Molecular Action of Insecticides on Ion Channels - ACS Publications

Chapter 14

Chloride-Channel Gene Probes from Cyclodiene-Resistant and -Susceptible Strains of Blattella germanica 1

Downloaded by CORNELL UNIV on August 30, 2016 | http://pubs.acs.org Publication Date: May 9, 1995 | doi: 10.1021/bk-1995-0591.ch014

Koichiro Kaku and Fumio Matsumura Department of Environmental Toxicology and Department of Entomology, University of California, Davis, CA 95616

The DNA and amino acid sequence of the membrane spanning region of a G A B A receptor of the German cockroach (Blattella germanica) has been identified, along with information on the nature and the specific site of mutation in a cyclodiene resistant strain (LPP strain). In this resistant strain the mutation has occurred at the most conserved, lower M2 cylinder region involving a G to T conversion, resulting in an amino acid change of alanine (GCC) residue to serine (TCC). The site, furthermore, coincides with the most conserved region of all G A B A receptor subunits and the expected C1 transporting segment constituting the innermost surface of the channel opening. The deduced sequence of the German cockroach G A B A receptor differs from that of the Drosophila mainly in the connecting region between M and M . -

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It has been known for a long time that many insect species are capable of developing high levels of specific resistance to highly toxic cyclodiene insecticides (1-2). Dr. A.W.A. Brown, a founding father of insecticide resistance studies, has described the cyclodiene resistance problem as "a delight for geneticists and a nightmare for biochemists." Indeed, the mode of inheritance of the resistance gene was found to follow a straightforward Mendelian model, but the mechanism by which such a resistance phenomenon is phenotypically expressed remained a mystery for the long time period from the early 1960s to the 80s. Early efforts included studies on lipid differences, search on resistance antagonists, metabolism, tissue distribution and uptake studies as well as cross-resistance studies including that to DDT, organophosphates and carbamates. These studies have clearly established that thisresistancemechanism is effective to most chemically defined cyclodiene insecticides and lindane, but not to any other types of commercially used pesticides. Since this group of chemicals includes very metabolically stable insecticides, such as heptachlor epoxide, and since metabolism and insect uptake studies could not 1

Current address: K-I Chemical Research Institute Company Ltd., Shizuoka, Japan

0097-6156/95/0591-0216$12.00A) © 1995 American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Cl-Channel Gene Probes

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reveal any difference between resistant and susceptible strains within the same species, the type of resistance developed here has been called "target insensitivity." Electrophysiological data clearly supported such a view as isolated, resistant nervous systems required higher concentrations of cyclodienes to show symptoms of excitation than control nerves. Interestingly, Brown (2) mentioned the anecdotal observation that pre-resistant populations among malaria mosquitoes were already present in Africa, even before dieldrin was used there. The level of pre-resistant population has been said to be in the order of several percent among Anopheles mosquito larvae. These observations suggest that a certain biochemical change must have taken place in the nervous system and that such a change is stable in a given population (i.e., is not giving the resistant individuals selective disadvantages against other stresses). The main question has been what the actual main target of these insecticides is. Without this knowledge we have no idea about the site of change in the resistant nervous system. In 1982 Dr. Ghiassudin and I (3) formulated a hypothesis that the target of this group of insecticides could be the G A B A receptor, based upon the observation that heptachlor epoxide and lindane could prevent GABA-induced increase in C1" uptake by isolated nerve cords and coaxial muscles from the American cockroach. Also helpful in this regard was the incipient observation made in our laboratory that cyclodiene resistant German cockroaches showed cross-resistance to picrotoxin, whose action mechanism at that time was being established to act on the G A B A receptors of the mammalian central nervous system. By using H-dihydropicrotoxinin as an artificial ligand we were able to clearly establish that the picrotoxinin binding site in the resistant nervous system is less affected by dieldrin than that of the susceptible counterpart. Subsequent mechanistic studies (3-8) have shown that these insecticides are specific antagonists of the G A B A action on G A B A receptors in both mammals and insects, and that their binding site within the receptor is probably identical to that of picrotoxinin, a well known naturally occurring antagonist of G A B A . In the case of the German cockroach, cyclodiene resistance was shown to have evolved as a result of a very specific change in the biochemical properties of the G A B A receptor itself (3,6,8). Such a change in this target site gives the homozygous individuals 10- to 100-fold resistance to these insecticides (6). The G A B A receptors of several cyclodiene-resistant species have been shown to exhibit less binding affinity to cyclodienes (9,10) or specific radioligands (11, 12) including H-dihydropicrotoxinin (6) as compared to each of their susceptible counter parts. Therefore, these insects obviously offer us a unique opportunity to understand the site of action of these chemicals. Recently ffrench-Constant et al. (13) have identified the DNA sequence of a G A B A receptor from Drosophila melanogaster (14) from a cloned DNA. This G A B A receptor differs considerably from the mammalian forms, and was named Rdl Subsequentiy Henderson et al. (14) have also cloned and sequenced another G A B A receptor gene, LCCH3 from Drosophila melanogaster cDNA libraries, which has been termed as a Psubunit based on the overall similarity to mammalian P subunits and that of Lymnaea stagnalis, the first G A B A receptor identified in an invertebrate species (15). In more recent papers ffrench-Constant et al. (16) and Thompson et al. (17) have shown that a mutation occurred in cyclodiene resistant Rdl type G A B A receptor to cause an Ala - Ser shift (at 302) in Drosophila and Aedes aegypti, respectively. 36

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Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS

MATERIALS AND METHODS The strains of German cockroaches (Blattella germanica) used for this work have been described elsewhere (9,10). The cyclodiene-resistant strains were occasionally selected by using dieldrin to maintain the homozygous resistant individuals. The LPP strain was genetically selected from the original chlordane-resistant London strain (10) by backcrossing to the CSMA-susceptible strain for 8 generations, each time selecting for hetero zygous resistance, and subsequent self crossing and selection for homozygous resistant individuals.


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Isolation of poly A mRNA. Poly A mRNA was isolated (18) from adult German cockroaches (CSMA and LPP strain) (10). Heads and thoraxes were collected from 100 adult German cockroaches (50 males and 50 females) combined, and immediately frozen under liquid nitrogen. Approximately 3 g of tissues were ground in mortar with pestle under liquid nitrogen, and the slurries were transferred into 50 ml cell culture tubes. Immediately thereafter nitrogen was evaporated, and 40 ml lysis buffer [0.2 M NaCl, 0.2 M Tris-HCl (pH 7.5), 1.5 mM MgCl , 2% SDS, 200 ug/ml Proteinase K (Boehringer Mannheim) in DEPC-treated H 0] was added, followed by immediate homogenization by Polytron® for 15-30 sec. The homogenates were incubated at 45°C for 2 hr with inter mittent agitation. The residue in the lysate, mainly the cuticle of cockroaches, was separated by low speed centrifugation, and the supernatant was transferred into a sterile 50 ml cell culture tube. The NaCl concentration of the lysate (0.2 M) was adjusted to that of binding buffer" (0.5 M NaCl) with 60 ul of 5 M NaCl in the same buffer per ml lysate. 80 mg of Oligo(dT) cellulose (GIBCO BRL/Iife Technologies, Gaitherburg, MD) which was equilibrated to the same concentration of binding buffer [0.5 M NaCl, 0.01 M TrisHCl (pH 7.5)] was mixed with the lysate, followed by incubation for 1 hr at room temper ature with intermittent agitation. The treatment of poly A mRNA bound oligo(dT) cellulose and the elution scheme were identical to that of Badley et al. (18). The precipitated poly A mRNA was pelleted by centrifugation at 10,000 x g for 10 min, washed with ice-cold 75% ethanol, vacuum dried for 30 min and dissolved in 54 ul of DEPC-treated H 0 . Yields of extracted poly A mRNAs were approximately 42 ug both from C S M A and LPP strains. 2

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Preparation of cDNA. Both mRNAs (38.5 ug) were reverse-transcribed into first strand cDNAs in 200 ul of reaction buffer containing 20 mM Tris-HCl (pH 8.4 at 25°C), 50 mM KC1, 5 m M MgCl , 0.01% gelatin [wt/volj, 1 mM deoxynucleotide triphosphates (Pharmacia LKB), 200 units RNAsin (cloned, Promega), 3000 units M - M L V reverse transcriptase (GIBCO BRL/life Technologies) and 1 ug oligo(dT) -adapter primer (5'G A C T C G A G T C G A C A T C G A T T T T T - T T n T n T r n T - 3 ' ) (Frohman et al., 1988). The reaction mixture was incubated at room temperature for 15 min, followed by incubation at 37°C for 1.5 hr and 95°C for 5 min. The mixture was then quickly chilled on ice and stored at -20 °C until further use. A l l primers were synthesized on Model 391 D N A Synthesizer (Applied Biosystems, Foster City, CA) and purified by gel filtration using Sephadex-50. 2

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Preparation of double stranded DNA by PCR. Each 1 ul cDNA mixture was combined in a 50 ul reaction mixture with 5 ul 10 X PCR buffer [100 m M Tris-HCl (pH 9.0 at 25°C), 500 mM KC1, 1.5 mM MgCl , 1% Triton X-100], 0.2 m M deoxynucleotide triphosphates (Pharmacia L K B Biotechnology), 1 unit Taq DNA polymerase (Promega), 2


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and 2.5 pg of both upstream and downstream degenerate primers or 5 pmol of specific primers. The mixture was overlaid with mineral oil. The PCR condition used was denaturation 95°C for 1 min, annealing 55°C for 2 min and extension 72°C for 3 min for 40 cycles in a DNA Thermal Cycler (Precision Scientific, Chicago, EL). 9 pi of each amplified product was run on a 2% agarose gel (FMC BioProducts, Rockland, ME) made with 1 X TBE (0.45 M Tris-HCl, 0.45 M boric acid, 10 mM EDTA) and stained with ethidium bromide. The desired PCR fragments were exercised from agarose gel and cut into pieces using a sterile blade, rinsed by ddH 0 twice, frozen and thawed in 100 pi ddH 0 (repeated twice), and left in a refrigerator overnight. 3 pi of the eluted D N A solution was re-amplified in 150 pi reaction mixture with 15 pi of 10 X PCR buffer, containing 0.2 mM deoxynucleotide triphosphates, 3 units Taq D N A polymerase, and 7.5 pg of both upstream and downstream degenerate primers or 15 pmol of specific primers. The PCR conditions were identical to the above except the annealing time was 1 min. In the second round PCR, three to six identical batches were amplified at the same time to prepare a large quantity of samples for DNA sequencing reaction or enzymatic restriction. The mass-produced PCR solution was collected in a 1.5 ml microcentrifuge tube and the mineral oil removed with chloroform. The water layer (450-900 pi) was concentrated to 35-40 pi by Centricon-100® (Amicon) at 1,000 x g for 30 min at 4-10°C. The concentrated DNA fragment was electrophoresed on 1% agarose gel made with 1 X TBE and excised from the gel, followed by purification by using QIAEX matrix (QIAGEN) according to the manufacturer's protocol. The DNA was eluted with ddH 0 and the concentration of the purified DNA was estimated by GelMarker® (Research Genetics, Huntsville, AL) as a quantitative standard on a 2% agarose gel. Usually 5-10 pg DNA was obtained from 450 |jl reaction mixture under our standard experimental condition. 2


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D N A sequencing reaction of P C R amplified double-stranded DNA. Sequenase® Version 2.0 System (United States Biochemical, Cleveland, OH) and DNA polymerase I (Klenow fragment) Promega) (20) were used for DNA sequencing reaction. D N A template-primer solution was made to 10 pi including the purified double-stranded D N A (approximately 0.5-2.0 pmol), primer (30 times the amount of DNA for a specific primer or approximately 1 pg for degenerate primer) and 2 pi of 5 X Sequenase Buffer [200 mM Tris-HCl (pH 7.5), 100 mM MgCl , 250 mM NaCl], which was boiled for 3 min and quickly cooled in dry ice-ethanol bath. The rest of DNA sequencing procedure was identical to that of Schuurman and Keulen (1991). Electrophoresis using 1 X TBE system was performed for 1.75 hr or 4.75 hr at 60 W on 7.4 M urea/6% Long Ranger gel (AT Biochem, Malvern, PA, 42 x 36 x 0.02 cm) with STS-45 Gel Electrophoresis Unit (IBI, New Haven, CT). The gel was dried at 60-80°C for 2 hr on Whatman 3 M M paper under vacuum and exposed to Kodak XAR-5 film (Rochester, NY) for 8 to 72 hr. 2

Strategies for identification of the G A B A receptor genes through P C R approach. The general flow diagram of the approach adopted is shown in Fig. 1. In brief, Step I. Primer A , B and C as shown were used to obtain DNA fragments designated as PCRDNA A - C and B-C. Step II. Since the DNA fragment amplified by the combination of primer B and primer C (i.e., PCR-DNA- B-C) contained two kinds of subunits of G A B A receptor genes, it was digested by several restriction endonucleases for analysis of D N A sequencing. One type possessed Rsa I recognition site but no Sfu I recognition site, whereas the other type possessed Sfu I recognition site but no Rsa I recognition site. The mass-produced PCR-DNA B-C (approximately 3 pg) was incubated at 37° C for 1 hr in


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MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS iral loop

•a a 100

200

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M4

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g-flanWng region (Amino acid number)

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c=fr primer A PCR-DNA A-C 3

primer B

PCR-DNA B-C

primer C

primer G PCR-DNA G-ADAPT I adapter primer > primer F PCR-DNA F-ADAPT I adapter primer

(

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D

.—s primer H PCR-DNA H-ADAPT adapter primer 4) ^

P C R - D N A I-J

Primer J

1

Primer J

^ primer L P C R - D N A L - K Qldl 1)

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1

Primer K

^ primer M P C R - D N A M - K (Rdl 2)

II

^ ,

Primer K

^primer L

PCR-DNA L-J (tM3)

I

1

Primer J

s primer M P C R - D N A M-J(tf

Molecular Action of Insecticides on Ion Channels - ACS Publications

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