Nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Their biophysical and pharmacological properties are critically determined by their subunit composition. nAChR can either be assembled as [alpha]7, [alpha]8 and [alpha]9 homopentamers or heteropentamers (different subunits). Preceding investigations from our laboratory have revealed the presence of mRNA coding for the nAChR subunits [alpha]3, [alpha]5, [alpha]7, [beta]2 and [beta]4 in the mouse superior cervical ganglion (SCG). In order to investigate the involvement of nAChR subunits in disease and their role in addiction to nicotine a variety of mice having targeted deletions of one (single knockout) or more of the five subunits (double/triple knockout) expressed in the SCG have been generated so far. These knockout animals are vital and show no obvious phenotype.
Nevertheless these knockout (KO) animals would be excellent tools to investigate the distinct pharmacological properties of each nAChR type under native conditions if the exact subunit composition were known.
Therefore the main aim of my studies is the investigation of the subunit composition of heteropentameric nAChR in the mouse SCG to accompany results of our laboratory from the 'pharmacological fingerprinting'.
Subunit specific antibodies are essential prerequisites for the analysis of the subunit composition of nAChR subtypes. However, most of the commercially available antibodies give false positive results when tested on the tissue of KO animals and are therefore not suited for most common techniques, such as immunolocalisation (Moser et al. 2007).
Therefore we generated subunit specific antibodies, extensively characterised on transfected HEK-293 cells, brain tissue and tissue collected from KO animals. With these antibodies immunoprecipitation (IP) experiments using [3H] labelled epibatidine, a highly specific agonist for heteropentameric nAChR, were performed to investigate the subunit composition in SCG of 18 day old C57/BL6 wild-type (WT) and KO mice.
Results from IP show that the subunits [alpha]3 and [beta]4 are predominant subunits and [alpha]5 and [beta]2 are expressed as minor constituents. In WT, [alpha]5 single KO and [beta]2 single KO animals [alpha]3 and [beta]4 subunits are expressed in every functional receptor. Additionally the overall number of receptors in [alpha]5 single KO, [beta]2 single KO and [alpha]5[beta]2 double KO mice is not significantly different compared to WT. In contrast, [beta]4 single KO and [alpha]5[beta]4 double KO animals express significantly less nAChR (< 13 % of controls). The [beta]2 subunit is expressed in comparable levels in all animals (approximately 15 %) except in [beta]2 single and [alpha]5[beta]2 double KO animals. [alpha]5, however, is not expressed in nAChR in [beta]4 single KO animals. Therefore these animals express only [alpha]3[beta]2 nAChR. The [alpha]4 subunit is not expressed at all.
Finally the IP data on the nAChR subunit composition of WT mice combined with mRNA data, IP data from KO mice and data from membrane receptor binding from WT and KO mice which indicates that nAChR subunits do not compensate for a missing subunit reveals the expression of the following nAChR in the SCG of WT mice: [alpha]3[beta]4 (55 %), [alpha]3[beta]4[alpha]5 (24 %) and [alpha]3[beta]4[beta]2 (21 %).