The role of subunit interfaces in the function of nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChR) containing α4 and β2 nAChR subunits are the most prevalent type of nAChR in the brain, where they modulate an assortment of physiological functions such as cognition, mood, reward and analgesia. α4β2 nAChRs have been implicated in a wide range of diseases such as depression, Alzheimer’s, Parkinson’s, Schizophrenia and a type of familial epilepsy. Also, α4β2 nAChRs are necessary and sufficient for the rewarding and reinforcing effects of nicotine. The α4β2 receptors assemble in two functional forms, (α4β2)2α4 and (α4β2)2β2. These two receptors have different pharmacological properties, which is partly accounted for by the presence of an additional agonist site at the signature α4/α4 interface of the (α4β2)2α4 nAChR. The canonical agonist sites of these receptors function asymmetrically, even though they are structurally equivalent. These findings suggested that the fifth subunit (α4 in the (α4β2)2α4 and a β2 in the (α4β2)2β2 may asymmetrically modulate the agonist sites. The impact of the fifth subunit on receptor function was investigated by using concatenated (α4β2)2α4 and (α4β2)2β2 nAChRs expressed heterologously in Xenopus oocytes in combination with mutagenesis and functional analysis. Concatenated receptors permit the expression of only one type of receptor stoichiometry and the mutations can be introduced in defined subunits of the pentamer. The overall aim of this PhD study was to advance our understanding of how the signature β2/β2 and α4/α4 interfaces of (α4β2)2β2 and (α4β2)2α4 nAChRs respectively affect the function of neighbouring α4β2 nAChRs. In (α4β2)2α4 receptors, the presence of an additional site at the α4(+)/α4(-) interface underlies differences in sensitivity to ACh but the interface flanking agonist sites also modulate the function of this site. For the (α4β2)2α4 receptors, this thesis examined the effects of mutations introduced in the fifth subunit or flanking subunits on Zn2+ potentiation of (α4β2)2α4 receptors. Zn2+ potentiation of the agonist responses of the (α4β2)2α4 receptors is mediated by a site located on the fifth subunit (α4). It was found that Zn2+ potentiation is inhibited by alanine substitutions of amino acids linking the fifth subunit to neighbouring agonist sites. For the (α4β2)2β2 receptors, the findings suggest that residues in loop B (W176, T177) and loop E (L146 and F144) of β2(+)/β2(-) interface link to the canonical agonist site anticlockwise to β2(+)/β2(-) to modulate maximal agonist responses. Long-range coupling analysis with the modulator β2(+)/β2(-) residues and a reported mutation in the ion channel, β2L9’T, showed these residues functionally couples, thus suggesting that the modulation of maximal agonist currents likely occur at the gating domain. Overall, it is proposed that the fifth subunit allosterically communicates with adjacent subunits to modulate agonist binding site function. These findings may lead to the development of stoichiometry- or interface-specific α4β2-selective drugs