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 sites. One is in subunit interfaces of the extracellular N-terminal domain. The other is in the transmembrane domain close to the channel gating machinery. This thesis focuses on a positive allosteric modulator of the human α4β2 nAChR, desformylflustrabromine (dFBr), which was found to exert its potentiating effects on this receptor by binding to a site in the transmembrane region of the α4 subunit. α4β2 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 subtype has been shown to be sufficient and necessary for the rewarding and reinforcing properties of nicotine. In addition, α4β2 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 α4β2 and α2β2 nAChRs that displays selectivity against all other nAChRs. Using functional mutagenesis and structural modelling, the molecular basis for the selective potentiation of α4β2 nAChRs has been identified. The potentiating binding site of dFBr is located in the top-half of a transmembrane cavity between the M3 and M4 helices of the α4 subunit. α4Y309, α4F312 and α4L617 influence dFBr potentiation in accord with a role in dFBr binding. Alanine substitutions of these residues annulled dFBr potentiation and experiments using MTSET showed 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 conserved 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-terminal) and the Cys loop residues F167 and F170 of the α4 subunit play central roles in transducing dFBr binding to potentiation of the ACh responses of the α4β2 nAChR. Whilst the residues that contribute to the dFBr binding site in the α4 are conserved across all nAChR subunits, except for α7, the post-M4 region is not. It is this region that determines the selective potentiating effects of dFBr on α4β2 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.
Alcaino-Ayala, C
Faculty of Health and Life SciencesDepartment of Biological and Medical Sciences
Year: 2015
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