Ligand binding properties of acetylcholinesterase determined with fluorescent probes

Mooser, Gregory; Sigman, David S.

doi:10.1021/bi00708a010

Cited by 111 publications

(73 citation statements)

References 17 publications

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“…This model can also account for the modulatory e ects of MIA at the rat D 2(long) dopamine receptor (Hoare & Strange, 1996). The model is also appropriate for the allosteric e ects of methoctramine and pentamethylene-bis(4-diphenylacetoxymethylpiperidine) at cardiac muscarinic acetylcholine receptors (Waelbroeck, 1994) and has also been applied to modulation of acetylcholinesterase (Mooser & Sigman, 1974). A theoretical interpretation of the model has also been carried out (Tomlinson & Hnatowich, 1988).…”

Section: Discussionmentioning

confidence: 99%

Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues

Hoare

Coldwell

Armstrong

et al. 2000

British J Pharmacology

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, Reading RG6 6AJ1 The modulatory e ects of the allosteric e ectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D 1 , D 2 , D 3 and D 4 dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2 In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D 3 subtype for acceleration of radioligand dissociation. 3 In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D 1 , D 2(short) , D 2(long) and D 3 receptors were described by Hill coe cients exceeding unity and data were ®tted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4 At the D 4 subtype, Hill coe cients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coe cient for MIA at the D 4 subtype was less than unity and data could be ®tted well by the allosteric/competitive model, but it was not possible to de®ne unambiguously the modulatory mechanism. For this e ect a better de®nition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5 MIA reduced the potency with which dopamine stimulated [ 35 S]-GTPgS binding at the D 2 receptor. The e ects of MIA could be described by the allosteric/competitive model with e ects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

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Section: Discussionmentioning

confidence: 99%

Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues

Hoare

Coldwell

Armstrong

et al. 2000

British J Pharmacology

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“…When acetylcholinesterase is inactivated irreversibly with methanesulphonyl fluoride (see Methods), there is no effect on 1/Toff. This might be expected if methanesulphonyl fluoride blocks the enzyme's catalytic activity without blocking the peripheral site where tubocurarine binds (Mooser & Sigman, 1974;Taylor & Lappi, 1975). Therefore we used another method to eliminate tubocurarine binding to the acetylcholinesterase in the cleft.…”

Section: 'mentioning

confidence: 99%

The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft.

Armstrong¹,

Lester²

1979

The Journal of Physiology

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SUMMARY1. The kinetics of tubocurarine inhibition were studied at the post-synaptic membrane of frog skeletal muscle fibres. Acetylcholine (ACh) and (+ )-tubocurarine were ionophoresed from twin-barrel micropipettes, and the membrane potential of the muscle fibre was recorded intracellularly. Tubocurarine-receptor binding was measured by decreases in the response to identical pulses of ACh. 2. The responses to both ACh and tubocurarine had brief latencies and reached their maxima rapidly. It is suggested that under these conditions the kinetics of tubocurarine action are not slowed by diffusion in the space outside the synaptic cleft.3. After a pulse of tubocurarine, recovery from inhibition proceeds along a roughly exponential time course with a rate constant, l/T0! -0. 5 see -1. This rate constant does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25).4. After a sudden maintained increase in tubocurarine release, the ACh responses decrease and eventually reach a new steady-state level. Inhibition develops exponentially with time and the apparent rate constant, 1/Ton, is greater than 1/Tof!. When the steady-state inhibition reduces the ACh response to 1/n of its original level, the data are summarized by the relation, 1/Ton = n(1/Toff).5. When the ACh sensitivity is reduced with cobra toxin, both I/Ton and 1/TO!! increase. Thus, the kinetics of tubocurarine inhibition depend on the density of ACh receptors in the synaptic cleft. 6. After treatment with collagenase, part of the nerve terminal is displaced and the post-synaptic membrane is exposed directly to the external solution. Under these circumstances, 1/Tofr increases more than tenfold.7. Bath-applied tubocurarine competitively inhibits the responses to brief ionophoretic ACh pulses with an apparent equilibrium dissociation constant, K = 0 5 /IM. 8. In denervated frog muscle fibres, extrasynaptic receptors have a lower apparent affinity for tubocurarine. After a pulse of tubocurarine, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the synapse.9. It is suggested that each tubocurarine molecule binds repeatedly to several

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“…A second, "peripheral," anionic site exists, so named because it appears to be distant from the active site (5,6). Bisquaternary AcChoEase inhibitors are believed to derive their enhanced potency, relative to similar monoquaternary ligands (7), from the ability to span the two "anionic" sites, .14 A apart (8,9). The three-dimensional structure of AcChoEase reveals that, like other serine hydrolases, it contains a catalytic triad (10).…”

mentioning

confidence: 99%

Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.

Harel

Schalk

Ehret‐Sabatier

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

874

754

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Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures ofcomplexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and Its m-hydroxyl displays bilbrcated hydrogen bonding to two members of the catalytic triad, Ser-200 and Hls-440. In a complex with tacrine, the acridine Is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium Is oriented along the narrow gorge leading to the active site; one quaternary group Is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This dose Interaction was confirmed by photoaffnity labeling by the photosensitive probe 3H-labeled p-(N,Ndimethylamlno)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active dte. Labeling ofTrp-279 was also observed. One mole oflabel is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role ofaromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

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Ligand binding properties of acetylcholinesterase determined with fluorescent probes

Cited by 111 publications

References 17 publications

Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues

Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues

The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft.

Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.

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Ligand binding properties of acetylcholinesterase determined with fluorescent probes

Cited by 111 publications

References 17 publications

Regulation of human D1, D2(long), D2(short), D3and D4dopamine receptors by amiloride and amiloride analogues

Regulation of human D1, D2(long), D2(short), D3and D4dopamine receptors by amiloride and amiloride analogues

The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft.

Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.

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Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues

Regulation of human D₁, D_2(long), D_2(short), D₃and D₄dopamine receptors by amiloride and amiloride analogues