Molecular and Cell Biological Characterisation of Neuronal Nicotinic Acetylcholine Receptors

Molecular and cell biological characterisation of neuronal nicotinic acetylcholine receptors (nAChRs) provides an insight into their functional roles and potential as therapeutic targets for neurological disorders. Nicotinic receptors are oligomeric ligandgated ion channels, comprising five subunits. Twelve vertebrate neuronal nAChR subunits (a2-al0 and (32-|34) have been cloned to date, with considerable diversity observed in nAChR subunit composition. Heterologous expression of cloned subunits is a powerful method for investigating ion channel receptor pharmacology and subunit composition, but achieving efficient expression of some nAChRs in cultured cell lines has proved difficult. In this study, chimeras containing the N-terminal domain of the nAChR subunits, fused to the C-terminal region of the 5-hydroxtryptamine type 3 receptor subunit, 5HT3A, were constructed to overcome some of the challenges of recombinant nAChR expression. When combinations of wild-type and chimeric subunits were expressed in human embryonic kidney tsA201 cells, inclusion of nAChR/5HT3A chimeras enhanced the expression of nAChRs containing each of the a2, a3, a4, a6 , a7, a9, alO, p2 or (34 nAChR subunits, determined by detection of radioligand binding sites. This was particularly significant for a6or a9/alO-containing nAChRs, as radioligand binding to wild-type nAChRs containing these subunits was not detected in tsA201 cells. A detailed pharmacological characterisation of receptors containing a9/5HT3A + alO/5HT3A chimeras in tsA201 cells via competition binding suggested that the chimeras provide suitable models for characterisation of wild-type nAChRs. Radioligand binding to intact cells and enzyme-linked assays to detect epitope-tagged subunits expressed in transfected cells, suggested that ot9/5HT3Acontaining receptors were expressed at the cell surface in high levels. Comparison of radioligand binding to nAChR subtypes containing combinations of wild-type and chimeric a2-a6 and P2-J34 subunits implicated the Nand C-terminal domains of both the a and p type nAChR subunits in subunit oligomerisation events and provided an insight into nAChR assembly.