(2) acetylcholine receptor-preferring antagonist AF-DX 384 to increase the area of attachment with the allosteric site. Addition of only the phthalimido moiety of W84 gave an allosteric enhancer of NMS binding. Thus, a new lead structure for the development of allosteric enhancers of NMS binding has been discovered.

Allosteric Regulation↗
PubMed1999

Binding, partial agonism, and potentiation of alpha(1)-adrenergic receptor function by benzodiazepines: A potential site of allosteric modulation.

Benzodiazepines, a class of drugs commonly used to induce anesthesia and sedation, can attenuate intracellular calcium oscillations evoked by alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation in pulmonary artery smooth muscle cells. We postulated a direct action of benzodiazepines in modulating alpha(1)-AR function at the receptor level. Benzodiazepines bound to each of the cloned alpha(1)-AR subtypes (alpha(1a)-, alpha(1b)-, or alpha(1d)-AR) on COS-1 cell membranes transiently transfected to express a single population of alpha(1)-AR subtype. The ability of benzodiazepines to alter alpha(1)-AR signal transduction was investigated by measuring total inositol phosphate generation in rat-1 fibroblast cells, stably transfected to express a single alpha(1)-AR subtype. By themselves, benzodiazepines displayed partial agonism. At alpha(1b)-ARs and alpha(1d)-ARs, the maximal inositol phosphate response to phenylephrine was potentiated almost 2-fold by either midazolam or lorazepam (100 microM). At alpha(1a)-ARs, diazepam, lorazepam, and midazolam all increased the maximal response of the partial agonist clonidine at these receptors, whereas the response to the full agonist phenylephrine was unaltered or inhibited. The potentiating actions of midazolam and its partial agonism at alpha(1)-ARs was blocked by the addition of 1 microM prazosin, an alpha(1)-AR antagonist, and not by a gamma-aminobutyric acid(A)-receptor antagonist. These studies show that benzodiazepines modulate the function of alpha(1)-ARs in vitro, and this is the first report of a potential allosteric site on alpha(1)-ARs that may be therapeutically useful for drug design.

Adrenergic alpha-Agonists↗
PubMed1985

Calcium-dependent allosteric modulation of the giant hemoglobin from the terrestrial oligochaete, Eisenia foetida.

The effect of calcium and magnesium ions on the oxygen equilibrium of Eisenia hemoglobin was investigated by using an automatic oxygenation apparatus. On addition of calcium chloride (20 mM, pH 7.5), oxygen affinity and cooperativity (nmax) of the hemoglobin increased markedly (p 50:3.82 mmHg, nmax :9.76). The effect of magnesium on the oxygen equilibrium was weaker than that of calcium. The top asymptotes of the oxygen equilibrium curve shifted to the left by adding cations whereas the bottom asymptotes remained almost unchanged. The free energy of heme-heme interaction (delta GR,T) also increased remarkably. These results imply the binding of calcium to Eisenia hemoglobin in the oxygenated form and its physiological role in modulating the oxygen affinity and cooperativity.

Allosteric Regulation↗
PubMed2001

Hexamethonium-type allosteric modulators of the muscarinic receptors bearing lateral dibenzazepine moieties.

Alkane-bisammonium compounds carrying lateral phthalimido substituents are known to have a high affinity for the allosteric binding site of the acetylcholine M2 receptor. The purpose of this study was to replace the lateral phthalimido moieties with rigid tricyclic skeletons of a large volume in order to learn more about the function of the lateral heterocycles. In addition, methyl groups were introduced into the lateral connecting chains. Allosteric inhibition of the dissociation of [3H]N-methylscopolamine from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. The phthalimido groups could be replaced with dibenzazepine moieties without any loss in potency. Interestingly, the additional methyl group in the lateral spacer seems to have a significant influence on the allosteric behaviour.

Allosteric Regulation↗