The potential subunits involved in two subtypes of α-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons

Insect Biochemistry and Molecular Biology 81 (2017) 32e40

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The potential subunits involved in two subtypes of a-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons Huahua Sun a, 1, Yang Liu a, 1, Jian Li a, b, Xinzhu Cang a, Haibo Bao a, Zewen Liu a, * a

Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China b Changzhou Entry-Exit Inspection and Quarantine Bureau, Longjin Road 1268, Changzhou 213022, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 September 2016 Received in revised form 22 November 2016 Accepted 30 November 2016 Available online 20 December 2016

The american cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurons provide an native tool to analyze the functional and pharmacological properties of ion channels and membrane receptors, such as nicotine acetylcholine receptors (nAChRs). Here the imidacloprid-activated nAChR subtypes were examined in DUM neurons by the patch-clamp technique and the potential subunits involved in important subtypes were analyzed by combining with RNA interference (RNAi) technique. Imidacloprid exerted agonist activities on one subtype in a-Bgt-sensitive nAChRs and another subtype in a-Bgt-resistant nAChRs, in which the a-Bgt-resistant subtype showed much higher sensitivity to imidacloprid than the a-Bgt-sensitive subtype, with the difference close to 200-fold. In a-Bgt-resistant nAChRs, nicotine exerted the agonist activity on two subtypes (nAChR1 and nAChR2), although imidacloprid only activated nAChR1. RNAi against Paa3, Paa8 and Pab1 significantly reduced both imidacloprid- and nicotine-activated currents on nAChR1. In contrast, RNAi against Paa1, Paa2 and Pab1 decreased nicotine-activated currents on nAChR2. The results indicated that, in a-Bgt-resistant nAChRs, Paa3, Paa8 and Pab1 might be involved in the subunit composition of nAChR1, and Paa1, Paa2 and Pab1 in nAChR2. In summary, from the present study and previous reports, we deduced that there are at least three nAChR subtypes that are sensitive to imidacloprid in the cockroach DUM neurons. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Periplaneta americana Dorsal unpaired median (DUM) neuron Nicotinic acetylcholine receptor Subunit composition

1. Introduction Neuronal nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels expressed in the vertebrate and invertebrate nervous systems, which mediate fast cholinergic synaptic transmission (Matsuda et al., 2001; Sattelle, 1980). They are pentameric transmembrane complexes containing five coassembled subunit proteins (Millar, 2003). Many nAChR subunits have been identified and characterized in insects, by advances in genome sequencing and molecular cloning (Hermsen et al., 1998; Huang et al., 1999; Jones et al., 2005, 2006; Liu et al., 2009). Ten nAChR subunits have been identified in the model insect species Drosophlia melanogaster (Da1-Da7 and Db1-Db3) and a similar level of nAChR subunit diversity has been revealed in other insect species (Adams et al., 2000; Baumann et al., 1990; Jones et al., 2007;

* Corresponding author. E-mail address: [email protected] (Z. Liu). 1 These authors contributed equally in this study. http://dx.doi.org/10.1016/j.ibmb.2016.11.009 0965-1748/© 2016 Elsevier Ltd. All rights reserved.

Millar and Denholm, 2007; Sawruk et al., 1988, 1990; Schulz et al., 1998). The abundance of nAChRs within the insect central nervous system (CNS) has led to the development of neonicotinoid insecticides, such as imidacloprid, targeting these receptors (Tomizawa et al., 2000). In order to faithfully reflect the functional and pharmacological properties of insect nAChRs, several techniques were tried in studies on insect native nAChRs, such as radioligand binding and co-immunoprecipitation (Li et al., 2010). The cells from insect nervous system also provide precious tools to study functional properties of insect native nAChRs, such as the american cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurons (Tan et al., 2007). DUM neurons, located along the dorsal median line of ganglia and isolated from the sixth abdominal (A6) ganglion, are commonly used as biomedical models to study the biophysical and pharmacological properties of ion channels on cytomembrane (Grolleau and Lapied, 2000). These neurons express a wide variety of ion channels, such as nAChRs. For example, on the cockroach DUM neurons, several nAChR subtypes were characterized, such as nAChR subtypes sensitive and resistant to a-bungarotoxin (a-Bgt)

H. Sun et al. / Insect Biochemistry and Molecular Biology 81 (2017) 32e40

(Courjaret and Lapied, 2001; Salgado and Saar, 2004). On a-Bgtresistant nAChR subtypes, nicotine and acetylcholine activated two subtypes, but imidacloprid only acted on one subtype as agonist (Bodereau-Dubois et al., 2012; Courjaret and Lapied, 2001). These studies showed the diversity of insect nAChRs on the cockroach DUM neurons. However, the subunit compositions for these nAChR subtypes are still unknown, which limited the studies and applications of DUM neurons on the interaction of insect nAChRs and compounds, including neonicotinoids and other insecticides targeting on insect nAChRs. Here, combining with RNA interference (RNAi) and patch-clamp technique, the potential nAChR subunits of two a-Bgt-resistant nAChR subtypes were characterized in P. americana DUM neurons. 2. Materials and methods 2.1. Insects and chemicals The male adult Periplaneta americana were purchased from the Feitian Medicinal Animal Co. Ltd. (Danyang, Jiangsu, China). They were reared indoors at room temperature 28 ± 1
C, humidity 70e80% and 12/12 h light/dark cycles, respectively. Imidacloprid, nicotine, a-bungarotoxin, and d-tubocurarine were purchased from Sigma-Aldrich (St. Louis, Mo, USA), and a-conotoxin ImI was from American Peptide Company (Sunnyvale, CA, USA). 2.2. Amplification of cDNA The central nervous system were vivisected from adults of P. americana and collected into tubes, frozen with liquid nitrogen and stored at 80
C for total RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). First-strand cDNAs were synthesized with the reverse transcriptase XL (AMV) (Takara, Kyoto, Japan) and oligo dT18, using the following cycling parameters: 42
C for 60 min, then 95
C for 5 min. The first-strand cDNA (1 mg) was used as a template for PCR. Degenerate primers were designed from the conserved regions of insect nAChR a1, a2, a3, a6, a8 and b1 subunits respectively (Table S1). PCR reactions were performed with 0.1 mM dNTPs, 5 mM oligonucleotide primers, and 1.0 unit of Go-taq DNA polymerase (Promega, Madison, WI, USA) in a total volume of 20 mL. Thermal cycling conditions were 94
C for 3 min followed by 20 cycles of 94
C for 40 s, 52
C (0.5
C/cycle) for 40 s, and 72
C for 30 s, then followed by 15 cycles of 94
C for 40 s, 42
C for 40 s, and 72
C for 30 s. The last cycle was followed by final extension at 72
C for 8 min. The full-length cDNA was obtained by the rapid amplification of cDNA ends (RACE) according to the 5'and 3'full RACE Core Set (Takara, Kyoto, Japan) protocol with gene-specific primers (Table S1). The resulting PCR products were cloned into pGEM TEasy vector (Promega, Madison, WI, USA). Several independent sub-clones were sequenced from both directions. Sequence homology was determined using the NCBI BLAST online services at http://www.ncbi.nlm.nih.gov/BLAST. Protein alignments were performed using the ClustalW program http://www.ebi.ac.uk/services/ . 2.3. DUM neurons preparation The DUM neuron cells were isolated from the sixth abdominal (A6) ganglion of the adult male cockroaches (Lapied et al., 1989, 1990b; Salgado and Saar, 2004). The A6 ganglions were incubated 25 min at 37
C in cockroach physiological saline (185 mM NaCl, 3.0 mM KCl, 4 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.2, adjusted with NaOH) supplemented with collagenase (type IA, 1 mg/mL, Sigma, St. Louis, MO, USA). Then the ganglions were

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washed three times with cockroach physiological saline and mechanically dissociated by repetitive gentle suctions through a Pasteur pipette. The cell suspension was filtrated into cockroach physiological saline containing 5 mM CaCl2 plus 10% fetal calf serum (GIBCO-BRL, Gaithersburg, MD, USA), with 50 IU/mL penicillin and 50 mg/mL streptomycin (GIBCO-BRL, Gaithersburg, MD, USA) through 100 mM mesh sieve strainer and incubated at 37
C. All operations were conducted under sterile conditions at room temperature (25
C). The DUM neuron cells were used for RNAi after 12 h. 2.4. DUM neurons RNAi For in vitro transfection, FAM labeled short interfering RNA (siRNA, 22e23 nt siRNAs) duplexes were chemically synthesized and purified by GenePharma Co., Ltd (Shanghai, China). The sequences for each strand with symmetric 3 TT overhangs were listed in Table S2. After synthesis, the siRNAs were diluted to 20 mM with DEPC treated water and kept at 80
C until use. Transfection was performed according to HiPerFect Transfection Reagent (Qiagen, Dusseldorf, NW, Germany) manufacturer. The siRNA (1 mL) and HiPerFect Transfection Reagent (3 mL) were added into 200 mL cockroach physiological saline containing 5 mM CaCl2 and incubated for 5e10 min at room temperature (25
C). Then 200 mL DUM neuron cells cultures were transferred to 24-well plates and incubated with siRNA. The negative control provided by GenePharma Co., Ltd (Shanghai, China) was performed with the same concentration of siRNAs for control genes (control siRNA) and all transfections had at least three repetitions. The DUM neuron cells were maintained in an incubator containing 5% CO2 at 37
C. The transfection efficiency was assayed in the dark environment within 12 h or 24 h after transfection. The culture plates were washed three times with cockroach physiological saline to remove siRNAs and the transfection was observed at white light and 488 nm fluorescence. Under the fluorescence microscope (Olympus IX71, Olympus corporation, Tokyo, Japan), the numbers of the cells exhibited fluorescence and no stained ones were counted (3 horizons/well) and the transfection efficiency was calculated with the following equation: transfection efficiency ¼ (fluorescent marked cells/total cells)  100%. 2.5. Quantitative real time PCR (qRT-PCR) The treatment group (treated with siRNA for subunit gene) and the control group (treated with control siRNA) cells (400 mL) were washed three times with PBS and collected into the 1.5 mL centrifuge tubes for RNA extraction at the interval of 12 h. Total RNA was extracted from DUM neuron cells using RNeasy Micro Kit (Qiagen, Dusseldorf, NW, Germany) according to the manual instructions. DNA contaminations were removed by treating RNA extractions products with RNase-free DNase (Qiagen, Dusseldorf, NW, Germany). The first-strand cDNA was synthesized according to the manufacturer's instructions of PrimerScript RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan) with 1 mg total RNA, and immediately stored at 80
C until use. qRT-PCR was performed using SYBR Green Supermix (TaKaRa, Kyoto, Japan) following the manufacturer's instructions on a 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) with gene-specific primers (Table S3). The amplification conditions for the real-time PCR were as following: 95
C for 30 s, followed by 40 cycles of 95
C for 5 s and 60
C for 34 s. Non-template reaction (replacing total RNA by H2O) was set up as negative controls and each reaction had three biological repetitions and three technical repetitions. Two reference genes (b-actin (AY116670.1) and 18s-rRNA (JF499922)) were validated experimentally for each treatment, with the geometric mean

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H. Sun et al. / Insect Biochemistry and Molecular Biology 81 (2017) 32e40

of the selected genes then used for the normalization (Vandesompele et al., 2002). To calculate the relative expression levels of target genes, the 2DDCt method was used (Livak and Schmittgen, 2001). All analysis of fluorescence data was conducted using 7500 Software v.2.0 (Applied Biosystems, Carlsbad, CA, USA).

2.6. Patch-clamp recording In 4 days after RNAi, the treatment and control group DUM neurons (30e50 mm in diameter) were transferred to the extracellular fluid (200 mM NaCl, 3.1 mM KCl, 5 mM CaCl2, 4 mM MgCl2, 10 mM HEPES buffer and 1 mM atropine, and pH was adjusted to 7.4 with NaOH) for electrophysiological recording. The induced currents in DUM neuron cells were recorded using the patch-clamp technique in the whole-cell recording configuration under voltage-clamp mode at different membrane potentials (as mentioned in different places) with an Axopatch 200B Patch-clamp amplifier (Axon Instruments, Foster City, CA) (Courjaret and Lapied, 2001; Hamill et al., 1981; Thany, 2009). Pipettes were pulled from Clark borosilicate glass GC150TF (Warner Instruments, Hamden, CT,

USA) with the resistance of 1.0e2.0 MU when filled with standard pipette solution (160 mM Kþ/D-gluconic acid, 10 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM KF, 3 mM ATP Mg, 10 mM EGTA, 20 mM HEPES, pH was adjusted to 7.4 with KOH). The liquid junction potential between extracellular and intracellular solutions was always corrected before the formation of a gigaohm seal (>1 GU). Current data was recorded by Digidata 1320A digitizer (Molecular Devices, Sunnyvale, CA, USA). Drugs were applied by pneumatic pressure ejection (15 psig, 300 ms, Miniframe, Medical System Corporation, USA) through a glass micropipette positioned in solution within 50 mm from the recorded cells. The steady-state recordings were made 5 min after the setting of the whole cell recording configuration and agonist applications were performed at 2-min intervals. To ensure reproducible current amplitudes among DUM neuron cells, the capacitance ranged between 200 and 250 pF in the experiments. These data were recorded and analyzed by pClamp10 software (Axon Instruments, Foster City, CA).

2.7. Curve fitting and statistical analysis Concentration-response curves were fitted with the Hill

Fig. 1. The transfection of FAM labeled siRNA with HiPerFect Transfection Reagent. A and B were the transfection observation in 12 h after siRNA application. A was photographed by white light, and B was photographed by 488 nm fluorescence. C and D were the transfection observation in 24 h after siRNA application. C was photographed by white light, and D was photographed by 488 nm fluorescence. E and F were the observation of non-transfected group at white light and 488 nm fluorescence.

H. Sun et al. / Insect Biochemistry and Molecular Biology 81 (2017) 32e40

equation for the monophasic sigmoid curve:

I ¼ Imax

.h

 i 1 þ EC50 =xÞnH

where I is the response, Imax is the maximum response, EC50 is halfmaximal activation concentration, x is agonist concentration and nH is Hill coefficient. The biphasic dose-response curves were fitted by using a “double” Hill equation:

.h h .h h  nH1 i i þ Imax_2 1 I ¼ Imax_1 1 þ EC50_1 = x   nH2 ii þ EC50_2 x where I is the response, Imax_1 and Imax_2 are the maximum responses for the first and second receptor subtypes, EC50_1 and EC50_2 are the half-maximal activation concentrations for the first and second receptor subtypes, and nH1 and nH2 are the Hill coefficient for the first and second receptor subtypes. Antagonist was added in the internal pipette solution immediately before use. Values were plotted against the concentrations of the antagonist on a logarithm scale and fitted with an equation: y ¼ 1/[1 þ (IC50/[ antagonist])nH], where nH is the Hill coefficient. Differences in values were analyzed by one-way ANOVA with at

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least three repeats. Differences between values were assessed statistically using a LSD pair wise comparison of means at a significance level of 0.05. 3. Results 3.1. Cloning of six nAChR subunits in P. americana Using RT-PCR with general primers designed on insect nAChR subunits and RACE techniques, six putative nAChR subunit genes (a1, a2, a3, a6, a8 and b1) were cloned from P. americana with full open reading frames and denominated as Paa1 (GeneBank accession number: KP725463), Paa2 (KP725464), Paa3 (KR021292), Paa6 (KP725465), Paa8 (KP725466) and Pab1 (KP725467), respectively (Fig. S1). The deduced amino acid sequences of these subunit genes showed high similarities to their orthologous genes from other insect species. The constructed phylogenetic tree and cluster analysis were listed in Fig. S2. Additionally, typical conserved motifs of nAChR subunits were also observed, such as four conserved transmembrane regions (TM1-TM4) and three agonist binding sites (Loop A, B, C in a subunits, and Loop D, E, F in b1 subunits) (Fig. S1). It should be noted that this work began in 2010 and in order to obtain more in-depth results, we took a long time to finish it. Other researchers had submitted several of P. americana nAChR subunit gene sequences to GenBank before us. Although high similarities

Fig. 2. The relative expression of subunit genes at different times after the transfection of siRNA for subunit genes or control siRNA, using b-actin and 18s-rRNA as reference gene. Data were means of at least three independent experiments ± SEM. A, Relative levels of Paa1 gene at different times at interval of 12 h. B, Relative levels of different subunit genes at 24 h and 96 h.

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H. Sun et al. / Insect Biochemistry and Molecular Biology 81 (2017) 32e40

Fig. 3. Agonist potency of imidacloprid on DUM neuron cells with or without aBungarotoxin (a-Bgt) application. Imidacloprid and a-Bgt was applied by pressure ejection from a patch pipette. A and B, Representative inward currents activated by imidacloprid at 0.1 mM concentration with or without a-Bgt (0.5 mM). C and D, Representative inward currents activated by imidacloprid at 10 mM concentration with or without a-Bgt (0.5 mM). E, Dose-response curves for imidacloprid with or without 0.5 mM a-Bgt, and the putative dose-response curve (dashed line) for the imidaclopridactivate currents that were inhibited by a-Bgt. Points are means of 5e6 independent experiments ± SEM.

were found between sequences in this study and those submitted to GenBank by other researchers, there are a few differences between amino acid sequences in various regions, such as loop A of Paa2 (JQ585634, the Genbank accession number for the sequence from the other study) and TM4 of Paa3 (JQ585635), and we got a longer 30 UTR of Paa1 (JQ609337). 3.2. SiRNA transfection and RNAi efficiency analysis In order to develop RNAi technique against nAChR subunits in DUM neurons, the transfection efficiency of siRNA delivery into neuron cells was first examined. The FAM labeled siRNAs were thought to be successfully transfected into DUM neuron cells if the fluorescence was observed. The calculated transfection efficiency was above 60% at 12 h and 24 h, which was thought to be enough for further RNAi efficiency evaluation (Fig. 1). In order to test the changes in gene expression, neurons were collected at interval of 12 h after transfection and qRT-PCR was conducted to investigate the efficiency of RNAi. The RNAi efficiency was the reduction of gene expression in siRNA treated neurons compared to that in neurons treated by the control siRNA at the

Fig. 4. Influence of RNAi against different subunit genes on agonist or antagonist potencies on isolated DUM neuron. A, Current-voltage relationships of imidaclopridinduced current amplitudes at different steady state holding potentials of neurons with RNAi against Paa3, Paa8 or Pab1. Imidacloprid concentration was 200 mM. B-D, Current-voltage relationships of nicotine-induced current amplitudes at different steady state holding potentials of neurons with RNAi against Paa1, Paa2 and Pab1, with or without a-conotoxin ImI (10 mM). In C and D, the curves were only presented between 30 and 20 mV. Nicotine concentration was 20 mM. Points are mean s of 5e6 independent experiments ± SEM.

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that all subunits had high RNAi efficiencies at 96 h (69e84%), which were higher than that at 24 h. As prediction, RNAi against a specific subunit gene only caused the expression decrease for this subunit, but not obviously changed the expression of other subunit genes tested in this study (Fig. S3). 3.3. Imidacloprid agonist acting subtypes in a-Bgt-sensitive and -resistant nAChRs

Fig. 5. Representative currents activated by imidacloprid and dose-response curves of imidacloprid on isolated DUM neurons. A, Representative currents activated by imidacloprid (200 mM) on DUM neurons following RNAi with siRNA for different subunit genes or control siRNA, at the steady state holding potential of 70 mV. B and C, Doseresponse curves of imidacloprid on DUM neurons following RNAi with siRNA for Paa1, Paa2 and Paa3 (B), or Paa6, Paa8 and Pab1 (C), using RNAi with control siRNA as the control. Points are means of at least 5 independent experiments ± SEM.

same time. The expression levels of Paa1 gene were significantly lower in neurons treated with Paa1 siRNA than that of control siRNA at all test times from 12 h to 132 h, with the biggest RNAi efficiencies close to 78% at 72e96 h (Fig. 2A). From the preliminary tests, the changes in mRNA levels of other subunits following RNAi were similar to that of Paa1 (data not shown), and detailed results were especially given at 24 h and 96 h (Fig. 2B). The results showed

In order to test the subunit composition of some important nAChR subtypes, the properties of these subtypes were investigated first. On DUM neurons isolated from the cockroach A6 ganglion, imidacloprid induced currents were recorded with the patchclamp technique (Fig. 3). Because 1 mM atropine was added into the extracellular fluid to block the possible currents from the muscarinic acetylcholine receptors (Lapied et al., 1990a), these current was believed from the nicotinic acetylcholine receptors activated by imidacloprid applications. Application of a-bungarotoxin (0.5 mM or higher concentrations) could not inhibit the currents activated by 0.1 mM imidacloprid (Fig. 3AeB), but significantly reduced the currents from 10 mM imidacloprid (Fig. 3CeD). These preliminary results indicated that imidacloprid acted on the a-Bgtresistant nAChR subtypes at low concentrations and on both a-Bgtsensitive and -resistant nAChR subtypes at high concentrations. In order to confirm this indication, dose-response curves were obtained on DUM neurons applied by imidacloprid at different concentrations, and with or without a-Bgt. A biphasic dose-response curve was obtained when only imidacloprid was applied (Fig. 3E), with Imax and EC50 values on the first subtype of 191.3 ± 27.1 pA (Imax_1) and 82.7 ± 13.4 mM (EC50_1), and on the second subtype of 425.7 ± 51.9 pA (Imax_2) and 15.8 ± 2.3 mM (EC50_2). When a-Bgt (0.5 mM) was applied, the monophasic curve for imidacloprid was obtained with Imax and EC50 values of 184.6 ± 25.5 pA and 74.32 ± 12.9 mM, which were close to Imax_1 and EC50_1 on the first nAChR subtype when only imidacloprid was applied. The second nAChR subtype that was sensitive to imidacloprid was completely blocked by a-Bgt (the dashed line in Fig. 3E). It should be noted that the a-Bgt-sensitive nAChR subtype (EC50_2 ¼ 15.8 ± 2.3 mM) with much lower sensitivity to imidacloprid than that resistant to a-Bgt (EC50_1 ¼ 82.7 ± 13.4 mM from the biphasic dose-response curve, and EC50 ¼ 74.32 ± 12.9 mM from the monophasic curve). So, we were more interested in the a-Bgtresistant nAChR subtypes on DUM neurons. 3.4. Influence of RNAi against nAChR subunits on agonist potencies on a-Bgt-resistant nAChR subtypes As reported in previous studies (Bodereau-Dubois et al., 2012; Courjaret and Lapied, 2001), nicotine acted on two different subtypes of a-Bgt-resistant nAChRs, nAChR1 and nAChR2, on DUM

Table 1 Agonist potency of imidacloprid and nicotine, and antagonist potency of a-conotoxin IMI on DUM neurons following RNAi with different siRNA. Imidacloprid at 70 mV

RNAi-CK RNAi-Paa1 RNAi-Paa2 RNAi-Paa3 RNAi-Paa6 RNAi-Paa8 RNAi-Pab1

Nicotine at 70 mV

Imax (pA)

EC50 (mM)

Imax (nA)

186.52 ± 22.39 a 190.74 ± 28.63 a 174.39 ± 31.42 a 66.04 ± 11.23 c 170.26 ± 26.84 a 103.35 ± 15.01 b 50.37 ± 8.030 c

75.45 ± 11.04 a 80.10 ± 14.92 a 77.31 ± 9.67 a 85.27 ± 15.14 a 89.92 ± 14.05 a 321.32 ± 41.16 b 76.13 ± 9.62 a

1.24 1.33 1.27 0.52 1.20 0.83 0.44

± ± ± ± ± ± ±

0.14 0.21 0.16 0.08 0.19 0.11 0.05

a a a c a b c

114.36 123.02 122.13 130.29 121.82 300.06 115.93

a-conotoxin ImI at 20 mV

Nicotine at 20 mV

EC50 (mM) ± ± ± ± ± ± ±

14.40 15.84 18.51 16.36 18.94 45.57 18.34

EC50 (mM)

Imax (nA) a a a a a b a

0.97 0.65 0.44 1.02 0.99 0.95 0.41

± ± ± ± ± ± ±

0.10 0.07 0.04 0.11 0.13 0.08 0.05

a b c a a a c

15.92 36.06 16.13 14.50 13.73 15.14 18.05

± ± ± ± ± ± ±

2.03 3.72 2.65 2.71 2.90 3.12 2.86

IC50 (mM)a a b a a a a a

1.38 ± 0.24 a 13.03 ± 2.51 b 1.62 ± 0.25 a 1.45 ± 0.28 a 1.59 ± 0.33 a 1.76 ± 0.41 a 1.64 ± 0.29 a

Data are means of at least five independent experiments (different batches of DUM neurons) ± SEM. Different letters in the same column show significant differences at 0.05 level. a At 20 mV potential, a-conotoxin ImI acted as the antagonist to inhibit the currents caused by nicotine.

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neurons, while imidacloprid only acted on nAChR1 as an agonist, which was perfectly repeated in this study (Fig. S4). Imidacloprid activated responses between 90 and þ 20 mV, which was strongly reduced by d-tubocurarine (Fig. S4A). Nicotine activated responses in a biphasic aspect, between 90 and 30 mV and between 30 and þ 20 mV, which could be inhibited by d-tubocurarine (Fig. S4B) and a-conotoxin ImI (Fig. S4C), respectively. With the combination of RNAi and electrophysiological technique, the influence of RNAi against Paa1, Paa2, Paa3, Paa6, Paa8 or Pab1 on imidacloprid potencies were evaluated. The electrophysiological recording was conducted in 96 h (4 days) after RNAi because of the greatest drop of mRNA levels (Fig. 2) and protein levels (preliminary results, data not shown). At the membrane potential from 90 to þ20 mV, RNAi against Paa3, Paa8 and Pab1 resulted in the significant decrease in imidacloprid-activated currents (Fig. 4A), but this decrease was not found on DUM neurons following RNAi against Paa1, Paa2, and Paa6 (data not shown). Similarly, RNAi against Paa3, Paa8 and Pab1 caused similar decrease in nicotine-activated currents, but only at the membrane potential from 90 to 30 mV. In contrast, at the membrane potential from 30 to þ20 mV, RNAi against Paa1 (Fig. 4B), Paa2 (Fig. 4C) and Pab1 (Fig. 4D) resulted in the significant decrease in nicotine-activated currents. Another finding was, at the membrane potential from 30 to þ20 mV, that only RNAi against Paa1 could decrease the inhibition potency of a-conotoxin ImI against nicotine (Fig. 4B). In order to present the influences of RNAi against different subunit genes on agonist or antagonist potencies, the changes in Imax, EC50 and IC50 values were compared among DUM neurons at the fixed membrane potentials. RNAi against Paa3, Paa8 and Pab1 resulted in big decrease in currents activated by imidacloprid (200 mM) at 70 mV, among which RNAi against Paa8 made the biggest change (Fig. 5A). Although the influence of RNAi against Paa3 and Pab1 on Imax values was bigger than that against Paa8, only RNAi against Paa8 caused a significant increase in EC50 value (Fig. 5BeC). At 70 mV, the influence pattern of RNAi on nicotineactivated currents was identical to that of imidacloprid, such as the significant influence on peak amplitudes from RNAi against Paa3, Paa8 and Pab1, and EC50 increase only in RNAi against Paa8 (Table 1). RNAi against Paa1, Paa2 and Pab1 resulted in the significant decrease in nicotine-activated currents at þ20 mV (Fig. 6A). The influence of RNAi against Paa2 and Pab1 on Imax values was bigger than that against Paa1, but only RNAi against Paa1 caused a significant influence on EC50 value among RNAi against three subunit genes (Fig. 6BeC). RNAi against Paa1, Paa2 and Pab1 also showed significant influences on the efficacies for a-conotoxin ImI to antagonize nicotine at þ20 mV, such as the current amplitudes evoked by nicotine when co-applied with a-conotoxin ImI (Fig. 7). Another finding was that only RNAi against Paa1 caused a dramatic increase (9.4-fold) in IC50 value for a-conotoxin ImI to antagonize nicotine (Table 1). 4. Discussion As one of the most important targets of insecticides, insect nAChRs attract worldwide attention (Tomizawa and Casida, 2001). In order to characterize the pharmacological properties of insect nAChRs, different techniques were tried, among which the recombination of insect nAChRs in heterologous systems were thought the best way. Several heterologous expression systems, such as Xenopus oocyte and Drosophlia S2 cells, have been used for the in-depth studies of recombinant insect nAChRs (Millar, 1999, 2003). Unfortunately, we are still not able to generate functional insect receptors with all insect nAChR subunits themselves in these

Fig. 6. Representative currents activated by nicotine and dose-response curves of nicotine on isolated DUM neurons. A, Representative currents activated by nicotine (20 mM) on DUM neurons following RNAi with siRNA for different subunit genes or control siRNA, at the steady state holding potential of þ20 mV. B and C, Dose-response curves of nicotine on DUM neurons following RNAi with siRNA for Paa1, Paa2 and Paa3 (B), or Paa6, Paa8 and Pab1 (C), using RNAi with control siRNA as the control. Points are means of at least 5 independent experiments ± SEM.

systems (Bertrand et al., 1994; Lansdell and Millar, 2000). As a replacement, people try to study the function and the combination of insect nAChRs using the native techniques, such as the radioligand binding and co-immunoprecipitation (Li et al., 2010). However, these semi-native techniques only give some indirect evidences to deduce the properties of insect nAChRs, such as the subunit composition. The more native techniques to study insect nAChRs are from the neuron cells isolated from insect CNS. As a representative example, the american cockroach dorsal unpaired median (DUM) neurons were widely used to characterize the pharmacological properties of insect nAChRs and the interactions between nAChRs and other proteins. For example, the direct evidences showed that imidacloprid acted on both a-Bgt-sensitive and -resistant nAChR subtypes on the cockroach DUM neurons (Bodereau-Dubois et al., 2012; Courjaret and Lapied, 2001; Salgado and Saar, 2004). However, until now, there are few studies to analyze the subunit composition for these nAChR subtypes, which limits the application of DUM neurons in interaction analysis

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Fig. 7. Inhibition curves of a-conotoxin IMI on the responses evoked by nicotine (200 mM) on DUM neurons at the steady state holding potential of þ20 mV. DUM neurons were treated with siRNA for Paa1, Paa2 and Paa3 (A), or Paa6, Paa8 and Pab1 (B), using RNAi with control siRNA as the control. Points are means of at least 5 independent experiments ± SEM.

between nAChRs and insecticides and further in insecticide development. Here, combining with RNA interference (RNAi), a powerful and widely used tool for the analysis of gene function in insects (Caplen et al., 2001), we tried to identify the potential subunits in two aBgt-resistant nAChR subtypes on the cockroach DUM neurons, in which imidacloprid acted on one subtype. Imidacloprid could evoke inward currents at 70 mV with the amplitude up to 580 pA, in which only a part of each current could be blocked by a-Bgt. Because 1 mM atropine was added into the extracellular fluid to block the possible currents from the muscarinic acetylcholine receptors or mixed nicotinic-muscarinic acetylcholine receptors (Lapied et al., 1990a), the results indicated that imidacloprid acted on the a-Bgt-sensitive nAChR subtype as an agonist. In a previous study, imidacloprid was found to inhibit the ACh-activated currents on the a-Bgt-sensitive nAChRs (nAChD receptors), possibly acting as an antagonist (Salgado and Saar, 2004). So, on a-Bgt-sensitive nAChRs, imidacloprid had two target subtypes, exerting the agonist activity on one subtype and the antagonist activity on another subtype. After blocking a-Bgt-sensitive nAChRs by 0.5 mM a-Bgt, imidacloprid acting subtypes were analyzed on a-Bgt-resistant nAChRs. In accord with previous studies (Bodereau-Dubois et al., 2012; Courjaret and Lapied, 2001), nicotine exerted the agonist activity on two subtypes (nAChR1 and nAChR2) of a-Bgt-resistant nAChRs, but imidacloprid only activated one subtype nAChR1 as an agonist.

When comparing imidacloprid potencies as agonists on subtypes belonging to a-Bgt-sensitive and a-Bgt-resistant nAChRs, the aBgt-resistant subtype (EC50 ¼ 74.32 ± 12.85 mM) was much more sensitive to imidacloprid than that of a-Bgt-sensitive subtype (EC50 ¼ 15.83 ± 2.34 mM), with 213-fold difference based on EC50 values. So, from whole cell aspect, the sensitivity of the cockroach DUM neurons to imidacloprid would be more reflected on the aBgt-resistant nAChR subtype, which led us to analyze the potential subunit composition of a-Bgt-resistant nAChR subtypes. RNAi against Paa3, Paa8 and Pab1 resulted in the significant decrease in imidacloprid- and nicotine-activated currents at the membrane potential of 70 mV, a potential among the range to define nAChR1 of a-Bgt-resistant nAChRs. RNAi against these three subunits could reduce imidacloprid-activated currents at all tested potential between 90 and þ20 mV. Because imidacloprid only activated currents on nAChR1 as an agonist, it appears reasonable to conclude that Paa3, Paa8 and Pab1 are involved in the subunit composition of nAChR1. Similarly, RNAi against Paa1, Paa2 and Pab1 resulted in the significant decrease in nicotine-activated currents on nAChR2. RNAi against Paa1 could dramatically change the potency of a-conotoxin ImI as an antagonist to inhibit nicotineactivated currents on nAChR2. The results indicated that Paa1, Paa2 and Pab1 were involved in the subunit composition of nAChR2. Interestingly, in the brown planthopper (Nilaparvata lugens), a hemipteran insect, Nla3, Nla8 and Nlb1 were included in one receptor, and Nla1, Nla2 and Nlb1 in another receptor, which

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constituted the higher and lower affinity binding sites for imidacloprid (Li et al., 2010). A difference is that the receptor containing Nla1, Nla2 and Nlb1 may be activated by imidacloprid as an agonist, because imidacloprid was a full agonist on the recombinant receptors containing Nla1, Nla2 and rb2 (rat b2 subunit) expressed on Xenopus oocytes (Liu et al., 2009). From the present study and previous reports, there are at least three nAChR subtypes that are sensitive to imidacloprid, including one agonist acting subtype and one antagonist acting subtype in aBgt-sensitive nAChRs, and one agonist acting subtype in a-Bgtresistant nAChRs. However, in insect CNS, only two binding sites with different affinities for imidacloprid have been identified in insects from Hemiptera, such as Aphis craccivora (Kd ¼ 0.8 nM and 21 nM), Myzus persicae (Kd ¼ 0.14 nM and 12.58 nM), Nephotettix cincticeps (Kd ¼ 4.3 pM and 1.23 nM)and N. lugens (Kd ¼ 3.5 pM and 1.5 nM) (Li et al., 2010; Lind et al., 1998), and from Orthoptera with one species, Locusta migratoria (Kd ¼ 0.2 nM and 8.9 nM) (Wiesner and Kayser, 2000). In other insect species, including P. americana (Kd ¼ 3.14 nM), single binding site was identified (Lind et al., 1998). Comparing to single binding site for imidacloprid in P. americana CNS and two sites for some insect species, at least three nAChR subtypes that are sensitive to imidacloprid revealed the high diversity of nAChRs expressed in the cockroach DUM neurons. The abundance and diversity of nAChRs in the cockroach DUM neurons make it an ideal tool to analyze the pharmacology, diversity and evolution, and other properties of insect nAChRs. Acknowledgements This work was supported by National Natural Science Foundation of China (31322045), Jiangsu Science for Distinguished Young Scholars (BK20130028), and National Key Technology Research and Development Program (2012BAD19B01). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2016.11.009. References Adams, M.D., Celniker, S.E., Holt, R.A., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185e2195. Baumann, A., Jonas, P., Gundelfinger, E.D., 1990. Sequence of D alpha 2, a novel alpha-like subunit of Drosophila nicotinic acetylcholine receptors. Nucleic Acids Res. 18, 3640. Bertrand, D., Ballivet, M., Gomez, M., Bertrand, S., Phannavong, B., Gundelfinger, E.D., 1994. Physiological properties of neuronal nicotinic receptors reconstituted from the vertebrate beta 2 subunit and Drosophila alpha subunits. Eur. J. Neurosci. 6, 869e875. Bodereau-Dubois, B., List, O., Calas-List, D., Marques, O., Communal, P.Y., Thany, S.H., Lapied, B., 2012. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidaclopridinsensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides. J. Pharmacol. Exp. Ther. 341, 326e339. Caplen, N.J., Parrish, S., Imani, F., Fire, A., Morgan, R.A., 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U. S. A. 98, 9742e9747. Courjaret, R., Lapied, B., 2001. Complex intracellular messenger pathways regulate one type of neuronal alpha-bungarotoxin-resistant nicotinic acetylcholine receptors expressed in insect neurosecretory cells (dorsal unpaired median neurons). Mol. Pharmacol. 60, 80e91. Grolleau, F., Lapied, B., 2000. Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity. J. Exp. Biol. 203, 1633e1648. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85e100. Hermsen, B., Stetzer, E., Thees, R., Heiermann, R., Schrattenholz, A., Ebbinghaus, U., Kretschmer, A., Methfessel, C., Reinhardt, S., Maelicke, A., 1998. Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression.

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