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 ligand-gated ion channels, comprising five subunits. Twelve vertebrate neuronal nAChR subunits (2-10 and 2-4) 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 2, 3, 4, 6, 7, 9, 10, 2 or 4 nAChR subunits, determined by detection of radioligand binding sites. This was particularly significant for 6- or 9/10-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 9/5HT3A + 10/5HT3A 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 9/5HT3A- containing receptors were expressed at the cell surface in high levels. Comparison of radioligand binding to nAChR subtypes containing combinations of wild-type and chimeric 2-6 and 2-4 subunits implicated the N- and C-terminal domains of both the alpha and beta type nAChR subunits in subunit oligomerisation events and provided an insight into nAChR assembly.

The role of small and intermediate conductance calcium-activated potassium channels (SK and IK channels) in dorsal root ganglion (DRG) neurones was examined. Sixteen antibodies raised against human or rat SK/IK channel peptide epitopes were tested for their ability to stain cells expressing channel protein. Of sixteen antibodies, 12 (6 to SKI, 1 to SK2, 2 to SK3 and 3 to IK) were deemed suitable for immunohistochemistry in human or rat tissue. Real-time quantitative PCR (qPCR) of rat DRG cDNA was performed to examine SK/IK expression levels. DRG neurones produce mRNA for all SK/IK channels and these mRNA levels were found to increase during development. Antibody staining experiments using DRG neurones cultured from different aged animals produced a positive stain with the anti-SK3 antibody only. The number of cells that stained positively and the intensity of staining for SK3 increased with age. To investigate possible functional roles for SK/IK channels sensory neurones, action potential afterhyperpolarisations (AHPs) were recorded from cultured DRG and nodose cells. The majority of these AHPs proved to be insensitive to the SK channel blocker UCL 1848. Attempts to block medium duration AHPs in DRG cells using IK and calcium channel blockers, also failed in most cases, suggesting that some other potassium conductances) are responsible. The possibility that SK3 is functional at the terminals of primary afferents was examined next. Spinal cord slices stained with SK/EK channel antibodies revealed positive SK3 staining in the outer laminae of the dorsal horn, where small and large diameter DRG fibres are expected to terminate. In vivo experiments (done by Dr Rie Suzuki, Department of Pharmacology, UCL) using UCL 1848 and l-ethyl-2- benzimidazolinone (1-EBIO an SK channel opener) showed that SK channels are likely to be active at these terminals where they have a functional role in mediating innocuous mechanical and nociceptive responses.

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Unlike many nicotinic subunits, the al nicotinic subunit and the 5-HT3A subunit are able to form functional homomeric receptors. Despite sharing this ability, they have differences in cell-surface expression in human embryonic kidney (HEK) cells, with 5HT3A subunits forming a large number of functional receptors, whereas the al subunit forms few or no correctly folded receptors as assayed by radioligand binding with 125I a-BTX. A series of chimeras between al and 5HT3A were constructed to investigate which domains of the subunits were important for folding and cell surface expression. Only chimeras that contained the region from the beginning of Ml to the end of M3, and M4 domain of the 5HT3A subunit were able to form correctly folded receptors. The chimeras that gave high levels of radioligand binding were also found to be functional using whole-cell patch-clamp recording. Functional characteristics were examined, and differences were found in single channel conductance and desensitization. Chimeras with the large cytoplasmic loop and the extracellular N-terminal region of al had larger single channel conductances than the 5HT3A receptor, with the inclusion of the al large cytoplasmic loop and the N-terminal domain increasing the conductance by approximately 10 and 2 fold respectively Co-expression of al with RIC3 (a protein originally identified in C. elegans) in HEK cells, results in high levels of cell surface expression. These receptors were shown to be functional, with whole cell responses from 20-300 pA. showing fast desensitization (time constant of 66 13 ms) and strong inward rectification. RIC3 was also shown to increase the functional expression of the a8 and the rat a3p2 nAChRs, which rarely formed detectable functional receptors when expressed alone. On co-expression with RIC3 almost all cells expressing a8 responded giving an average response of 240 pA, and all cells expressing a3 32 responded giving an average response of 99 pA. C. elegans RIC3 has been shown to increase the functional expression of the human 5HT3A receptor by 168 %. Human RIC3 had no effect on human 5HT3A and in fact decreased the functional expression of the murine 5HT3A by 59 %.

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