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Determination of the Residues in the Extracellular Domain of the Nicotinic ? Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

ABSTRACT: Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the ?2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the ?4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine. Chimeric constructs of the ?2 and ?4 subunits located two regions in the extracellular amino-terminal domain of the subunit: the E loop (a loop of the transmitter-binding domain) and a region closer to the amino-terminus that collectively could completely determine the different effects of physostigmine. Point mutations then identified a single residue, ?2(I92) versus ?4(R92), that, when combined with transfer of the E loop, could convert the inhibition seen with ?2 subunits to potentiation and the potentiation seen with ?4 subunits to inhibition. In addition, other point mutations could affect the extent of potentiation or inhibition, indicating that a more extensive set of interactions in the amino-terminal domain plays some role in the actions of physostigmine.

SUBMITTER: Jin X

PROVIDER: S-EPMC5548365 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Determination of the Residues in the Extracellular Domain of the Nicotinic α Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

Jin Xiaochun X Germann Allison L AL Shin Daniel J DJ Akk Gustav G Steinbach Joe Henry JH

Molecular pharmacology 20170619 3

Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the α2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the α4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine. Chimeric constructs of the α2 and α ...[more]

PMID: 28630263

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Project description:Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the ?7 nAChR extracellular ligand-binding domain and the transmembrane domain of ?1 glycine receptor (?7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at ?7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on ?7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for ?-bungarotoxin and similar snake ?-neurotoxins also targeting ?7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Dataset Information

Determination of the Residues in the Extracellular Domain of the Nicotinic ? Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

Publications

Determination of the Residues in the Extracellular Domain of the Nicotinic <i>α</i> Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

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