| Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. In mammals and other vertebrates, nAChRs are expressed at both the neuromuscular junction and within the CNS (Central Nervous System) and PNS (Peripheral Nervous System). In insects, though not expressed at the neuromuscular junction, nAChRs play an important role in the insect CNS, where ACh is the major excitatory neurotransmitter. Many insect nAChR subunits have been identified and characterized by molecular cloning and genome sequencing. In the last two decades, studies have focused on the heterologous expression of functional insect nAChRs. 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. Some proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperones, regulators and modulators.In the present study, we co-expressed four nAChRs a subunits of brown planthopper, Nilaparvata lugens, with rat β2subunit in Xenopus oocytes and characterized the hybrid nAChRs using electrophysiological recordings with a two-electrode voltage clamp and radioligand binding assay. Co-immunoprecipitation was used to examine the native nAChRs subunit composition. We also reported the identification of two members of the Ly-6/neurotoxin superfamily in the brown planthopper and their action as the modulators on insect nAChRs were evaluated in the Xenopus oocytes heterologous expression system. The mechanism of these two modulators effecting on the agonist potency was also discussed.1. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nlal and Nla2Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nAChRs and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlal and Nla2cloned from the N. lugens, a major insect pest of rice crops in many parts of Asia. When Nlal or Nla2was co-expressed with the rat nAChR β2subunit in Xenopus oocytes, functional hybrid nAChRs could be generated, which is consistent with previous studies. The difference in EC50values for ACh from oocytes expressing Nlal/rP2and Nla2/rβ2nAChRs was less than two fold (EC50=37±6μM and EC5o=62±10μM, respectively; n=9). Whereas EC50values for imidacloprid were61±7μM (n=9) and870±76μM (n=13) for Nlal/rβ2and Nlα2/rβ2, respectively.In oocytes co-injected with the Nlal, Nla2, and rP2subunit cRNAs, the experimental dose-response curve differed from theoretical dose-response curves predicted for mixed populations of Nla1/rβ2and Nla2/rβ2nAChRs, and no plateau was detected in the experimental dose-response data, which provided evidence for a single population of ’triplet’ Nla1/Nla2/rβ2nAChR. Increasing the ratio of the P2subunit resulted in reduced maximal currents, but no significant difference in EC50values was detected, which also proved that one ’triplet’ nAChR was generated. In addition, evidence has also been obtained from immunoprecipitation study to indicate that Nlal and Nla2subunit co-assembled in N. lugens native nAChR.2. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nla3and Nla8Genome sequence analysis has provided information concerning subunit diversity of nAChRs from several insect species. Phylogenetic analysis suggests that the ’DP2group’ contains ’a’ type subunit in most insect species including a8subunit. There have been no reports of the successful functional heterologous expression of any nAChR subunit from the insect a8/DP2subunit group previously. But there is the evidence to suggest that the Dβ2subunit might be able to co-assemble with two other subunits into a triplet nAChR. Here we cloned the nAChRs a8subunit of N. lugens (GenBank accession number FJ481979) and demonstrated for the first time the incorporation of an insect nAChR subunit from the a8/Dβ2group into a functional recombinant nAChR.The full length of N. lugens nAChRs a8subunit is2036bp, including1617bβ ORF which code538amino acid, and share close sequence similarity with nAChR a8subunits from honey bee Apis mellifera, the malaria mosquito Anopheles gambiae, and the red flour beetle Tribolium castaneum. Heterologous expression demonstrated that when Nla8was co-expressed with rat β2subunit or co-expressed together with Nla3and rβ2, functional nAChRs were detected. However, in oocytes co-injected with the Nlα3, Nlα8, and rβ2subunit cRNAs, much larger responses were detected than those in oocytes expressing Nla8/rβ2nAChRs. The maximal agonist response (Imax) detected with imidacloprid in oocytes expressing Nla8/rβ2nAChRs was29.69fold higher than Imax in oocytes expressing Nla3/Nla8/rβ2nAChRs. EC50values for acetylcholine were95.8±11.7and17.6±3.7μM for Nla8/rβ2and Nla3/Nla8/rβ2nAChRs, respectively, and for imidacloprid values were127.9±19.3and3.2±0.5μM, respectively. These data indicated Nla3, Nla8, and rβ2subunit generated a’triple’nAChRs. Evidence has also been obtained from immuno-precipitation study to indicate that Nla3and Nla8subunit co-assemble in native nAChR.3. Identification of two Lynx proteins and the modulation on insect nAChRsSome proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperone, regulator and modulator. In present study, two members of the Ly-6/neurotoxin superfamily have been identified by blasting in EST database of N. lugens with Pr-lynxl (Pyrocoelia rufa) and named as Nl-lynxl and Nl-lynx2. Although Nl-lynxl and Nl-lynx2did not show very high similarities to Pr-lynx1(53%and50%) and vertebrate Ly-6/neurotoxin superfamily, such as Mus musculus lynxl (39%and41%) and lynx2(42%and44%), both Nl-lynx1and Nl-lynx2contained the signature cysteine-rich motif of the Ly-6/neurotoxin superfamily. Similar to Pr-lynxl, both Nl-lynxl and Nl-lynx2only increased agonist-evoked currents, but did not change the agonist sensitivity on nAChRs expressed in Xenopus oocytes. In Xenopus oocytes expressing Nlal/rβ2, the addition of Nl-lynx1or Nl-lynx2increased Imax of ACh to3.56-fold and3.25-fold respectively, and increased Imax of imidacloprid to1.72-fold and1.51-fold respectively. Radioligand binding assay determined that co-expression of Nl-lynx1or Nl-lynx2with Nlal/rβ2only increased the maximum binding (Bmax), but did not change the binding affinity (Kd). Further studies showed that the co-expression of Nl-lynxl or Nl-lynx2did not change the desensitization properties of Nlal/rβ2or the expression levels of Nlalor rβ2subunit on oocyte surface. So, the higher efficiency of protein folding and subunit assembly might be the important reasons for the significantly higher level of binding detected with co-expression of Nlal/rβand Nl-lynxl or N1-lynx2.The present study shows that co-expression of two insect nAChRs a subunits is able to increase agonist response of hybrid receptors, but functional nAChRs could not be detected in Xenopus oocytes without mammalian β subunit, even with nAChRs modulator Nl-lynx1or Nl-lynx2, which suggests additional accessory proteins need to be identified for heterologous expression of functional insect nAChRs. So, we have tried to identify more proteins of native nAChRs complex by co-immunoprecipitation, SDS-PAGE electro-phoresis and LC-MS/MS. Although the number of proteins identified is too large to find useful information, the method we used was proved to be feasible. |
