Identification And Function Analysis Of Nicotinic Acetylcholine Receptors Regulators In Locusta Migratoria Manilensis

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that carry out the fast actions of the neurotransmitter acetylcholine (ACh). In vertebrate, nAChRs have been involved in many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and schizophrenia. In insect, nAChRs have been exploited as molecular targets to develop insecticides, such as neonicotinoids and spinosad insecticides. Over the past 30 years,it has become clear that the activity of nAChRs is dependent on their interaction with a host of proteins. These proteins regulate a wide range of functions, including receptor folding,assembly, maturation,and trafficking to and from the cell surface, as well as how they directly modulate functional characteristics such as sensitivity to insecticides. For example, protein kinase A could affect the sensitivity of neurons to insecticides. Therefore, the identification and function analysis of nAChRs associated proteins would bring insights into the treatment of diseases and open novel strategies for optimizing insect pest control, including the management of insecticide resistance and development of new insecticide. Large number of nAChRs associated proteins was identified in vertebrate, and the functions of nAChRs have been well understood in the context of their interactions with these associated proteins.However, only four protein families were found to regulate the function of insect nAChRs,including RIC-3, Lynx, sodium-potassium pump and protein kinases.As an ideal model, Locusta migratoria manilensis has been used as materials in the interaction of nAChRs and associated proteins. Atranscriptome of locust neurons was created to obtain the potential genes encoding ion channels and nAChRs associated proteins. The selective activities of Lynx proteins on different nAChRs were studied further in Xenopus oocytes using electrophysiological recordings and radioligand binding assay. The locust dorsal unpaired median (DUM) neurons with RNA interference (RNAi) were used to investigate the function nAChRs associated proteins, including ubiquilin-1, PICK1 and CRELD2 which play important roles in receptor assembly, trafficking and clusters of insect nAChRs. The variation of neurons membrane potential and the inward current that determined by the voltage-sensitive fluorescent dye and patch-clamp technique showed these three proteins were the regulators of nAChRs in insects. Ubiquilin-1 and PICK1 regulated insect nAChRs negatively, but CRELD2 regulated receptors positively.1. The transcriptome of the Locusta migratoria manilensis neuronsNeurons expressing a wide variety of ion channels are the basic units of insect nervous system. The electrical activity of the neurons is dependent on opening and closing of these channels. The nerve impulse conduction is carried by the flux of Na+ K+, Ca2+ and Cl-.nAChRs are not only the important ligand-gated ion channels in insect neurons, but also the targets of several classes of insecticides. Therefore, the locust neuron is becoming an important material for the study of insect nAChRs. Comparing to Periplaneta americana neurons, one ideal material widely used in insect nAChRs studies, it was found that the fat attached to the ventral nerve cord of L. manilensis was less and easier to clean. The number of DUM isolated from one ventral nerve cord of L.manilensis (28± 5) was more than that of P. americarna (8±1). In L.manilensis, more DUM neurons with appropriate size were used in electrophysiological experiments than that in P. americana. Meanwhile, L. manilensis DUM had some advantage to culture in vitro because of fewer impurities in cell suspension than that of P. americana. Therefore, we choose the locust neurons as the experimental material in the following study.The gene information released in NCBI is general few, although the genomic sequencing of the locust was completed,but without annotation. For example, only four nAChR subunits of the locust have been reported in NCBI. In this study, the neurons were isolated from the ventral nerve cord by mechanical dissociation and enzymatic treatment, and then purified by discontinuous density gradients. Three discontinuous density gradients for the locust neuron separation were obtained through a series of tests. After centrifugation, the neurons accounted at least for 80% of total cells. With Illumina sequencing to obtain the transcriptome of locust neurons,a large number of complete and partial sequences expressing in nervous system specifically were identified, including 11 putative nAChR subunits, 3 putative ?-aminobutyric acid (GABA) gated anion channels and 7 putative glutamate-gated chloride channels (GluCls). Meanwhile, associated proteins which may be related to the composition and function of nAChRs, such as Lynx proteins, 14-3-3 protein, RIC-3, ubiquilin-1, CRELD2 and PICK 1 were identified.The expression profiles of some important genes encoding nAChRs associated protein,such as Lynx, ubiquilin-1, PCIK1 and CRELD2, were determined by real-time quantitative PCR. The results showed that lynx genes were highly expressed in the nervous system,suggesting that Lynx proteins might function specifically and importantly in nervous system.The expression levels of ubiquilin-1, PCIK1 and CRELD2 genes were not with the tissue-specificity, suggesting that these proteins might be involved in several different biological processes.2. Selective action of Lynx proteins on different nAChRs in the locustThe full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. In Nilaparvata lugens,Lynx proteins significantly increased the maximum agonist-evoked inward currents through nAChRs and showed selectivity among nAChRs subtypes. Here, three Lynx proteins, Loc-lynxl, Loc-lynx2 and Loc-lynx3, were identified in the locust. The selectivity of these Lynx proteins among insect nAChRs was interpreted in detail, such as different agonists, binding sites or modulation patterns.Co-expression with Lynx resulted in a dramatic increase in agonist-evoked macroscopic currents on nAChRs Loc?l/r?2 and Loc?2/r?2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc-lynx1 and Loc-lynx3 only modulated nAChRs Loc?1/r?2, while Loc-lynx2 modulated Loc?2/r?2 specifically. The results indicated that the selectivity of Lynx proteins was from different nAChR subtypes. Meanwhile, Loc-lynxl and Loc-lynx3 enhanced acetylcholine (ACh) Imax to 3.18-fold and 2.93-fold of that of Loc?1/r?2 alone, and the enhanced ratios of imidacloprid and epibatidine Imax were 4.31-fold and 6.47-fold, 4.18- and 5.82-fold, respectively. Therefore, the selectivity was from various agonists because the effects of Loc-lynx1 and Loc-lynx3 on imidacloprid and epibatidine were significantly higher than that on ACh. Among three Lynx proteins, only Loc-lynxl significantly increased[3H]epibatidine binding on Local/r?2. The results indicated that Loc-lynxl had different modulation patterns in nAChRs compared to Loc-lynx2 and Loc-lynx3. Taken together, these findings indicated that three Lynx proteins were nAChRs modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns.3. Identification and function analysis of nAChRs regulators in the locustIn insects, protein kinases affected the sensitivity of neurons to insecticides by regulating nAChRs function. The finding revealed the relation among nAChRs, nAChRs associated proteins and insecticide sensitivity. It was reported that the resistance to imidacloprid in a selected strain of N. lugens was associated with a reduction in expression levels of nAChR subunit Nla8. It is important to identify the regulator of insect nAChRs, for the mechanism study and management strategy of insecticide resistance and the development of new insecticide. In this study, we used siRNA to silence the expression of ubiquilin-1,CRELD2 and PICK1 in the cultured L. manilensis DUM neurons. After the expression of these genes was silenced successfully, DiBAC4(3) fluorescent dye and the patch-clamp technique were used to test the variation of neurons membrane potential and the inward current of the agonists.The result showed that, at 48 h, the RNAi efficiency of ubiquilin-1, CRELD2 and PICK1 were 79.4%, 73.4% and 85.6%, respectively. Treated with the same dose of imidacloprid, the fluorescence intensity detected by DiBAC4(3) was higher on DUM neurons following RNAi against ubiquilin-1 and PICK1 than that on the control neurons, with enhanced ratios of 1.47-and 1.48-fold. By contrast, the fluorescence intensity on DUM neurons following RNAi against CRELD2 was lower than that on the control neurons (72.53% of control neurons). In patch-clamp recording, when ACh and imidacloprid were applied on DUM neurons following RNAi against ubiquilin-1 and PICK1, the inward currents increased significantly when compared to control neurons. However, the inward currents of DUM neurons following RNAi against CRELD2 decreased. Taken together,these results demonstrated that the three proteins were regulators of insect nAChRs. The regulation of ubiquilin-1 and PICK1 on the receptors was negative, but the regulation of CRELD2 was positive.Meanwhile, on the locust DUM neurons, two pharmacologically distinct types of nAChRs were differentially evoked by low dose and high dose of agonists, respectively. The low dose induced the currents of a-bungarotoxin-resistant nAChRs specifically, and the high dose induced the currents on both a-bungarotoxin-resistant and a-bungarotoxin-sensitive nAChRs. It is interesting that ubiquilin-1 regulated two types of nAChRs, but PICK1 and CRELD2 only regulated the ?-bungarotoxin-resistant nAChRs.