ACS SYMPOSIUM SERIES 591 Molecular Action of Insecticides on Ion Channels 1 0 0 w 1.f 059 J. Marshall Clark, EDITOR 5- 9 University of Massachusetts 9 1 k- b 1/ 2 0 1 0. 1 oi: org 5 | d cs.99 a1 bs.9, 12 | http://pun Date: May Devbeylo tpheed D frivoimsio an s yomf Apgorsioucmhe mspiocanlsso red 2, 20catio at the 207th National Meeting uly 2Publi of the American Chemical Society, J San Diego, California, March 13-17, 1994 American Chemical Society, Washington, DC 1995 In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. QP 801 .I48M65 1995 copy 1 Molecular action of insecticides on ion Library of Congress Cataloging-in-Publication Data Molecular action of insecticides on ion channels / J. Marshall Clark, editor. p. cm.—(ACS symposium series; 591) "Developed from a symposium sponsored by the Division of Agro­ -chemicals at the 207th National Meeting of the American Chemical Society, San Diego, California, March 13-17, 1994." 1 0 w0 Includes bibliographical references and index. 1.f 9 ISBN 0-8412-3165-6 5 0 95- 1. Insecticides—Physiological effect—Congresses. 2. Ion channels— 19 Congresses. I. Clark, J. Marshall (John Marshall), 1949 - . k- II. American Chemical Society. Division of Agrochemicals. ΙΠ. Amer­ 1/b ican Chemical Society. Meeting (207th: 1994: San Diego, Calif.) 2 IV. Series. 0 1 0. 1 QP801.I48M65 1995 oi: 574.87'5-4c20 95-6612 org 5 | d CIP cs.99 a1 bs.9, 12 | http://pun Date: May TChopisy briogohkt ©is p1r9i9n5te d on acid-free, recycled paper. 0o 2, 2cati American Chemical Society uly 2Publi Achlla pRteigrh itns tRhiess evrovleudm. eT inhdei caaptepse atrhaen cceo poyfr igthhet ocwodneer 'ast c tohnes ebnott tthoamt roefp rtohger afpirhsitc pcaogpeie so fo fe athche J chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. 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Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. 1995 Advisory Board ACS Symposium Series M. Joan Comstock, Series Editor Robert J. Alaimo Cynthia A. Maryanoff Procter & Gamble Pharmaceuticals R. W. Johnson Pharmaceutical Research Institute Mark Arnold University of Iowa Roger A. Minear 1 University of Illinois 0 0 w David Baker at Urbana-Champaign 1.f University of Tennessee 9 05 Omkaram Nalamasu 95- Arindam Bose AT&T Bell Laboratories 9 1 Pfizer Central Research k- b Vincent Pecoraro 1/ 2 Robert F. Brady, Jr. University of Michigan 0 0.1 Naval Research Laboratory 1 oi: George W. Roberts acs.org 1995 | d MChaermyE Edi. tC Caosmteplalnioyn NJoohrtnh CRa. rSolhinaap Sletayt e University 012 | http://pubs.on Date: May 9, MNAUanratitvirhoegnuraasrrilt eyBSt c.o iAfeE nW.l clCeiiss aFc ovonausnninadau atgito hMn adison UDCnooaniuvcte ugUrrlsraritbesyna tnAo aTf. - eICScllhhminnaoimotilshop g aiiegsn C orporation 2, 2cati uly 2Publi GUnuivnedrsai tIy. oGf Keoarngsa s LD.u PSoonmt asundaram J Madeleine M. Joullie Michael D. Taylor University of Pennsylvania Parke-Davis Pharmaceutical Research Lawrence P. Klemann William C. Walker Nabisco Foods Group DuPont Douglas R. Lloyd Peter Willett The University of Texas at Austin University of Sheffield (England) In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. Foreword THE ACS SYMPOSIUM SERIES was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of this series is to publish comprehensive books developed from symposia, which are usually "snapshots in time" of the current research being done on a topic, plus 1 some review material on the topic. For this reason, it is neces­ 0 0 w sary that the papers be published as quickly as possible. 91.f Before a symposium-based book is put under contract, the 5 5-0 proposed table of contents is reviewed for appropriateness to 9 9 the topic and for comprehensiveness of the collection. Some 1 k- papers are excluded at this point, and others are added to b 21/ round out the scope of the volume. In addition, a draft of each 0 0.1 paper is peer-reviewed prior to final acceptance or rejection. 1 oi: This anonymous review process is supervised by the organiz­ org 5 | d er^) of the symposium, who become the editor(s) of the book. cs.99 The authors then revise their papers according to the recom­ a1 bs.9, mendations of both the reviewers and the editors, prepare 012 | http://puon Date: May wvcaihemwoe crAphasa -erpceaeka r drstuh yaal ecrt, oe a poliynln ,lnc ylea ucnodedrsesi dgsau irinbynam lrt ihertvee it ssheviaoeo nrlucfsihm nh aeapls va.pe p aebVpreeese rrnbas namttdoiam dot hreri.ee g pienrdoaidlt ourrecs­­, 2, 2cati tions of previously published papers are not accepted. uly 2Publi J M. Joan Comstock Series Editor In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. Preface INSECTICIDES HAVE BEEN THE BOON and the bane of human societies. Their use has resulted in the most plentiful, least expensive, and most secure food source thus far produced by our agrarian efforts. As with all simplistic, directed approaches, the use of insecticides has had far- reaching and, in most cases, unforeseen environmental impacts. Nevertheless, it is unlikely that in the near future the quality or quantity 1 00 of foodstuff can be maintained in the absence of pesticidal chemicals, par­ pr 1. ticularly insecticides. Thus, our efforts as pesticide scientists should be 9 05 focused on research approaches that will result in the availability of novel 5- 9 pesticide products that have been evaluated in terms of what we have 9 1 k- learned from past faults in pesticide design and unmanaged overuse. b 1/ Molecular biological approaches have provided the means to funda­ 2 10 mentally change the way in which pesticidal chemicals are discovered and 0. 1 used. Technical knowledge for the production of genetically engineered org 5 | doi: csoroopn pwlailnl tsb eacnodm be ioetceocnhonmoliocgailclya l fepaessitb lceo. ntBroecl asutrsaet etghiee s laarlrgeea dmy aejxoirsitt ya nodf acs.199 currently used insecticides are neurotoxic and many are directed toxico- ubs.y 9, logically to ion-channel disruption, an extensive database is currently pa 12 | http://n Date: M macivdoaedislea lbssu letco hf o aars ssa esDssseD stshTme a enanddtv .t ahnTeth acugyes,cs ltoahdnei demn deoisss atm devanaydn utbaregi neisgn copofr nnotevrwiidb iumntgioo lwnece oullfl- asintru stdeecicethid­­ 0o 2, 2cati nologies. It is an appropriate time to assess the impact that these molec­ July 2 Publi umlaorle cauplparro paecshteicsi dhea vsec iehnacde .o n pest control and to evaluate the future of This scenario provided the idealistic focus for the organization of an ACS symposium entitled "Molecular Action and Pharmacology of Insecti­ cides on Ion Channels", upon which this book is based. Topics were chosen principally in terms of their contemporary relevance to insect pest control and concentrated on those aspects in which a molecular under­ standing of insecticide action was being actively researched. Chapters were then requested from researchers and scholars drawn equally from academia and industry. These researchers are leading experts in their chosen areas of study and present a truly international group from Canada, Israel, Japan, the United Kingdom, and the United States. This book is divided into three sections, each concerned with the action of insecticides on a major ion-channel family: voltage-sensitive channels, ligand-gated channels, and biopesticide-induced channels. ix In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. Presented in this volume are recent advances in how we envision molecu­ lar binding sites of insecticides on ion channels and molecular alterations of these sites that result in site-insensitive resistance mechanisms. Addi­ tionally, new receptor-ion channels and mosaic receptor organizations are described as novel sites of insecticide action and perhaps as means of genetically managing the development of insecticide resistances in the future. I thank the authors for their presentations in the symposium and for their contributed chapters that encompass this volume. In particular, I extend my deepest appreciation to the many expert colleagues who pro­ vided helpful and necessary critical reviews. I thank Anne Wilson of ACS Books for all her help, suggestions, and encouragement; and Margaret Malone, Amity Lee-Bradley, and Julia Connelly of the Department of 1 0 Entomology, University of Massachusetts, for endless organizational and 0 1.pr editorial concerns. Their efforts and the generous financial support of 9 5 the Division of Agrochemicals of the American Chemical Society; FMC 0 95- Corporation; Ciba-Geigy Corporation; Miles, Inc.; Mycogen Corporation; 9 k-1 NOR-AM Chemical Corporation; Nissan Chemicals of America Corpora­ 1/b tion; and Rollins, Inc., made this book possible. 2 0 1 0. 1 J. MARSHALL CLARK oi: Department of Entomology org 5 | d University of Massachusetts acs.199 Amherst, MA 01003 ubs.y 9, pa 12 | http://n Date: M January 17, 1995 0o 2, 2cati uly 2Publi J x In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. Chapter 1 Ion-Channel Diversity Present and Future Roles in Insecticide Action J. Marshall Clark 1 Department of Entomology, University of Massachusetts, 0 0 h Amherst, MA 01003 c 1. 9 5 0 5- 9 9 Insecticides have played an enormous role in the development of 1 k- pesticide and environmental sciences. In our attempts to understand b 21/ the toxic action of insecticides on target organisms, we have 0 0.1 elucidated many fundamental physiological processes and recently 1 oi: have began the study of these interactions at the molecular level. cs.org 995 | d pGriovbeanb tlyh en eafifveec tnivoetn teoss h aavned ewnivdies-isopnreeda dth ues een ovfi rionnsmecetnictiadl ecso, nitc ewrnass ubs.ay 9, 1 tnheagta titvhee auspseec tos fo ft hinesseec ticchiedmesi caanlds thheaisr ucsaeu hseadv.e rNeseuvletretdh einle as sm, uthche pa 12 | http://n Date: M mtfoatoxeri ceo acdnoymdn apdmleeitgcers aa donafd te ironinvg,io rrooanunmsd eu nnptdraoel brcsloteanmntadsm inaigns asoonfct stih,a etteh dteo ixrw iecinotvhki irnoeenxtimtcesne nsaitnvadel 0o 2, 2cati overuse, such as resistance. July 2 Publi many inBteercaacuts ed itrheec tllayr gwei tmh aijoonri tcyh aonfn ienlsse cinti cnieduerso anrse annedu reoltsoexwich earned, an extensive research data base now exists that is available for assessment. Thus, the most enduring contribution of insecticides such as DDT, the cyclodienes, etc., may be in providing well-studied models to assess the advantages or disadvantages of new molecular biotechnologies as they become available for use in pesticide science. This aspect, of course, was the focus of the ACS symposium presentations that served as the template for the following chapters which comprise this volume. The past fifty years of agricultural productivity have been largely the result of the widespread availability of synthetic chemicals, such as fertilizers and pesticides. These compounds have allowed the farmer to protect the high quality 0097-6156/95/0591-0001$12.75/0 © 1995 American Chemical Society In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. 2 MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS food and fiber produced with synthetic fertilizers that are necessary to feed and clothe an ever increasing human population. Currently, the world market value for pesticides is approximately $23 billion with herbicides, insecticides and fungicides accounting for 47, 28 and 22% of this total cost, respectively. (1). Even with a projected growth rate of 10% per annum, the market share for biologicals has been estimated at 2 to 10% of the total pesticide market share by the year 2000 (2). Thus, synthetic chemical pesticides will remain the primary method of efficacious pest control into the next century and, at least in some fashion, well into the future. As impressive as our progress has been in agriculture, we now face enormous challenges as pesticide scientists. In five years, we will begin the 21st century with an estimated world-wide population of 6 billion people (3). This population is likely to double in only a few decades resulting in tremendous pressure for agriculture to be even more productive. The past excessive and indiscriminate use of nonselective pesticides has made these challenges even more 1 00 difficult (4). We now are all aware and concerned about the short and long term h 1.c effects of pesticides in the environment. Societal response to these concerns has 9 5 resulted in increasingly difficult and costly development and registration processes 0 95- for new and novel pesticides. More disconcerting, perhaps, than even the 9 1 environmental impact that certain pesticides have produced, is the ever increasing k- 1/b incidence of pests that are resistant to pesticides and the loss of these chemicals as 02 effective pest control agents. Thus, at precisely the time we should be developing 1 0. more selective, biodegradable, efficacious, and environmentally-benign chemical 1 oi: pesticides, we are not even keeping pace with those that are being lost through org 5 | d regulatory bannings or pest resistance problems. cs.99 From the above scenario, it follows that the pesticide scientists' most a1 ubs.y 9, serious task at hand is to begin anew the development of innovative and rational 12 | http://pn Date: Ma mppelaescatiencdsi d feoo nrd etchsrieog png , eplnraoecttkiec c,ot ifob nsioe cl(eh3c)et.mi viiTctyah,le ,a sanend d a dovvpaehnra-crumessea .c moluMosgto ircaeav leo mdidipf fhpearreseivnsic oenuses e odfafs u ttlaotsr gbeinet 0o versus nontarget organisms. Additionally, more natural products should serve as 2, 2cati molecular models for the design of new synthetic but biodegradable pesticides. uly 2Publi Recent advances in molecular biology have provided a fundamentally novel J manner in which we analyze the genetic, physiological, and developmental properties of organisms. Molecular approaches have revolutionized the study of pesticide mode of action, the determination of selective toxicities of target versus nontarget organisms, and the elucidation of mechanisms of pest resistance (4). The availability of such molecula. biological tools has greatly increased our basic understanding of allied sciences including pharmacology, neurobiology, ion channel and receptor biology, signal transduction, and developmental biology (5). From these basic studies, we now have a wealth of new information applicable to many applied aspects of pesticide science. The technical know-how for the production of genetically-engineered crop plants and bibtechnological pest control strategies already exists and will soon be economically feasible (4). Obviously, it is In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. 1. CLARK Ion-Channel Diversity 3 an appropriate time to assess the impact that these molecular approaches have had on pest control and to evaluate the future of molecular pesticide science. Molecular Basis of Toxicity of Insecticides on Ion Channels Investigation into the toxic action of insecticidal chemicals has provided much of the leadership on how we conduct, evaluate, and use mode of action studies on pesticides and on their environmental impact. Over the past 25 years, these studies have elucidated new and novel sites of action in insects. Knowledge pertaining to insecticide receptors and xenobiotic metabolism have allowed the more efficient design of efficacious analogues. These findings, together with pharmacological and genetic information, have lead to a fundamentally new way in which insecticides are being used to suppress the selection of resistant insects. Such approaches are now well known as resistance management strategies. 1 0 h0 The reasons for these early advancements in the insecticide aspects of c 1. pesticide science are many. Included in these are that insect pests are particularly 9 05 numerous and potentially devastating to many essential crop systems. Therefore, 5- 9 we allotted a large portion of our effort to find ways to control them. Also, insects 9 1 k- are advanced physiologically and present complex developmental systems, such as b 1/ the nervous and endocrine systems, that are easily targeted for chemical disruption. 2 10 Of particular importance is the role that Drosophila melanogaster has played in 10. cytological, developmental, and molecular genetic studies in insect and pesticide oi: science (5). org 5 | d Pesticide chemists were enormously successful in producing a variety of acs.199 extremely cheap and effective insecticides by applying the wealth of information ubs.y 9, obtained from relevant animal studies in neurobiology, pharmacology and pa 12 | http://n Date: M bmdeeov daeuclotliapvtmoe rebsn yto adfl ivsbariuroiplootuignsyg .i othnOe c fih tnahsneensceetsl e' faunrnelyrcv tpiooruonsds .us yctBsst,ee cmtha ueas nhed uo gifen t hmpiasar,jt oitcrhuietlyrae rw , neborywe a fecoxtuiinnsgtds ataos uly 22, 20Publicatio rmdoeebvtueaslboto ptlomisxeminc,ot lrooegfc ietchpatelo sdre a tbiaoi onb laocshgeay on,n ne etnhl-vedi irsroterncumtcetedun rteian-las ceftcaitvtiecit iyda ernsed.l atiBimoenpcsaahcuitpsse,s , satoonmxd iec roeoksfii nsttheatenisccese , J neurotoxic insecticides are no longer widely used due to environmental concerns (e.g., DDT, cyclodienes, etc.), their most enduring contribution will most likely be in providing a historical data base to review and to assess the advantages of various molecular biological tools as they become available for use in pesticide science. Ion Channels Perturbed by Insecticides Neuronal ion channels certainly have been the most widely exploited group of ion channels in terms of providing a site of action for existing insecticidal chemicals, however, they are far from the only type of ion channel available for such use. As more ion channel pharmacology and molecular sequence data is analyzed, it has become more and more evident that many ion channels, regardless of their tissue location, share many topological and functional similarities. Such similarities, can In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. 4 MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS even extend to considerable amino acid and nucleic acid homologies (6). Our understanding of the basic structure and function of ion channels has been revolutionized certainly by the use of molecular biology. Specifically, molecular cloning and sequence analysis of complementary DNAs (cDNAs) have provided primary structures of ion channels which have allowed the first biochemical-based interpretation of the tertiary structure of the encoded proteins. Cloned DNAs can be overexpressed in various cells. Function can then be assigned directly to specific proteins or polypeptides or indirectly by the use of specific antibodies. Additionally, oligonucleotide-directed mutagenesis can be used to site-specifically mutate cDNAs which allows us to test biophysical interpretations concerning channel kinetics, ion-selectivity, ligand binding domains, etc (6). Thus, our studies on the molecular action of insecticides on ion channels is a start to our understanding of these processes and not an end. Such investigations 1 will inevitably elucidate the nature of vulnerable sites for pest insect control which 0 h0 will be of value in parallel research for the control of weeds and microbial c 1. pathogens and vice versa. Molecular knowledge of the target sites of insecticides, 9 05 natural products, and pharmaceuticals, will be used in the design of new pesticides 5- 9 and in the search of new and novel sites of action. 9 1 k- b 1/ Voltage-Sensitive Sodium Channels, The voltage-sensitive (or voltage-gated) 2 10 sodium channel is a transmembrane protein complex that is essential in the 0. 1 generation of action potentials in excitable cells, in particular those associated with oi: the all-or-nothing electrical impulse carried by nerve cell axons (7). On receiving a org 5 | d depolarisation signal, the permeability of the sodium channel rapidly increases for acs.199 approximately 1 millisecond. The rising phase of the action potential is due to this ubs.y 9, increased Na+ permeability and the process that causes it is termed activation. The 12 | http://pn Date: Ma pachcetraimnvnaeetailbo inwl itiypll ruospcueobsnss e dqeuaplesonolta lyrdi seadteteirocmnrei.n aesIsfe dietnhp eoa l arlraeissteas tidoarnnad mp eavrtsoiicls ttasfg,a esth heido epnpe eramnnddeea nbrceiletiu tryon fo ft Not haea+ uly 22, 20Publicatio niitn oanrccetotiuvnardntuisoc ntit noagn da s dtaenttoeenr mcooivnneedrs u ctthhtieen grn aetesxt taa tne2d-. 3v omlTtahigleleiss ede cetopwennodd s.ep nrcoTech eosifss eNpsa,r +o cpaeecsrtsmi veaiastib oitnlei rtyma neadds J inactivation, allow the voltage-sensitive sodium channel to exist in any one of three distinct functional states; active, inactive, or resting. Although both inactive and resting channels are nonconducting, they are very different in how they enter the "active" conducting state upon depolarisation. An inactivated channel is recalcitrant to depolarisation and must first return to the resting state by repolarization prior to being activated. The primary structures of at least five distinct sodium channels have been reported (8). These are: 1) the sodium channel from the electric organ of the eel Electrophorus electricus, 2-4) three separate sodium channels from rat brain (Types I, II & III); and 5) the ul sodium channel from rat skeletal muscle. All sodium channels possess an a subunit that is approximately 1,800-2,000 amino acids in length. The eel electroplax sodium channel is comprised only of an a subunit whereas the rat skeletal channel has both a and p-1 subunits. The rat brain sodium In Molecular Action of Insecticides on Ion Channels; Clark, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.