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PubMed1984

Effects of temperature and allosteric modulators on [3H] nitrendipine binding: methods for detecting potential Ca2+ channel blockers.

The effects of incubation temperature and allosteric modulators were studied on [3H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca2+ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [3H]nitrendipine binding in a temperature-dependent manner (37 degrees greater than 25 degrees greater than 4 degrees C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37 degrees C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [3H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca2+ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

N-methyl-D-aspartate receptor-mediated changes in thermal nociception: allosteric modulation at glycine and polyamine recognition sites.

The effects of allosteric modulators of the N-methyl-D-aspartic acid receptor ion-channel complex on the nociceptive tail-flick reflex were studied in awake rats. Intrathecal administration of D-serine (100 fmol-1 mumol) but not L-serine or glycine to the lumbar spinal cord produced a facilitation of the tail-flick reflex at doses > or = 1 pmol (maximum at 0.5-1 min). Intrathecal pretreatment with the glycine modulatory site antagonist 7-chlorokynurenic acid (3 pmol) blocked both D-serine-produced and N-methyl-D-aspartate-produced facilitation of the tail-flick reflex. D-serine-produced facilitation was also blocked by intrathecal pretreatment with a N-methyl-D-aspartate receptor ion-channel blocker, MK 801 (100 fmol), or with an alternate substrate for nitric oxide synthase, NG-nitro-L-arginine-methyl ester (100 nmol). Intrathecal administration of spermine (0.01 nmol-3 mumol) produced biphasic effects on tail-flick latency accompanied by mechanical hyperesthesia and vocalization at greater doses. Spermine-produced facilitation (maximum with 0.01 nmol to 1 nmol at 1 min) was blocked by intrathecal pretreatment with MK 801 (100 fmol), NG-nitro-L-arginine-methyl ester (100 nmol) or the polyamine modulatory site antagonist, arcaine (10 nmol). Spermine-produced inhibition (maximum with 300 nmol at 2 min) was blocked by intrathecal administration of MK 801 (1 nmol). Intrathecal administration of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoic acid (1 nmol), blocked inhibition and uncovered a facilitation produced by 1 mumol spermine. In addition, spermine produced multi-stage motor effects (immediate- and late-onset). Intrathecal pretreatment with MK 801 (1 nmol) blocked only the immediate-onset motor effects while the late-onset motor effects were selectively blocked by pretreatment with the kappa opioid receptor antagonist, nor-binaltorphamine (200 nmol). Taken together, these data suggest that D-serine and spermine facilitate nociceptive transmission by positive allosteric modulation of the N-methyl-D-aspartate receptor ion-channel. Furthermore, activation of the N-methyl-D-aspartate receptor is also necessary to elicit the immediate-onset motor effects and inhibition of the tail-flick reflex produced by greater doses of spermine. Because kappa opioid receptors appear to be involved, the spermine-produced late-onset motor effects may involve endogenous dynorphin release.

Allosteric Regulation↗
PubMed1991

Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gamma-aminobutyric acid-gated Cl- currents.

gamma-Aminobutyric acid (GABA)-activated Cl- currents in neonatal rat cortical neurons and in cultured cells engineered for the expression of specific molecular forms of the GABAA receptor alpha, beta, and gamma subunits, were recorded with the patch-clamp technique in the whole-cell configuration. The effects of various allosteric modulators of GABAA receptors were determined. Diazepam and clonazepam showed greater efficacy as positive modulators of GABA-elicited currents in alpha 2 beta 1 gamma 2 or alpha 3 beta 1 gamma 2 receptors than in alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Alpidem was more efficacious at alpha 1 beta 1 gamma 2 or alpha 2 beta 1 gamma 2 receptors than at alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Conversely, zolpidem was equally efficacious for all these receptors except for alpha 5 beta 1 gamma 2. Both imidazopyridines (alpidem and zolpidem) were virtually ineffective at modulating the GABA response of alpha 5 beta 1 gamma 2 receptors and in almost all the receptors assembled from alpha 1, alpha 2, alpha 3 or alpha 5 subunits together with beta 1 and gamma 1 subunits. The beta-carboline derivatives methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and methyl-beta-carboline-3-carboxylate (beta-CCM) elicited a positive allosteric modulation of alpha 1 beta 1 gamma 1 or alpha 2 beta 1 gamma 1 receptors, whereas they acted as negative allosteric modulators at nearly all other receptors tested, as they do in cortical neurons. Although the positive allosteric modulation by beta-carbolines never exceeded a doubling of the GABA response, DMCM was more efficacious at alpha 1 beta 1 gamma 1 receptors and beta-CCM was more efficacious at alpha 2 beta 1 gamma 1 receptors. DMCM was inactive at alpha 3 beta 1 gamma 1 receptors, whereas beta-CCM was virtually inactive at alpha 5 beta 1 gamma 1 receptors. The benzodiazepine 4'-chlorodiazepam, which is a negative modulator resistent to flumazenil inhibition, acted at all the various GABAA receptors that contained a gamma subunit.

Allosteric Regulation↗
PubMed1996

Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors.

The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [3H]N-methylscopolamine from muscarinic M2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M2 receptors in the [3H]N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.

Allosteric Regulation↗
PubMed2004

A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain.

We found that N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca(2+) assays 7- to 8-fold with EC(50) values in the 400 to 800 nM range, and at 10 microM shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4- to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3'-difluorobenzaldazine (DFB) affected [(3)H]quisqualate binding to mGluR5, but although DFB partially competed for [(3)H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 microM) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 microM CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.

Allosteric Regulation↗
PubMed2004

Regulation of M2 muscarinic acetylcholine receptor expression and signaling by prolonged exposure to allosteric modulators.

The effects of prolonged exposure of M(2) muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3'-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [(3)H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 microM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 microM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M(2) mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.

Alcuronium↗
PubMed1995

Alpha-scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine.

At least six topologically separated neurotoxin receptor sites have been identified on sodium channels that reveal strong allosteric interactions among them. We have studied the allosteric modulation induced by veratridine, binding to receptor site 2, and brevetoxin PbTx-1, occupying receptor site 5, on the binding of alpha-scorpion toxins at receptor site 3, on three different neuronal sodium channels: rat brain, locust, and cockroach synaptosomes. We used 125I-AaH II, the most active alpha-scorpion toxin on vertebrates, and 125I-Lqh alpha IT, shown to have high activity on insects, as specific probes for receptor site 3 in rat brain and insect sodium channels. Our results reveal that brevetoxin PbTx-1 generates three types of effects at receptor site 3:1) negative allosteric modulation in rat brain sodium channels, 2) positive modulation in locust sodium channels, and 3) no effect on cockroach sodium channel. However, PbTx-1 activates sodium channels in cockroach axon similarly to its activity in other preparation. Veratridine positively modulates both rat brain and locust sodium channels but had no effect on alpha-toxin binding in cockroach. The dramatic differences in allosteric modulations in each sodium channel subtype suggest structural differences in receptor sites for PbTx-1 and/or at the coupling regions with alpha-scorpion toxin receptor sites in the different sodium channels, which can be detected by combined application of specific channel modifiers and may elucidate the dynamic gating activity and the mechanism of allosteric interactions among various neurotoxin receptors.

Allosteric Regulation↗
PubMed2005

Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators.

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.

Actins↗
PubMed2004

Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists.

Allosteric modulation of [3H]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [3H]strychnine and glycine bindings (alpha and beta, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (beta-alanine, taurine and gamma-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 microM propofol. Further, propofol showed positive cooperativity (beta < 1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [3H]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity (beta >> 1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.

Alanine↗
PubMed2006

The positive allosteric modulator GS39783 enhances GABA(B) receptor-mediated inhibition of cyclic AMP formation in rat striatum in vivo.

We studied the effects of the positive allosteric modulator GS39783 on GABA(B) receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABA(B) receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABA(B) agonist R(-)-baclofen inhibited cyclic AMP formation stimulated by a water-soluble forskolin analogue in a concentration-dependent manner (EC50 7.3 microM, maximal inhibition 40%). The selective GABA(B) antagonist CGP56999 reversed R(-)-baclofen-induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(-)-baclofen (1 microM), significantly decreased cyclic AMP formation in a dose-dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABA(B) receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(-)-baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABA(B) receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein-coupled receptor-positive allosteric modulator.

Animals↗
PubMed2001

The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation.

Most biogenic amine G protein-coupled receptors contain a conserved aspartic acid residue positioned near the intracellular side of the second transmembrane-spanning (TMS) domain that is the primary site of allosteric modulation by sodium ions and pH. Recently, zinc ions and amiloride derivatives were found to allosterically modulate antagonist binding to dopamine receptors. In the current study, the wild-type D4 dopamine receptor showed an 8-fold decrease in zinc affinity in the presence of 120 mM NaCl, but the binding of zinc to the neutral TMS2 D4-D77N mutant was completely sodium-insensitive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wild-type D4 receptor was virtually unaffected by sodium. In addition, the binding affinity for MIA was essentially unchanged in the presence of an IC(50) concentration of zinc and vice versa. Furthermore, MIA binding affinity was decreased 4-fold for the D4-D77N mutant and increased 30-fold for the TMS3 mutant D4-M107V, even though the binding affinity for zinc was similar to the wild-type D4 background for both mutants. These findings demonstrate for the first time the existence of three distinct sites of allosteric modulation within a G protein-coupled receptor.

Amiloride↗
PubMed2001

Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors.

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.

Alcuronium↗
PubMed2003

Multiple molecular determinants for allosteric modulation of alternatively spliced AMPA receptors.

Positive allosteric regulation of glutamate AMPA receptors involves conformational changes that can attenuate receptor desensitization and enhance ion flux through the channel pore. Many allosteric modulators (e.g., cyclothiazide and aniracetam) preferentially affect the flip (i) or flop (o) alternatively spliced isoform of AMPA receptors, implicating residues in the flip-flop domain as critical determinants of splice variant sensitivity. Indeed, previous mutational analyses have demonstrated that the differential sensitivity to cyclothiazide and aniracetam depends on a single amino acid, Ser (flip) and Asn (flop), suggesting that this residue may be solely responsible for differences in modulation of AMPA receptor isoforms. The present studies tested this hypothesis by investigating the molecular determinants of modulation of AMPA receptor splice variants by a structurally distinct compound, LY404187, which displays strikingly different and opposing kinetics of allosteric regulation characterized by a time-dependent enhancement in potentiation of homomeric GluR1-GluR4i and a time-dependent reduction in potentiation of GluR1-GluR4o. Site-directed mutagenesis of residues in the flip-flop domain of GluR2 revealed that, although exchange of Asn775 for Ser in GluR2o was sufficient to confer the GluR2i phenotype of potentiation, the corresponding mutation, Ser775Asn, in GluR2i did not impart the GluR2o response. In fact, the GluR2o kinetics of modulation depended on a novel set of substitutions in GluR2i, including Thr765Asn, Pro766Ala, and Val779Leu in combination with Ser775Asn. Collectively, these results show that, unlike cyclothiazide and aniracetam, the residues that confer splice variant differences in modulation by LY404187 are not identical and indicate that allosteric regulation of AMPA receptors can arise from multiple molecular determinants.

Allosteric Regulation↗
PubMed2005

6-Methylflavanone, a more efficacious positive allosteric modulator of gamma-aminobutyric acid (GABA) action at human recombinant alpha2beta2gamma2L than at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors expressed in Xenopus oocytes.

6-Methylflavanone acted as a positive allosteric modulator of gamma-aminobutyric acid (GABA) responses at human recombinant alpha1beta2gamma2L, alpha2beta2gamma2L and alpha1beta2 GABA(A) receptors expressed in Xenopus laevis oocytes. It was essentially inactive at rho1 GABA(C) receptors. The EC50 values for 6-methylflavanone for the positive modulation of the EC(10-20) GABA responses were 22 microM, 10 microM and 6 microM and the maximum potentiations were 120%, 417% and 130% at alpha1beta2gamma2L, alpha2beta2gamma2L and alpha1beta2 GABA(A) receptors respectively. Thus 6-methylflavanone was much more efficacious as a positive modulator at alpha2beta2gamma2L than at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors. This may be significant since diazepam-induced anxiolysis is considered to be mediated via alpha2-containing GABA(A) receptors, while sedation is thought to be mediated via alpha1-containing GABA(A) receptors. We have previously reported that 6-methylflavone (1-100 microM) produced positive allosteric modulation at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors with no significant difference between the enhancement seen at either receptor subtype. In the present study, 6-methylflavone was tested at alpha2beta2gamma2L GABA(A) receptors and found to maximally potentiate the EC(10-20) GABA response by 183+/-39% which is similar to that previously observed for 6-methylflavone at alpha1beta2gamma2L GABA(A) receptors. Thus, 6-methylflavone did not show a preference for alpha2beta2gamma2L over alpha1beta2gamma2L GABA(A) receptors in terms of efficacy. Compared to 6-methylflavone, 6-methylflavanone is more efficacious as a positive allosteric modulator at alpha2beta2gamma2L GABA(A) receptors, and less efficacious at alpha1beta2gamma2L GABA(A) receptors. This may represent a relatively unique type of selectivity for positive modulators of GABA-A receptor subtypes based on efficacy as distinct from potency. As was previously shown for 6-methylflavone at alpha1beta2gamma2L GABA(A) receptors, the positive modulation of GABA responses at alpha1beta2gamma2L and alpha2beta2gamma2L GABA(A) receptors by 6-methylflavanone was insensitive to antagonism by flumazenil, indicating that this action is not mediated via "high-affinity" benzodiazepine sites.

Allosteric Regulation↗
PubMedDate not supplied

Allosteric modulation of native cochlear P2X receptors: insights from comparison with recombinant P2X2 receptors.

Extracellular adenosine 5'-triphosphate (ATP)-gated ion channels assembled from P2X receptor subunits exhibit subunit-selective allosteric modulation by protons and divalent cations. In voltage-clamped guinea-pig cochlear outer hair cells (OHC) and Deiters' cells (DC), H(+) and Cu(2+), but not Zn(2+), enhanced the P2X receptor-mediated inward currents. Acid pH (6.5) potentiated OHC ATP-gated currents by 45%. Co-application of Cu(2+) (1-40 microM) with ATP increased the response by 20%. In DCs, ATP-gated currents were potentiated 85% by acid pH, and 70% by Cu(2+). Alkaline pH inhibited ATP-gated inward currents by 73% in OHCs and 85% in DCs. Zn(2+) was either ineffective (1-10 microM) or inhibitory (40-400 microM). Recombinant rat P2X(2) receptor-mediated inward currents in XENOPUS oocytes displayed allosteric modulation that was different from the native guinea-pig cochlear P2X receptors. The oocyte ATP-gated inward current was potentiated 450% by shifting from pH 7.5 to pH 6.5, and 130% with 40 microM Cu(2+). The enhanced response to ATP with acid pH and Cu(2+) is a signature of the P2X(2) subunit. In contrast to native guinea-pig cochlear cells, extracellular Zn(2+) (40 microM) increased the recombinant ATP-gated inward current by 200% in oocytes. These results suggest that the positive allosteric modulation of cochlear OHC and DC ATP-gated ion channels by protons and Cu(2+) arises in part from the P2X(2) receptor subunit, with additional regulatory elements.

Adenosine Triphosphate↗
PubMed2000

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors.

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.

Alcuronium↗
PubMed2004

Sites of positive allosteric modulation by neurosteroids on ionotropic gamma-aminobutyric acid receptor subunits.

Neurosteroids are known as allosteric modulators of ionotropic gamma-aminobutyric acid (GABA) receptors. Here, we investigated sites of positive allosteric modulation by allotetrahydrodeoxycorticosterone (5alpha-THDOC) at GABA receptors using the technique of chimeragenesis and the Xenopus oocyte expression system. Our findings have demonstrated that the region from transmembrane segment (TM) 4 to the C-terminus of the GABA(A) receptor alpha1 subunit is crucial for the action of 5alpha-THDOC, but insufficient for the action of another neurosteroid allopregnanolone, suggesting that a specific region critical for neurosteroid action at GABA receptors exists in the domain between TM4 and the C-terminus of GABA receptor subunits.

Allosteric Regulation↗
PubMed2000

Allosteric modulation of nicotinic receptors as a treatment strategy for Alzheimer's disease.

Impairment of the central cholinergic system has a pivotal role in the cognitive decline observed in patients with Alzheimer's disease (AD). One of the most prominent cholinergic deficits is the reduced number of nicotinic acetylcholine receptors (nAChR) in the brain. Since these receptors are important for memory and learning, enhancing nicotinic neurotransmission is a promising treatment strategy for AD. The two most common approaches to correcting these cholinergic deficits are to increase the synaptic availability of acetylcholine (ACh) by inhibiting acetylcholinesterase (AChE), or to mimic the effects of ACh (nicotinic agonists) by acting directly on nicotinic receptors. Clinical studies suggest that AChE inhibitors produce only short-term symptomatic improvement. Similarly, long-term use of nicotinic agonists may induce desensitization of nicotinic receptors, leading to tolerance and therefore limiting the duration of efficacy. Allosteric modulation of nAChR is a novel approach, which circumvents the development of tolerance. Allosteric modulators bind to a site on nAChR that is different to the binding site of the natural agonist, ACh. This allosteric interaction amplifies the actions of ACh at post- and presynaptic nAChR. In particular, presynaptic nAChR are capable of modulating the release of ACh and other neurotransmitters, such as glutamate, serotonin and GABA, which may contribute to symptoms of the illness. Allosteric modulation of nAChR could therefore produce significant therapeutic benefit in AD. One of the most potent of these allosteric modulators is galantamine. As well as modulating nAChR, galantamine inhib- its AChE. The extent to which the clinical benefits of galantamine are attributable specifically to its nicotinic effects is uncertain and requires further investigation. However, galantamine maintains patients' level of cognitive and daily function for at least 1 year, which has not been reported for other AChE inhibitors. Galantamine's modulatory effects on nAChR may influence transcriptional regulation, resulting in an increased synthesis of nAChR. This may account for galantamine's sustained efficacy.

Alzheimer Disease↗