Calcium Channel as a New Potential Target for Insecticides - ACS

Chapter 10

Calcium Channel as a New Potential Target for Insecticides 1

1

1

1

2

1

L . M. Hall , D . Ren , G . Feng , D . F . Eberl , M. Dubald , M. Yang , F. Hannan , C . T. Kousky , and W . Zheng Downloaded by UNIV OF TEXAS AT DALLAS on July 11, 2016 | http://pubs.acs.org Publication Date: May 9, 1995 | doi: 10.1021/bk-1995-0591.ch010

1

1

1

1

Department of Biochemical Pharmacology, State University of New York at Buffalo, Buffalo, N Y 14260-1200 Rhone Poulenc A G , P.O. Box 12014, 2 T W Alexander Drive, Research Triangle Park, N C 27709 2

Pharmacological, electrophysiological, and ligand-binding studies have indicated the presence of diverse voltage-sensitive calcium channels in insects which appear to be pharmacologically distinct from those of vertebrates. To define the molecular structure of these channels, we have used a polymerase chain reaction (PCR) cloning strategy to identify and sequence a cDNA encoding an α subunit of Drosophila calcium channels. Quantitative Northern blotting studies have shown that this subunit is most highly expressed in legs and heads. Genetic analysis has demonstrated that a premature stop codon in the α subunit causes an embryonic lethal phenotype demonstrating that function of this subunit (which is the target for organic calcium channel blockers) is required for survival of the organism. The structural differences between this insect calcium channel and those from vertebrates may prove useful for the design of new insect-specific calcium channel blockers. 1

1

Voltage-sensitive calcium channels play two important roles in all excitable cells. First, they play a key role in control of cell excitability by either contributing to the shape of regenerative action potentials or by generating the action potential in cells which lack sodium channels. The second major role of calcium channels is to serve as the link which transduces a depolarization into the nonelectrical processes that are controlled by excitation. Some of these processes include: synaptic transmission, endocrine secretion, and muscle contraction. (See review by Bhle(l).) Calcium channels are ubiquitous and have been reported in excitable cells in species ranging from Paramecium to humans. We are interested in calcium channels as potential targets for the development of new insecticides. In determining their suitability as targets, we have used molecular genetic and pharmacological approaches to consider 4 questions: 1. What is the structure of insect calcium channels? 2. Are insect calcium channels structurally and pharmacologically different enough from mammalian calcium channels so that insect selective agents can be developed? 3. Are insect calcium channels accessible to insecticides? 4. What are the physiological consequences of blocking calcium channels in insects? 0097-6156/95/0591-0162$12.00/0 © 1995 American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

10. HALL ET AL.

Calcium Channel as a Target for Insecticides

163

Downloaded by UNIV OF TEXAS AT DALLAS on July 11, 2016 | http://pubs.acs.org Publication Date: May 9, 1995 | doi: 10.1021/bk-1995-0591.ch010

What is the Structure of Insect Calcium Channels? Voltage-dependent calcium channels in mammalian skeletal muscle are comprised of 5 subunits ( a i , n% P, y, and 8). A s reviewed by Hofmann and coworkersfi), the a i subunit itself forms the calcium conducting pore and also contains the binding sites for all known calcium channel blockers. The smaller p subunit enhances calcium current expression when coexpressed with the a i subunit in a variety of heterologous expression systems. In some expression systems the p subunit alters channel kinetics. The 02/6 subunits are encoded by a single gene. The two subunits are formed by posttranslational proteolytic processing. Coexpression of 01, ct2/8, and p cDNAs together is required for maximum calcium channel expression in some, but not all, expression systems. The addition of the y subunit to coexpression experiments generally has little affect on overall channel expression levels, but does shift the steady state inactivation of Ina by 40mV to negative membrane potentials (3). It is, however, not yet clear whether in vertebrates the y subunit is present in tissues other than skeletal muscle (2). Although molecular analysis of voltage-dependent calcium channels is just beginning in insects, gene cloning studies from our laboratory have provided evidence that calcium channels in the fruitfly Drosophila melanogaster have a similar subunit structure to the mammalian channels. We have sequenced complete c D N A clones encoding cti and p subunits (D. Ren, G. Feng, W . Zheng, D.F. Eberl, F. Hannan, M . Dubald and L . M . Hall, unpublished results). In addition, using reduced stringency PCR and probing Southern blots with a mammalian ct2 cDNA, we have obtained preliminary evidence for the existence of an 012 subunit in Drosophila. Because of the key role that a i subunits play in the function of calcium channels and their utility as targets for pharmacologically active agents in mammalian systems, we have focused our initial efforts on the characterization of this component Figure 1 illustrates the successful strategy used to clone the first a\ subunit from Drosophila. Initially, we designed a set of 9 PCR primer pairs from the most highly conserved regions of the known vertebrate calcium channel a i subunits focusing on those conserved regions which would also give the least codon degeneracy. These primer pairs were used to amplify from a genomic D N A template so that no assumptions were made concerning the time in development or the tissue of expression. PCR products of approximately the same size or larger than that predicted from the vertebrate c D N A sequences were sequenced directly to determine whether any encoded a peptide with amino acid sequence similarity to the vertebrate subunit. Codon preference analysis was used to identify introns. Three of the most promising PCR products were sequenced and one of these showed a high degree of similarity to the vertebrate a i subunit. The Drosophila genomic sequence did contain two small but easily identifiable introns of 59 and 60 base pairs. The positions of the successful primers are given as black boxes numbered 6 and 7 in Figure 1A. Once the PCR amplification product was shown to encode a portion of an a i subunit, this product was used to screen an adult head c D N A library to obtain the full coding sequence. Our previous calcium channel ligand binding studies (4) plus Northern analysis had suggested that this channel was expressed in significant amounts in adult heads which are enriched in neuronal tissue. In Drosophila as in other organisms, the a i subunit mRNA is very long (>8kb). To obtain the full open reading frame, it was necessary to isolate three overlapping c D N A clones ( N l , W8A, and SH22C) as shown in Figure I B . Although there was excellent match in most of the overlapping regions of these clones, there is a region of 149 nucleotides in the 3' end of clone W 8 A (indicated by the downward pointing arrow) which

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


164

MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS

Figure 1. Strategy for cloning and relationship of cDNA clones encoding a full length calcium channel a i subunit from Drosophila. (A) Cartoon showing the general structure of all a i subunits of voltage-sensitive calcium channels cloned to date including the a\ subunit from Drosophila melanogaster. The black boxes labeled 6 and 7 designate the positions of primers used for successful amplification of the Drosophila a\ subunit (B) c D N A clones used to obtain the full open reading frame for the Drosophila a\ subunit The small open boxes within clone SH22D indicate regions of known alternative splicing.



10. HALL ET AL.


165

shows no overlap with the sequence in SH22C. Sequence analysis of genomic clones has indicated alternatively spliced exons in this region. Additional regions of alternative splicing are indicated as open boxes in clone SH22D. These alternatively spliced regions provide a means to generate functional diversity of calcium channel subunits from a single gene. Complete sequencing of the a i subunit revealed that the Drosophila subunit is similar in overall plan to the mammalian homologues. This is revealed dramatically in the hydropathy plots shown in Figure 2. The Drosophila subunit contains 4 repeat regions labeled in Figures 1 and 2 as I, H, HI, and IV. Within each repeat there are 6 transmembrane domains labeled 1-6 (and referred to in the literature as S1-S6) which show structural similarity when compared among the 4 repeat regions. For example, the S4 regions all show positively charged amino acid side chains alternating every 3 to 4 amino acids. These positively charged side chains have been proposed to all lie on one side of an a helix and to constitute at least part of the voltage sensing mechanism of these channels. Another structural feature that is conserved between this Drosophila a\ subunit and those from other species is a hydrophobic loop (generally referred to as SSI and SS2) which is located between transmembrane segments S5 and S6 in each repeat segment. This region has been modeled as dipping part way through the membrane as shown in Figure 1A. This region contributes to the ion selectivity filter since changing single amino acid residues in these regions can shift channel selectivity from sodium to calcium or vice versa (5, 6). In this crucial region the Drosophila subunit contains the glutamic acid residues diagnostic of calcium channels. This, along with the high sequence similarity to vertebrate calcium channels (ranging from 63.4% to 78.3% similarity), firmly establishes this clone as encoding an insect calcium channel a i subunit. Are there structural and pharmacological differences between insect and vertebrate calcium channels which would allow the development of insect specific channel blockers? Although the general structural plan between insect and vertebrate calcium channels is conserved, there are many regional differences. One striking structural difference is revealed in the comparison of the hydropathy plots in Figure 2. It is apparent that the amino and carboxy terminal cytoplasmic domains are much longer in the Drosophila subunit than in the illustrated mammalian skeletal muscle homologue. When these sequences are compared at the amino acid level, there is no similarity at all in the insect sequence after about 225 amino acids upstream of the beginning of IS 1 and after 160 amino acids downstream of the carboxyl region IVS6. It is not yet clear whether these regions are functionally significant in the insect calcium channel. If they are, they might be targeted by membrane permeant agents to generate insect specific toxins. In mammals the very effective phenylalkylamine class of calcium channel blockers are thought to penetrate membranes and interact with the cytoplasmic domains of the a i subunit (7). Therefore, there is a precedent for designing highly effective, membrane permeant calcium channel blockers. A more complete and systematic analysis of the regional differences between the rabbit skeletal muscle subunit and the Drosophila subunit is given in Figure 3 where the cytoplasmic, transmembrane and extracellular domains have been compared sequentially across the molecule and the percent difference in amino acid sequence has been plotted. It should be noted that the first (highly nonconserved) 493 amino acids in the Drosophila have not been included in this analysis. This figure shows that in addition to the nonconserved, cytoplasmic amino and carboxy termini, there are numerous extracellular domains such as those between IS1 & IS2, IS3 & IS4, IVS1 & IVS2, and IVS3 & IVS4 which are only weakly conserved and therefore are possible targets for insect-specific agents which act extracellularly.



166


Figure 2. Comparison of hydropathy plots for a i subunits from rat brain type V (17) (panel B) with the Drosophila subunit (panel A). Hydropathy plots were determined using the method of Kyte and Doolittle (18) with the GeneWorks software. Up is hydrophobic and down (negative numbers) is hydrophilic.


10. HALL ET AL.


V//A

Cytoplasmic

H I

Transmembrane


1

III

I Extracellular

IV

Relative Length of Amino Acid Sequence

Figure 3. Comparison of regional sequence differences in calcium channel a i subunits from Drosophila versus rabbit skeletal muscle. A n amino acid pair was scored as different only if it represented a nonconservative substitution. The width of each bar is proportional to the length of the amino acid sequence except in the region labeled C T where the scale is 1/10 that of the rest of the chart The 4 repeat domains are designated I, n, HI, and IV. Within each repeat the proposed transmembrane segments (S1-S6) are designated as 1-6. N T = amino terminal segment (beginning with methionine 494). CT = Carboxy terminus. Note that parts of the "extracellular" loop between S5 and S6 form the membrane embedded SS1/SS2 loop shown diagrammatically in Figure 1 A .


167

168


The dihydropyridine class of calcium channel blockers are thought to act against the mammalian calcium channel a i subunit by blocking from the outside of the channel (8-10). From the point of view of amino acid sequence there appear to be numerous areas of differences. These gene cloning and sequencing studies provide a means to define the molecular basis for previously observed pharmacological similarities and differences between insect and mammalian calcium channels (4, 11-13). Some highlights of these differences are summarized in Table I below.


Table L Pharmacology of Calcium Channels Predominant in Drosophila Heads versus Mammalian Skeletal Muscle L-type Calcium Channels (4,13)

Drosophila Phenylalkylamines Dihydropyridines Benzodiazepines Tetrandrine

Sensitive Insensitive Insensitive Very sensitive

Mammalian L-tvpe Sensitive Sensitive Sensitive Sensitive

As summarized in Table I, at least some subtypes of both Drosophila and mammalian calcium channels are sensitive to the phenylalkylamine class of calcium channel blockers. Photoaffinity labeling coupled with immunoprecipitation studies have identified a peptide including the cytoplasmic domain immediately adjacent to IVS6 and extending into the transmembrane domain of IVS6 which is involved in binding this class of calcium channel blocker. Phenylalkylamines act from the cytoplasmic side of the channel (9). Our sequencing studies have shown that the Drosophila a\ subunit is completely conserved in the cytoplasmic portion of this peptide and shows only two conservative amino acid differences within the transmembrane domain of IVS6. Thus, in order to develop insect-specific agents targeted against this region of the channel, the ligand would have to have a domain of action that extended outside of this highly conserved area. In contrast, the predominant calcium channel activity expressed in Drosophila heads is insensitive to the dihydropyridines which are very effective at blocking mammalian L-type calcium channels. Since the cloned calcium channel we describe here has not yet been expressed, we do not know its exact pharmacological specificity. We do know that it is highly expressed in Drosophila heads and therefore is likely to encode the phenylalkylamine-sensitive, dihydropyridine-insensitive channel found in heads (4). Consistent with this idea is our finding that this Drosophila subunit shows many nonconservative amino acid substitutions in the regions thought to encode the dihydropyridine binding site. This binding site is thought to reside in part in the regions beginning in the extracellular domain between IHS5 & IHS6 and between IVS5 & IVS6 and extending into the adjacent S6 transmembrane domain (8-10) Thus, this drug binding site is thought to extend into the channel from die outside. Binding sites for the other two classes of calcium channel drugs (benzodiazepines and tetrandrine) shown in Table I have not yet been defined in any species. Chimeras between the insect and mammalian subunits could be used in expression studies to identify the position of these sites in the future. Of particular interest is the very high sensitivity of the insect calcium channel ligand binding to inhibition by tetrandrine, an active component found in the Chinese herb used to treat cardiac arrythmias (14). In nature tetrandrine might contribute to the defense


10. HALL ET AL.


169

mechanism of the plant by affecting pest insect calcium channels much the same way that the pyrethrins from chrysanthemums act on insect sodium channels to protect the plant from invaders. Thus, tetrandrine is potentially a useful lead compound for the development of effective insecticides targeted against insect calcium channels. Are insect calcium channels accessible to the actions of insecticides ?


We have taken two approaches to this question. One is to use quantitative Northern blotting and in situ hybridization to ask where this calcium channel oti subunit is expressed. The second approach is to ask whether Drosophila is susceptible to orally administered phenylalkylamines. As summarized in Table H , quantitative Northern blotting using mRNA prepared from adult Drosophila body parts shows that this a i subunit is most highly expressed in legs. (The level of calcium channel subunit m R N A has been standardized against ribosomal protein-49 [RP49] message (15).) The second highest level of expression is in heads which are enriched for neuronal tissue. This table also illustrates that there was more apparent heterogeneity in calcium channel message size classes in heads than in other body parts. The enrichment in heads was determined by combining the two different size classes. Enrichment of message expression in heads relative to bodies is generally indicative of a nervous system specific expression. Thus, this calcium channel is likely to show a distribution pattern similar to the para sodium channel which is a target for pyrethroid action (16). This sodium channel is expressed throughout the nervous system. The high level of expression in legs suggests that this channel may be susceptible to die action of insecticides which could penetrate through the legs as an insect walks across a surface. Neuronal specific expression of this subunit has been confirmed by whole mount in situ hybridization to embryos which shows widespread expression throughout the central nervous system. Table IL Quantitative Expression of Calcium Channel mRNA Tissue Body Head Head Leg

Calcium Channel ai mRNA size class 9.6"kb 10.2 kb 9.5 kb 9.5 kb

on subunit/RP49 0.21 0.18 0.16 1.23

To further address the issue as to whether these calcium channels are susceptible to action of blocking agents, we raised flies from egg to adult on media containing various amounts of the phenylalkylamine verapamil. This drug was designed for use in treating cardiac arrythmias in humans and so would not be expected a priori to be extremely effective against insect calcium channels. Nevertheless, as shown in Figure 4 we found a dose dependent killing effect with 100% lethality for wild-type flies at the highest verapamil concentration tested. Interestingly, we also observed a dramatic, drug-dependent delay in time required to mature from egg to adult. There was no obvious sexual dimorphism in the effects of verapamil on either the viability or the developmental delay.



170


M-OmM F-OmM M-5mM F-5mM

- D - M-10mM - O - F-10mM M-15mM F-15mM

M = Male F = Female 13

15 17 Days from Egg Lay

Figure 4. Effects of the calcium channel blocker verapamil on Drosophila survival and development. Wild-type Canton-S adults were allowed to lay eggs on standard Drosophila medium (19). Shortly after laying, groups of 50 eggs were transferred to fresh shell vials containing 7 ml of Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply Co.) prepared with an equal volume of 10% ethanol containing die indicated concentration of verapamiL Vials were incubated in total darkness at 25°C until adults began eclosing. The number and sex of the flies hatching each day was recorded. Two hundred eggs (-50% male, -50% female) were treated at each concentration.


10. HALL ET AL.


171


What are the consequences of functionally inactivating this specific ai subunit in insects? Although the above verapamil feeding experiment suggests that blockade of calcium channels will lead to lethality, the interpretation of this type of drug feeding experiment is complicated by the fact that we cannot be certain that the cloned cxi subunit is the target causing lethality. Since it requires high concentrations of drug administered in the food arid since we do not know what the actual concentration is at the target site, there is a concern that the lethality might be due to effects at a site other than the expected target. Interpretation is further complicated by the fact that in mammals, there are multiple genes encoding different a\ subunits (2). There is preliminary evidence for at least one additional gene in Drosophila encoding a different a i subunit (L. Smith and J.C. Hall, personal communication). From a simple feeding experiment we cannot tell whether the lethality is due to an effect on calcium channels. Even i f the killing is through a calcium channel mechanism, we cannot distinguish whether the lethality is due to effects on this channel or on another channel or due to effects on multiple channels. Indeed, with the multiplicity of neuronal calcium channel subtypes in mammals, it is an important question to determine whether they functionally overlap. We have used a genetic approach to address the question of the consequences of inactivating this specific a i subunit. First, we mapped the gene to a position on the left arm of the third chromosome using in situ hybridization of the cloned cDNA to salivary gland chromosomes. This initial localization was followed by mapping of the c D N A with respect to chromosome deletions to obtain a more precise location. This physical mapping of the locus was correlated with mutant analysis to determine which of the exising mutations in the region showed the same deletion mapping pattern as the cDNA. This approach allowed us to identify one candidate complementation group which causes embryonic lethality. D N A sequencing studies showed that one allele at this locus causes a premature stop codon in the cytoplasmic loop between IVS4 and IVS5. (See Figure 1.) This point mutation would result in the formation of a truncated protein lacking carboxy portion of homology region IV and the long carboxy terminal cytoplasmic tail. Since the genetic mutation specifically affects this particular a i subunit subtype, we can conclude that agents which inactivate this subunit have the potential to kill the insect as early as the late embryonic stage. For pest insects, i f an eggpermeable compound can be developed, it would have the potential to kill the insect before the destructive larval stages emerge. These experiments establish the utility of this subunit as a potential insecticide target. The availability of a complete cDNA encoding this subunit provides a basis for developing a heterologous expression system which can be used for the initial, rapid screening of test compounds for action on this target Calcium Channels as Targets for New Insecticides Our gene cloning and sequencing studies have shown that although there is enough sequence similarity to allow cloning of insect calcium channel subunits across species using appropriately designed PCR primers and reduced stringency conditions for amplifications, there are substantial regional differences between the insect and mammalian channels. These regions could be targeted to develop species-selective agents. The expression of these channels in peripheral structures such as legs offers the hope that these channels may be susceptible to compounds as the insect walks across a surface. The existing calcium channel blockers as well as natural products such as tetrandrine may provide useful lead compounds for the development of new insecticidal compounds which target this calcium channel subunit Screening for such new compounds will be facilitated by the expression of


172


this cloned gene in cells which can be readily mass produced for large scale screening.


Acknowledgments We thank Dr. Maninder Chopra for comments and help in the preparation of this manuscript. This work was supported by grants from NIH (HL39369) and the New York State Affiliate of the American Heart Association (92329GS) to L M H who is a Jacob Javits Scholar in Neuroscience. Salary support for G F was from NIH grant NS16204 and from a Pharmaceutical Manufacturers Association Predoctoral Fellowship. A part of this study was conducted under the BIOAVENIR program financed by R H O N E - P O U L E N C with the contribution of the Ministfcre de lEnseignement Supdrieur et de la Recherche. D F E was supported by a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada. C T K was supported by a Howard Hughes Predoctoral Fellowship and W Z received salary support from N I H grants HL16003 and GM42859 to D.J. Triggle. Literature Cited (1) Hille, B . Neuron 1992, 9, 187-195. (2) Hofmann, F.; Biel, M.; Flockerzi, V . Annu. Rev. Neurosci. 1994, 17, 399418. (3) Singer, D . ; Biel, M.; Lotan, I.; Flockerzi, V . ; Hofmann, F.; Dascal, N. Science 1991, 253, 1553-7. (4) Greenberg, R. M.; Striessnig, J.; Koza, A.; Devay, P.; Glossmann, H.; Hall, L . M. Insect Biochem 1989, 19, 309-322. (5) Heinemann, S. H.; Terlau, H.; Stuhmer, W.; Imoto, K.; Numa, S. Nature 1992, 356, 441-443. (6) Tang, S.; Mikala, G.; Babinski, A . ; Yatani, A . ; Varadi, G.; Schwartz, A . J. Biol. Chem. 1993, 268, 13026-13029. (7) Striessnig, J.; Glossmann, H . ; Catterall, W. A . Proc Natl Acad Sci U S A 1990, 87, 9108-9112. (8) Nakayama, H . ; Taki, M.; Striessnig, J.; Glossmann, H . ; Catterall, W . A . ; Kanaoka, Y . Proc Natl Acad Sci U S A 1991, 88, 9203-9207. (9) Striessnig, J.; Murphy, B . J.; Catterall, W . A . Proc Natl Acad Sci U S A 1991, 88, 10769-73. (10) Catterall, W. A.; Striessnig, J. Trends Pharmacol Sci 1992, 13, 256-62. (11) Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M . ; Berge, J. B.; Lazdunski, M. Biochemistry 1987, 26, 6311-6315. (12) Pelzer, S.; Barhanin, J.; Pauron, D.; Trautwein, W.; Lazdunski, M.; Pelzer, D . EMBO J 1989, 8, 2365-2371. (13) Glossmann, H.; Zech, C.; Striessnig, J.; Staudinger, R.; Hall, L.; Greenberg, R.; Armah, B . I. Br J Pharmacol 1991, 102, 446-452. (14) King, V . F.; Garcia, M. L.; Himmel, D.; Reuben, J. P.; Lam, Y.-K. T.; Pan, J.-X.; Han, G.-Q.; Kaczorowski, G. J. J Biol Chem 1988, 263, 2238-2244. (15) O'Connell, P.O.; Rosbash, M. Nucleic Acids Res. 1984, 12, 5495-5513. (16) Hall, L. M.; Kasbekar, D . P. In Book ; T. Narahashi and J. E . Chambers, Eds.; Plenum Press: New York, N Y , 1989. (17) Hui, A.; Ellinor, P. T.; Krizanova, O.; Wang, J. J.; Diebold, R. J.; Schwartz, A . Neuron 1991, 7, 35-44. (18) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132. (19) Lewis, E. B. Drosophila Inform. Serv. 1960, 34, 117-118. RECEIVED

October 19, 1994


Calcium Channel as a New Potential Target for Insecticides - ACS

Recommend Documents