katchamaon.pdf [6.46 mb] |Untersuchungen zur Quartärstruktur neuronaler nikotinischer Acetylcholinrezeptoren von Drosophila melanogaster|
|Fruitfly Drosophila melanogaster neuronal nicotinic acetylcholine receptor receptor subunit quarternary structure|
Studies on the quarternary structure of neuronal nicotinic acetylcholine receptors from the fruitfly Drosophila melanogaster Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central nervous system (CNS) of insects and play an important role in fast synaptic transmission. The structural complexity of nicotinic receptors is largely unknown. Data presented in this study indicate the existence of at least three different receptor complexes from the fruitfly Drosophila melanogaster. New antisera were raised against, the beta-subunit SBD (second beta-subunit from Drosophila). Together with previously available antibodies against alpha-subunits ALS (alpha-like subunit from Drosophila), Dalpha2 (Drosophila alpha2-subunit), Dalpha3 (Drosophila alpha3-subunit) and the beta-subunit ARD (AChR protein from Drosophila) they were used to characterise nAChR complexes existing in vivo. From eluates of alpha -Bungartoxin-affinity column, the apparent molecular weights of individual subunits could be determined on Western blots, i. e. 80 kDa for ALS, 65 kDa for Dalpha2, 105 kDa for Dalpha3, 60 kDa for SBD and 50 kDa for ARD. Immunohistochemical analyses on cryostat sections of adult Drosophila heads indicated that in the optic lobes the distribution of SBD is very similar to that of the other receptor subunits. In the medulla, layering of SBD immunoreactivity closely resembles that of ALS. In the distal lamina, in contrast, co-distribution of Dalpha3 and ARD, but not of ALS, Dalpha2 and SBD could be observed. Receptors were solubilised from head membranes and the presence of receptors was examined by [125I]alpha-Bungarotoxin binding assays. Immuno-precipitation experiments from head extracts showed that ALS, Dalpha2 and SBD can co-assemble into the same receptor complex. Co-immunoprecipitation experiments further confirmed that Dalpha3 and ARD co-assemble into a receptor complex physically distinct from the ALS-Dalpha2-SBD receptor. Moreover, the beta-subunits ARD and SBD can be detected in a third receptor complex with yet unidentified alpha-subunits. The present report provides an important contribution to our appreciation of the complexity of nicotinic receptors of the insect CNS.