Structural and molecular determinants of the sensitivity of alpha4beta2 nicotinic acetylcholine receptors to the allosteric ligand desformylflustrabromine

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. By binding to a site of the receptor distinct from the neurotransmitter binding site allosteric modulators alter the response of the receptors to their agonists. There are two major locations of allosteric modulator binding site . One is in subunit interfaces of the extracellular -terminal domain. The other is in the transmembrane domain close to the channel gating machinery. This thesis focu es on a po 1t1 e allosteric modulator of the human a4P2 nAChR, desformylflustrabromine (dFBr) which was found to e ert its potentiating effects on this receptor by binding to a site in the transmembrane region of the a4 subunit. a4P2 nAChRs are the most abundant nAChR type in the brain, where they modulate a range of brain functions such as mood cognition nociception and reward. This receptor ubtype has been shown to be sufficient and necessary for the rewarding and reinforcing properties of nicotine. In addition, a4P2 nAChRs have been implicated in aging-related cognitive dysfunction, Alzheimer's and Parkinson's diseases mood disorders and a rare type of family epilepsy. dFBr is a positive allosteric modulator of the a4P2 and a2P2 nAChRs that di pla s selecti ity against all other nAChRs. Using functional mutagenesis and structural modelling, the molecular basis for the electi e potentiation of a4P2 nAChRs has been identified. The potentiating binding site of dFBr is located in the top-half of a transmembrane ea ity b tween the M3 and M4 helices of the a4 subunit. a4Y309, a4F312 and a4L617 influence dFBr potentiation in accord with a role in dFBr binding. Alanine substitutions of the e residues annulled dFBr potentiation and experiments using MT ET howed that the residues in this putative site are accessible to MTSET and that dFBr competes with MTSET for the access to the cavity. These residues map to a highly con erved intra-subunit cavity in the pentameric ligand gated ion channel (pLGIC) family. In addition the effector system for the potentiating effects of dFBr was also identified. The post-M4 region (C-termina1) and the Cys loop residues FI 67 and F170 of the a4 subunit play central roles in transducing dFBr binding to potentiation of the ACh responses of the a4P2 nAChR. Whilst the residues that contribute to the dFBr binding site in the a4 are conserved across all nAChR subunits, except for a7, the post-M4 region is not. It is thi region that determines the elective potentiating effects of dFBr on a4P2 nAChR. This finding, together with recent data on the effect of propofol in bacterial and invertebrate evolutionary related pLGICs, suggest that for highly conserved transmembrane domain allosteric binding sites the effector machinery associated with these sites rather than the binding sites, define the receptor selectivity of the modulators.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Alcaino-Ayala, Constanza Alejandra

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/39ph-md18

OAI identifier oai:identifier

tle:6b36c65e-c673-4f75-b76f-b27ec343b0c2:d6bd9758-527a-46cd-bfe2-c433766e8fca:1