| Nicotinic acetylcholine receptors (nAChRs) are crucial neurotransmitter receptors in insect central nervous system, which play an important role in normal life activities of insects. Therefore, nAChR is an important target for many kinds of insecticides including neonicotinoids, Spinosyns, nereistoxin analogues and nicotine in insect pest control. Studies on the composition and pharmacological properties of insect nAChRwill benefit insecticides invention, rational use of existing pesticides and insect pest resistance management.It is a very effective way to study the composition and pharmacological properties of nAChR by constructing the recombination of it in heterologous expression system. However, although there are plenty of reports on nAChR research by reconstructing it in vitro, the recombinant receptor is either expressed with single subunit (only have the evidence of ligand binding assay) or expressed with subunit of mammal. So far, the heterologous receptor reconstructed only with insect nAChR subunits has been unsuccessful. Previously, it was believed that the unsuccessful heterologous expression was due to lacking the nAChR subunits which have not been found yet, but with the completion of genome sequencing of Drosophila and many other insects, it is realized that the hypothesis is wrong, cause there are only a subunit and (3 subunits which have been identified. Thus, another hypothesis has been advanced that the recombination of heterologous nAChR requires one or more associated proteins.In this paper, we take advantage of affinity chromatography and protein mass spectrometry to isolate and identify the associated proteins of nAChRs of Locusta migratoriamanilensis (Meyen). Some of the identified proteins are co-expressed with nAChR subunits in Xenopus oocytes and characterized using electrophysiological recordings with a two-electrode voltage clamp to analyze the function of the proteins and the effect to the function of nAChR. We are attempting to identify one or more key proteins that can make the successful expression of heterologous nAChR.1. The isolation and identification of the associated proteins of nAChRThe brain, ventral nerve cord and ganglion subpharyngeale which are vivisected from adults of locusta migratoria are used to isolate and identify the associated proteins. Affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography combine with tandem mass spectrometry are employed to isolate the associated proteins.The results showed that 1699 proteins have been identified.Among these proteins,68 proteins have no annotation; 460 proteins annotated either with unknown protein or with only a code, such as ACYPI000787 and AGAP000325-PA whichare hypothesis or predict proteins. The others that are clearly annotated belong to 241 different proteins or protein complex which including many protein that have reported interacting with nicotinic acetylcholine receptor. This provides vital clues to the subsequent researcher of the composition and function of nAChR.2. The gene cloning and expression vector constructing of associated proteins of nAChRFive sodium-potassium pump subunits genes of locusta migratoria were cloned throughRT-PCR and RACE technology.According to the results of homology analysis, they were named al (GenBank accession number KF813096),α2 (KF813097), β1 (KF813099), β2 (KF813100) and β3 (KF813098), Where α subunits have ATP phosphorylation sites, dephosphorylation of functional domains and hinge conserved sequences that are the characteristics of the sodium -potassium pump α subunit, while β subunits have the conserved cysteine loop and glycosylation sites which are characteristics of β subunit of the sodium-potassium pump.We alsocloned the gene of bh-04 protein.The cloned gene were subcloned into the pGH19 plasmid for the subsequent study of the effect on function of nAChR.3. Study on the effect of sodium-potassium pump on function of nAChRSodium-potassium pump is a transmembrane transporter protein complex, located in the cell membrane, and transport cations between intracellular and the extracellular environment against their electrochemicalgradients, maintaining cell potential. Recently studies have indicated that sodium-potassium pump could act as a signal transducer in addition to cations transporter. StudiesonC. elegansshows that sodium-potassium pump can regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner and another report shows that sodium-potassium pumpplay a role in regulation of skeletal muscle electrogenesis.These indicate that sodium-potassium pump has an important influence on function of nAChR.In this study we co-expressedaland β1 subunitof sodium-potassium pumpwithaland β1 subunitof nAChR in Xenopus oocytes to reveal the effect of sodium-potassium pump on nAChR.4. Reconstructing of the basic function complex of nAChR with all insect subunitsCurrently, it is demanded mammal β subunits of nAChR to recombine nAChR of insetsin vitro, and thus it is assumed that the reconstructing of nAChR with all insect subunits must have the association of one or more key associated proteins. Previous studies showed that associated proteins, such as RIC-3 and Lynx, have a great effect on the function of nAChR, however, they cannot associate to make a recombinant insect nAChR. In this paper, the associated protein bh-04 which has been identified previouslyco-expressed with al subunit and β1 subunit of insect nAChR in Xenopus oocytes and characterized by two-electrode voltage clamp electrophysiology system. The results showed that dose-response current can be detected, that is to say, functional nAChRs with all insect subunits were reconstructed with assistance of protein bh-04. And we re-estimated the effect of mutation of Y151S on nAChR, which takes place in a subunit of nAChR and causes target resistance against imidacloprid, one of neonicotinoids insecticides. |
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