The Pharmcological Characterstics Of Insect Nicotinic Acetylcholine Receptor βSubunits And Set-Up Of A New Expression System

Exogenous expression is an important method to study protein function. Many insect nicotinic acetylcholine receptor (nAChR) subunits have been identified and characterized by molecular cloning and genome sequencing. However, in the case of insects such as Drosophila with all subunits identified, considerable problems remain in identification of native subunit composition and even in generating functional insect nAChRs in heterologous expression systems.Nicotinic acetylcholine receptors (nAChRs) β subunits in insects were cloned by molecular techniques. To investigate the structure and function of insect nAChR β subunits and the possible mechanisms of neonicotinoids selectivity, the pharmcological properties of functional loops (D, E and F) and amino acids within loops of insect nicotinic acetylcholine receptor β subunits have been researched and analysised by electrophysiological techniques. Even when all insect a and β subunits were combined to generate a functional receptors, all tries are not successful. Previous studies showed that some proteins other than nAChRs themselves might play important roles in insect nAChRs formation, such as the chaperone, regulator and modulator, which indicates that more proteins should be expressed together to generate a functional insect nAChRs. To express more proteins together in frog oocytes, such as Xenopus oocytes, more cRNA or cDNA should be injected at the same time, which requires bigger capacities of oocytes. In this thesis, we are tyring to develop a new tool for exogenous expression of insect nAChRs and their accessary proteins, with big capacities. We have successfully set up the electrophysiology methods for Bufo bufo gargarizans (Chinese big toad) oocytes, including the indoor feeding methods, the factors affecting the reproduction of the big toad, the methods to obtain mature oocytes under artificial conditions, the injection of cRNA and electrophsiological recording. The endogenous expression of membrane channels in the big toad mature oocytes we tested and the exogenous expression of nAChRs were recorded.1. Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor P subunits influenced neonicotinoid selectivityTo investigate the mechanism of acetylcholine and neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F and amino acids within loops D, E and F of insect nicotinic acetylcholine receptor with two-electrode voltage clamp.The results indicated that the introduction of the insect-specific loops D, E and F, singly or together, into rat02subunit resulted in a leftward shift of the imidacloprid dose-response curves for hybrid nAChRs containing insect and mammalian subunits Nlal-β2(Nlal from the brown planthopper Nilaparvata lugens and02from rat) chimeras, reflecting decreases in EC50, compared to wildtype nAChRs Nlal-β2. By contrast, the influences on ACh potency were minimal or negligible. S131Y (R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F, reflecting in the significant leftward shifts in EC50to imidacloprid, but the influences on ACh potency were minimal, compared to wildtype nAChRs Nlal-β2. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity.2. Molecular cloning nicotinic acetylcholine receptor β1subunit from brown planthopper and A-to-I RNA editing in this subunitWe obtained the full-length cDNAs encoding nicotinic acetylcholine receptor P subunit in the Nilaparvata lugens, a major rice pest, by RT-PCR and RACE technology. The gene contain nicotinic acetylcholine receptor β subunit signature motifs, such as an extracellular N-terminal region with conserved residues within loops D-F which are involved in ligand binding, the cys-loop consisting of two disulphide bond-forming cysteines separated by13amino acid residues, four well-conserved transmembrane regions (TM1-4) and a variable intracellular region between TM3and TM4, and was denoted as N.lugens β(N1β1). Six A-to-I RNA editing sites were found in N1β1N-terminal domain, Among the six editing sites, four caused amino acid changes, in which the site2(E2) and site5(E5) caused an N to D change in loop D (N73D) and loop E (N133D) respectively. E2frequency was high in Sus (susceptible) strain and E5frequency was high in Res (resistant) strain. The frequency of the other sites is no change in the two different strains of N. lugens.3. The possible roles of A-to-I RNA editings in N1β1subunit in neonicotinoid sensitivityTo evaluate the influence of N73D in loop D and N133D in loop E by A-to-I RNA editing on neonicotinoid selectivity, loop D or loop E of N1β1was introduced into rat β2of N1αl-βnAChRs to construct the hybrid Nlal-β2LoopD or Nlal-β2LoopE nAChRs, and then N73D or N133D mutation was introduced into β2LoopD or β2LoopE to construct mutant Nlal-p2LoopD-N73D or Nlα1-p2LoopE-N133D. By expressing in Xenopus oocytes, N73D mutation in loop D significantly reduced the agonist potency of both ACh and imidacloprid, and the influence on ACh was more significant than on imidacloprid. In contrast, N133D mutation in loop E only showed significant effects on imidacloprid potency, and the influences on ACh potency were minimal or negligible. These results indicated, although E2and E5in loop D and E both caused an N to D change in important loops, their roles in imidacloprid sensitivity might be different.4. The biological characteristics of the big toadIn this section, the indoor feeding methods for the big toad, the factors affecting the reproduction of the big toad and the methods to obtain mature oocytes under artificial conditions were analyzed. When the environmental temperature was below15℃, the big toad is in a hibernation period, and adequate humidity and ventilation should be provided to keep such hibernation. When the environmental temperature was above15℃, the big toad start feeding Tenebrio molitor2or3times in a week at the early morning or dusk, and the water should be changed3to4times a week to ensure that the excrement of the big toad was immediately removed. The appropriate temperature indoors is18～22℃. When kept in in high temperature or under a dim environment, the Chinese big toad can not produce mature oocytes. The sufficient treatment by the low temperature was necessary for the oocyte maturation. After treatment by the low temerature of4～6℃for one week to one month and the injection of4000IU/kg CG (Chorionic gonadotropin) or8/kg PG (pituitary gland), the big toad toad could produce mature oocytes in3to5days.5. The endogenous expression of membrane channel proteins in the big toad oocytesThe endogenous expression of membrane channel proteins in the big toad oocytes was analysised by two-electrode voltage-clamp recording, in oocytes with or without follicular. The receptors for acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly) were tested, compared to the expression in Xenopus laevis oocytes and earlier findings in the big toad oocytes. The results showed that acetylcholine, gamma-aminobutyric acid and glycine could cause the obvious currents in the big toad oocytes with follicular membrane, with the current in response to1mM ACh, GABA and Gly in oocytes with follicular membrane was87.1±4.9nA,45.4±13.8nA and36.6±22.0nA. However, in the big toad oocytes without follicular membrane, no currents were detected when the application of ACh, GABA and Gly. The results indicated there was not the endogenous expression of the receptors for such chemcials in the big toad oocytes.6. The exogenous expression of membrane channel proteins in the big toad oocytesThe mRNA purified from American cockroach nerve cord and Caenorhabditis elegans was injected into oocytes of the big toad. The currents in oocytes without follicular membrane evoked by the application of ACh and GABA were recoded using the two-electrode voltage-clamp, compared to the expression in Xenopus laevis and earlier findings in the big toad oocytes. The obvious currents were observed in the big toad oocytes with the injection of exogenous mRNA when application of ACh and GABA. The current in response to1mM ACh and GABA in oocytes injected with American cockroach nerve cord mRNA was98.1±4.9nA and329.5±6.3nA. The current in response to1mM ACh and GABA in oocytes injected with Caenorhabditis elegans mRNA was84.1±13.8nA and89.5±7.9nA. Because there was not any currents were evoked by ACh and GABA in the big toad oocytes without any injection, the results showed the suscessful expression of exogenous ACh receptors and GABA receptors in the big toad oocytes. In future, more receptors and channels were tested in the big toad oocytes. According to the results at present, we could conclude, at least, that the big toad oocytes can be used as an expression tool for exogenous expression of ACh and GABA receptors.