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Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

ABSTRACT: The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (? subunit loop B) with regard to both of these parameters. ?Gly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. ?Gly153 is a "deactivation" hinge that maintains low values for these parameters. ?Trp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions ?Trp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse.

SUBMITTER: Purohit P

PROVIDER: S-EPMC3061369 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

Purohit Prasad P Auerbach Anthony A

Molecular pharmacology 20101129 3

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting ...[more]

PMID: 21115636

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Project description:Background and purposeThe binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage-clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat ?1?2?2L GABA(A) receptor.Experimental approachSimultaneous electrophysiological and site-specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally-sensitive fluorophore, Alexa 546 maleimide, at the ?1L127C site.Key resultsReceptors activated by GABA demonstrate a concentration-dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine-4-sulphonic acid, muscimol, ?-alanine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA-activated receptors by allopregnanolone or etomidate enhanced both the GABA-elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response.Conclusions and implicationsThe data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546-labelled ?1L127C?2?2L GABA(A) receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone.

Dataset Information

Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

Publications

Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site.

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