Ivermectin Interactions with Invertebrate Ion Channels - American

Chapter 17

Ivermectin Interactions with Invertebrate Ion Channels Susan P. Rohrer and Joseph P. Arena

Downloaded by CORNELL UNIV on August 30, 2016 | http://pubs.acs.org Publication Date: May 9, 1995 | doi: 10.1021/bk-1995-0591.ch017

Merck Research Laboratories, Department of Cell Biochemistry and Physiology, P.O. Box 2000, Rahway, NJ 07065

Ivermectin is a semi-synthetic analog of avermectin B . It is a potent anthelmintic/insecticide effective against a broad range of parasites. Ivermectin binding sites from nematodes and insects have been characterized b y e m p l o y i n g a Hivermectin binding assay and subsequently identified w i t h an I-azido-avermectin analog. Avermectin b i n d i n g proteins from the free l i v i n g nematode, Caenorhabditis elegans, and from the dipteran, Drosophila melanogaster, have been p u r i f i e d b y i m m u n o - a f f i n i t y chromatography u s i n g a m o n o c l o n a l a n t i b o d y against the l i g a n d , avermectin. Electrophysiological and pharmacological characterization of the chloride i o n channel target of ivermectin action in C. elegans has been accomplished by injecting C . elegans m R N A into Xenopus oocytes and studying the expressed chloride ion channel. The endogenous l i g a n d for the avermectin sensitive chloride channel expressed i n oocytes is glutamate. A t l o w concentrations, ivermectin potentiates the effect of glutamate o n the channel, w h i l e at h i g h concentrations, ivermectin causes irreversible opening of the channel. 1a

3

125

The avermectins are a family of macrocyclic lactones synthesized as natural fermentation products by the bacterium, Streptomyces avermitilis. The producing organism was isolated from a soil sample collected i n Japan as part of a collaborative agreement between Merck Research Laboratories and Kitasato Institute i n Tokyo (2). Potent anthelmintic activity of the avermectins was discovered when a fermentation broth generated from the soil sample was introduced into the diet of mice infected w i t h the intestinal nematode parasite, Nematospiroides dubius (2,3). Subsequent investigations revealed that this class of compounds also possessed potent insecticidal activity but lacked antibacterial or antifungal properties (4,5). 0097-6156/95/0591-0264$12.00/0 © 1995 American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

17. ROHRER& ARENA

Ivermectin and Invertebrate Ion Channels

265

Ivermectin (22, 23 d i h y d r o a v e r m e c t i n B i Figure 1), a semi synthetic avermectin analog (6), was introduced commercially i n 1981 and r a p i d l y became the d r u g of choice for treating a broad spectrum of conditions caused b y nematode and arthropod parasites (7,8). Ivermectin has been marketed for use against Dirofilaria immitis, the causative agent of heartworm disease i n dogs (9). In humans, ivermectin (Mectizan®) was shown to be effective i n the treatment and prevention of onchocerciasis (or river blindness) i n West Africa, and Central and South America (10,11). Abamectin (avermectin B i ) , developed for use as an insecticide i n crop protection programs, was introduced i n 1985 and has been shown to be effective against mites, leafminers, and lepidopterans (12). W i t h respect to the general mechanism of action, the avermectins are not unlike the anthelmintic compounds that preceded them. The organophosphates (13), pyrantel and morantel (14,15), levamisole and tetramisole (16) and the avermectins (17,18) a l l act b y interrupting neuromuscular transmission at some level. H o w e v e r , the avermectins are noteworthy for their extraordinary potency against a broad spectrum of endoparasites and ectoparasites combined w i t h a lack of toxicity to mammals, features which have contributed to the superior therapeutic i n d e x d i s p l a y e d b y the avermectins over p r e v i o u s l y d i s c o v e r e d anthelmintics. a

/


a

Ivermectin mode of action Early studies showed that ivermectin induced paralysis i n invertebrates was associated with increased cell permeability to chloride (for review see (19,20)). This suggested that the target of d r u g action was a chloride channel. The additional finding that picrotoxin blocked avermectinsensitive increases i n chloride conductance led to the suggestion that ivermectin interacted w i t h G A B A - g a t e d chloride channels (17,21,22). H o w e v e r , Duce and Scott (23) showed that avermectin increased the chloride conductance of locust leg muscle bundles which d i d not contain GABA-sensitive chloride channels. It was subsequently demonstrated that avermectins activated a glutamate-sensitive chloride conductance i n locust leg muscles (24). Its noteworthy that glutamate-gated chloride channels (or H-receptors) are also sensitive to picrotoxin (24,25,26). M o r e o v e r , Z u f a l l et al. (27) s h o w e d that avermectins activated a picrotoxin-sensitive multitransmitter-gated (glutamate, GABA, acetylcholine) chloride channel on crayfish muscle. These studies supported the hypothesis that avermectins modulate ligand-gated chloride channels. Identification of the target in nematodes and insects Isolation and characterization of the nematode specific ivermectin channel from the free l i v i n g nematode, Caenorhabditis elegans has been the

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


Figure 1. Structures of four different avermectin analogs used i n the described studies. Azido-avermectin and ivermectin-phosphate are both biologically active. Octahydroavermectin is biologically inactive.



17.

ROHRER & ARENA


267

primary focus of our research efforts. W e selected this animal as our model system because C . elegans is highly sensitive to avermectin (28). The ease w i t h w h i c h the organism can be maintained i n the laboratory and the feasibility of culturing massive quantities of worms for large scale experiments were additional reasons for choosing C. elegans over parasitic nematodes. M u c h of the biochemistry and molecular biology to be described (29,30£1 £2) was facilitated by our development of a protocol for cultivating C. elegans worms i n 500 g m quantities (33). In addition, the n e u r o m u s c u l a r system of C . elegans has been w e l l characterized developmentally, biochemically, and physiologically (34) and the neuronal "wiring diagram" is similar to that of parasitic nematodes, such as Ascaris suum (35,36). Specific, high affinity ivermectin binding sites have been identified and thoroughly characterized i n membrane preparations isolated from C . elegans (37). The affinities of a series of avermectin analogs for this binding site were determined and compared to the biological activity of each compound i n a C. elegans motility assay (Figure 2). The motility assay is performed i n liquid media and involves overnight incubation of C. elegans worms i n the absence or presence of d r u g (38). The strong correlation between the binding affinity and biological activity suggest that binding of ivermectin to this site mediates neuromuscular paralysis. H i g h affinity ivermectin binding sites also have been identified i n membranes prepared from several different arthropods i n c l u d i n g Drosophila melanogaster, Schistocerca americana (Figure 3), Spodoptera frugiperda, Heliothis zea and Liriomyza sativae (data not shown). A l l tissues tested were similar with respect to affinity for ^H-ivermectin but receptor density was variable. The density of b i n d i n g sites i n Drosophila head membrane preparations was ten fold greater w h e n compared to membranes from whole w o r m homogenates of C. elegans and the metathoracic ganglia neuronal membranes from Schistocerca americana were enriched 100-fold over C. elegans. (39): A l t h o u g h resistance to ivermectin has not presented itself as a problem i n the field thus far, the question of target site involvement i n the development of resistance could be addressed by comparing w i l d type and resistant organisms i n the ^H-ivermectin binding assay if, and when, it arises i n any of the arthropods listed above. Such a study was performed by comparing ivermectin sensitive and ivermectin resistant strains of the parasitic nematode, Haemonchus contortus (40). Membranes were prepared from L3 larvae of the w i l d type susceptible w o r m s and ivermectin resistant worms. Both tissue preparations exhibited h i g h affinity avermectin binding sites and the number of sites per m g of protein was the same indicating that resistance i n this particular strain was not due to a change i n the affinity of the receptor for the ligand or i n the number of receptors present on the membranes. This experiment also demonstrated that the membrane receptor from a parasitic target organism, was nearly identical to the C. elegans membrane receptor w i t h


268

MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS


250

0.1

0.25

0.5

1.0

2.5

K, (nM) Figure 2. Correlation between b i n d i n g affinities for a series of avermectin analogs and their efficacy i n the C. elegans motility assay. [1] ivermectin [2] avermectin B2a [3] avermectin B i 4"-0-phosphate [4] ivermectin-aglycone [5] ivermectin-monosaccharide [6] avermectin B i monosaccharide [7] 2-dehydro-4-hydro-a-2^-avermectin B i [8] avermec tin Bia-5-ketone. (Reproduced with permission from ref. 37. Copyright 1989 Elsevier Science Ltd.) a



17. ROHRER & ARENA


269

[3H-IVM] nM Figure 3. Characterization of avermectin binding sites o n membranes from C. elegans, Drosophila melanogaster, and Schistocerca americana. [A] Membranes were prepared by homogenizing whole C . elegans worms and isolating a 28,000 x g membrane preparation. Specific b i n d i n g of 1 2 5 i - i d o - A V M was measured i n the dark to a v o i d non-specific crosslinking of the ligand to other proteins. [B] B i n d i n g of ^ H ivermectin to D . melanogaster. Membranes were prepared b y homogenizing heads of adult flies and isolating a 28,000 x g membrane preparation. [C] ^ H - i v e r m e c t i n b i n d i n g to adult S. americana. Metathoracic ganglia were dissected from adult Schistocerca americana. N e u r o n a l membranes were homogenized and a 28,000 x g pellet prepared. (Reproduced with permission from ref. 32. Copyright 1992 S.P. Rohrer and from ref. 39. Copyright 1994 Insect Biochemistry and Molecular Biology). a z



270


respect to affinity for ivermectin (Kd) and receptor density or number of binding sites per m g of membrane protein (Bmax). Crude C . elegans and Drosophila membrane preparations have been useful for characterizing many aspects of the interaction of ivermectin w i t h the nematode and insect b i n d i n g sites respectively. H o w e v e r , purification and cloning of the nematode or insect receptors could lead to a more precise understanding of the mechanism of action and facilitate the establishment of new mechanism based screens for identification of novel compounds which interact with the same ion channel proteins. Because of the inherent difficulties associated w i t h isolation and cloning of i o n channel proteins, independent biochemical and molecular biological approaches toward obtaining the C. elegans and Drosophila ivermectinsensitive chloride channel have been taken. Biochemical isolation of ivermectin receptors from insect and nematode tissues The purification of the invertebrate ivermectin receptor was facilitated b y the use of an a z i d o - A V M analog as a photoaffinity probe (32). The c o m p o u n d s h o w n i n Figure 1 was synthesized (42) and found to be biologically active i n the C. elegans motility assay as well as the C . elegans ivermectin binding assay (Figure 3A) i n spite of the addition of the large substituent at the 4 -position. The ivermectin receptor was solubilized from C. elegans membranes w i t h Triton X-100 and then incubated i n the presence of the photoaffinity probe at room temperature i n the dark. After a one hour incubation, dextran coated, activated charcoal was added i n order to adsorb any unbound I Z S j - a ^ d o - A V M . The affinity ligand was crosslinked to the receptor by exposure to U V light and the result analyzed by autoradiography of an S D S - P A G E gel (Figure 4 A ) . Three C . elegans proteins w i t h molecular weights of 53, 47, and 8 k D a were radiolabeled. Increasing concentrations of unlabeled ivermectin (lanes 2-5) resulted i n elimination of the affinity labeling pattern. The l o w concentrations (10" M ) of ivermectin required to block the labeling pattern suggested that all three proteins were associated with the high affinity drug binding site. It is u n k n o w n whether the three labeled proteins represent non-identical subunits of a multi-subunit receptor, metabolic breakdown products of a larger precursor, or tissue specific forms of the receptor. M

8

The Drosophila proteins were affinity labeled while still b o u n d to the membrane (Figure 4B). Photoaffinity labeling of the Drosophila head membranes resulted i n identification of an apparent doublet i n the 45 k D a size range. Labeling was blocked by ivermectin at l o w concentrations, indicating that these two proteins are part of the high affinity drug binding site. A biologically inactive avermectin analog (3,4,8,9,10,11,22,23octahydroavermectin B i ) , d i d not block the labeling pattern, consistent w i t h the interpretation that the two proteins at 45 k D a were specific labeling products. a



17. ROHRER & ARENA


271

Figure 4. [Panel A ] Photoaffinity labeling of the C. elegans avermectin binding site. Autoradiography of a 5-20% polyacrylamide gel. Each lane contained 200 m g of Triton X-100 soluble membrane protein. C. elegans proteins were labeled with 1251-azido-AVM i n the presence of 0.0, 0.2, 0.8,2, or 20 n M ivermectin (lanes 1-5). [Panel B] Photoaffinity labeling of the Drosophila avermectin binding proteins. Autoradiography of a 5-20% gradient gel. In the presence of increasing concentrations of unlabeled ivermectin (0.1 n M , 1 n M , and 10 n M i n lanes 2-4 respectively) the labeling pattern was progressively blocked, indicating that the 45 k D a labeling product was associated with the high affinity site. The inactive analog, octahydroavermectin added at a concentration of 1 n M d i d not block labeling (Lane 5) indicating specificity of the labeling result shown i n Lane 1. (Reproduced w i t h permission from ref. 32. Copyright 1992 S.P. Rohrer and from ref. 39. Copyright 1994 Insect Biochemistry and Molecular Biology).


272


Purification of the photoaffinity labeled C. elegans and Drosophila melanogaster proteins was accomplished using a monoclonal antibody against avermectin B i to capture the 1 2 5 i - i d o - A V M labeled proteins (42). Preparative scale labeling and purification of the Drosophila proteins led to the recovery of picomole amounts of pure protein (Figure 5 and Table I) which may prove to be adequate for obtaining internal amino acid sequence information. a

a z

T A B L E I. Purification of the photoaffinity labeled Drosophila melanogaster protein


125i. Purification Step

Total Protein (mg)

Azido AVM Receptor (pmol)

Specific Act. (pmol/mg)

927

324a

0.35

1

100

ND

324^

ND

ND

ND

125

293

2.34

7

90

78

3391

9688

24

Head Membranes X - L i n k and Resuspend in Tris/SDSDTT S-300 G e l Filtration Protein A-3A61 Antibody Affinity

.023

b

Purification Recovery (fold) (%)

a p m o l of receptor covalently crosslinked to 1 2 5 i - A z i d o - A V M . Approximately 10% of total ligand bound became covalently crosslinked upon photolysis. b Based on Coomassie Blue staining of radiolabeled protein i n 10% SDSP A G E gels. (Reprinted with permission from ref. 42. Copyright 1994 The Biochemical Society and Portland Press.).

Electrophysiology, pharmacology and expression cloning of the ivermectin receptor Electrophysiological evaluation of C. elegans was not feasible because the tough cuticle prevents penetration w i t h microelectrodes and procedures for patch clamping of isolated neurons or muscle cells have not been established. Therefore, the Xenopus oocyte system was explored as a surrogate for expression of the C. elegans ivermectin gated channel. C . elegans poly ( A ) R N A ( m R N A ) was isolated and injected into Xenopus +



17. ROHRER & ARENA


273

Figure 5. Purification of the photoaffinity labeled avermectin binding proteins from Drosophila melanogaster. Two major proteins (Lanes 2 and 3) w i t h approximate molecular weights of 47 k D a and 49 k D a were obtained. A third protein with a molecular weight of 45 k D a is less heavily radiolabeled. The protein s h o w n i n lane 4 is mouse immunoglobulin heavy chain which co-elutes from the antibody affinity column along w i t h the avermectin binding proteins as a result of leaching from the column. (Reproduced with permission from ref. 42. Copyright 1994 The Biochemical Society and Portland Press.).



274


oocytes (20,29,30). W i t h i n 48-72 hours the protein translational and processing machinery of the oocyte expressed a functional ion channel from the nematode m R N A . Channel activity was measured using a standard two microelectrode voltage d a m p procedure (29^0). Application of ivermectin, or a water soluble derivative, 22,23-dihydroavermectin B i a 4"-0-phosphate ( I V M P O 4 ) , induces a s l o w l y activating, essentially irreversible increase i n membrane current (Figure 6). The irreversible nature of the response is consistent with the slow rate of dissociation for ivermectin observed i n binding studies (31,37). The ivermectin-sensitive membrane current displays a reversal potential, sensitivity to extracellular chloride, and current/voltage relationship consistent w i t h the flow of chloride through chloride channels. In addition, the current is blocked by high concentrations of picrotoxin (29). The ability of a series of avermectin analogs to activate current i n oocytes has been s h o w n to be directly correlated w i t h nematocidal efficacy and membrane b i n d i n g affinity (manuscript submitted). Ivermectin has no effect o n non-injected or water injected oocytes (Figure 6), a n d octahydroavermectin, the biologically inactive analog, does not activate the chloride channel expressed i n oocytes. These data strongly suggest that the channel expressed i n oocytes represents the target of neuromuscular paralysis i n the w o r m . Oocytes injected w i t h C. elegans m R N A also respond to the neurotransmitter glutamate (Figure 7). In contrast to the response to ivermectin, the glutamate-sensitive current activates rapidly, desensitizes in the continued presence of glutamate, and is rapidly and completely reversible. The glutamate-sensitive current is also carried by chloride and is blocked w i t h l o w affinity by picrotoxin (30). Ibotenate, a structural analog of glutamate activates current i n oocytes expressing C . elegans m R N A (Figure 7). Ibotenate cross-desensitizes w i t h glutamate, and is approximately four-fold more potent (EC50 70 p M compared to 300 p M for glutamate). Specific agonists for glutamate-gated cation channels such as kainate, N-methyl-D-aspartate ( N M D A ) , a-amino-3-hydroxyl-5-methyl-4isoxazole propionic acid ( A M P A ) , and quisqualate have no effect o n the chloride channel expressed from C. elegans m R N A (30). S e v e r a l l i n e s of e x p e r i m e n t a l data demonstrate that glutamate /ibotenate and ivermectin are acting on the same chloride channel. W h e n the concentrations of glutamate and I V M P O 4 that evoke maximal responses are coapplied, the response is only slightly larger than when either is applied alone (Figure 7). In addition, l o w concentrations of I V M P O 4 (< 10 n M ) that have no direct effect on membrane current, potentiate the response to sub-maximal concentrations of glutamate (Figure 7). This potentiation is due to a reduction in the EC50 and the H i l l coefficient of the glutamate dose-response curve (Figure 8). I V M P O 4 has the a d d i t i o n a l action of r e d u c i n g the p o p u l a t i o n of channels that desensitize i n the presence of glutamate (Figure 8). Finally, both glutamate a n d I V M P O 4 responses are b l o c k e d w i t h similar concentration of


17. ROHRER & ARENA

Water

C. elegans poly (A)+ R N A


275


IVM

IVM

IVMPO4

IVMPO4

100 nAI 10

sec

Figure 6. Ivermectin activates a membrane current i n Xenopus oocytes expressing C. elegans m R N A . Membrane currents were recorded from oocytes injected w i t h C. elegans m R N A (left) and w i t h water (right). Solid lines above current traces indicate the time of drug application. Concentrations of ivermectin and IVMPO4 applied were 1 \iM. Ivermectin or I V M P O 4 activated current i n >95 % of the oocytes injected w i t h C. elegans m R N A , but not i n oocytes injected w i t h water. (Reproduced with permission from ref. 29. Copyright 1991 Waverly).


276

A.


Glutamate 1 mM

IVMP0 1piM 4

Glutamate 1 mM

70 nA|^


10 sec B.

Ibotenate 200/iM

IVMPO4 1»iM

Ibotenate 200 uM

100 n A | ^ 6 sec Glutamate 100uM

C.

Glutamate 100 uM

IVMPO4 10nM

30 nA|__ 6 sec

Figure 7. I V M P 0 4 , glutamate, and ibotenate activate the same chloride current i n oocytes injected with C. elegans m R N A . A . Response of an oocyte to maximal concentrations of glutamate, I V M P 0 4 , and reapplication of glutamate (last trace). B. Response of an oocyte to maximal concentrations of ibotenate, IVMPO4, and re-application of ibotenate (last trace). C . After the control response to a submaximal concentration of glutamate (left), the oocyte was pretreated w i t h a concentration of IVMPO4 that failed to activate current (middle trace) for 3 min. Re-application of glutamate i n the presence of IVMPO4 resulted i n potentiation of the glutamate-sensitive current (last trace). (Reproduced w i t h permission from ref. 30. Copyright 1992 Elsevier Science.).


17. ROHRER & ARENA



Glutamate 50pM

•

0.001

277

Glutamate 1mM

i

i

0.01 0.1 1 Glutamate (mM)

i

10

F i g u r e 8. IVMPO4 shifts the concentration-response curve for g l u t a m a t e . The plot shows the n o r m a l i z e d glutamate-sensitive concentration response curve i n the absence (filled circles) and presence (open triangles) of 2 n M IVMPO4. The smooth curves represent fits to a modified Michaelis-Menten equation (30). IVMPO4 decreased the EC50 for glutamate from 300 to 85 p M , and changed the H i l l coefficient from 1.8 to 1.0 (smooth curves). The inset shows superimposed current traces i n the absence (filled circles) and presence of IVMPO4 (open triangles). (Reproduced w i t h permission from ref. 30. Copyright 1992 Elsevier Science.).



278


picrotoxin, and the same size range of the m R N A encodes for glutamate and ivermectin responses (29^0,43). The activity of ibotenate on the glutamate-gated chloride channels expressed from C . elegans m R N A suggests that the channel is similar to glutamate-gated chloride channels reported i n arthropods. Ibotenate gates glutamate-sensitive chloride channels present i n locust muscle ( H receptors), locust neurons, cockroach neurons, a n d lobster muscle (25,26,44,45,46,47). Half-maximal activation of these channels b y glutamate /ibotenate is i n the range of 10-500 | i M , similar to that for activation i n C . elegans-injected oocytes. Finally, it appears that the glutamate-gated chloride channels found on locust muscle are gated by avermectins (24). These comparative studies support the hypothesis that avermectins target glutamate-gated chloride channels. Obtaining electrophysiological responses i n Xenopus oocytes has enabled us to take an expression cloning approach toward isolating c D N A s for the C . elegans avermectin receptor (43,48,49). C. elegans m R N A was size-fractionated and then injected into Xenopus oocytes. Glutamate- and ivermectin-sensitive currents were expressed from m R N A i n the 1.8-2.0 kb size range. A c D N A library was synthesized from the active fraction. R N A has been synthesized in vitro from the pools of recombinant c D N A s and are b e i n g screened i n Xenopus oocytes for expression of an ivermectin-sensitive glutamate-gated chloride channel. Interactions of avermectins with other ligand-gated chloride channels Avermectins are highly selective for invertebrate receptors (37). However they have been s h o w n to act o n G A B A - g a t e d chloride channels i n vertebrate brain (37,50,51,52,53). W e have investigated the response to ivermectin using oocytes injected with rat brain m R N A ((54), Figure 9). There was no direct activation of GABA-sensitive chloride current w i t h ivermectin. H o w e v e r , ivermectin potentiated the response to l o w concentrations of G A B A , and reduced the fraction of current that desensitized during application of maximal G A B A concentrations. Except for the lack of direct activation, the interaction of ivermectin w i t h mammalian G A B A A receptors resembles the response observed i n oocytes injected with C . elegans m R N A . In oocytes expressing C . elegans m R N A , ivermectin potentiated the effect of glutamate whereas i n oocytes expressing rat brain, chick brain, or cloned G A B A A receptor m R N A , ivermectin potentiates the effect of G A B A (52,54). In both systems ivermectin causes a reduction i n the EC50 and the H i l l coefficient of the ligand dose-response curve and reduces the fraction of channels that become desensitized. Avermectins do not readily cross the blood brain barrier, so only very l o w concentrations ever come i n contact w i t h the vertebrate receptors. The combination of lower affinity for the vertebrate receptor (37) and compartmentalization of ivermectin may account for the l o w incidence of host toxicity w i t h this class of compounds. It has recently



17.

ROHRER & ARENA


0.01

0.1

1

10

100

279

1000

GABA (uM) Figure 9. IVMPO4 shifts the concentration-response curve for G A B A . Oocytes were injected with m R N A isolated from rat cerebral cortex, and recordings were made 2 days after injection. The inset shows superimposed current traces i n the absence and presence of IVMPO4 (open triangles). The plot shows the G A B A dose response curve i n the absence (filled circles) and presence (open triangles) of 1 p M IVMPO4. The smooth curves represent fits to a modified Michaelis-Menten equation (30). IVMPO4 decreased the EC50 for G A B A from 42 to 3 p M , and changed the H i l l coefficient from 1.8 to 1.0 (smooth curves).



280


been s h o w n that transgenic mice w i t h a disrupted m d r l a gene are 100 times more sensitive to ivermectin than are w i l d type mice (55). The m r d l a gene encodes for the major drug transporting P-glycoprotein i n the blood brain barrier (55). This result suggests that normal individuals expressing a functional m d r l a gene, are not subject to toxic side effects of ivermectin because they possess the ability to prevent drug accumulation in brain tissue by virtue of the P-glycoprotein efflux pump. Avermectins also have been shown to interact w i t h other members of the l i g a n d gated chloride channel family. They inhibit strychnine b i n d i n g to the m a m m a l i a n glycine-gated c h l o r i d e channels i n a noncompetitive fashion (56). In crayfish stomach muscle, avermectins activate a multi-transmitter-(glutamate, acetylcholine, G A B A ) gated chloride channel (27). Moreover, there is evidence for both potentiation and blockade of G A B A - g a t e d chloride channels i n arthropods (23,57), and blockade of the Ascaris body w a l l muscle G A B A - g a t e d chloride channels (58,59). Based on these accounts, avermectins appear to be a promiscuous class of compounds interacting w i t h many of the k n o w n ligand-gated chloride channels. It remains to be determined whether the effects of direct channel activation, blockade, a n d / o r potentiation of ligand-gated responses can be correlated with the biological activity of the avermectins. Future studies The biochemical isolation and expression cloning approaches outlined earlier are being applied i n parallel i n order to obtain the C . elegans and Drosophila avermectin receptor genes. C l o n i n g of the genes that encode chloride channels gated by glutamate from both nematodes and insects w i l l broaden our understanding of the physiological importance of these channels and the similarity of these channels to other ligand-gated chloride channels. It w i l l also a l l o w us to determine the native configuration and conformation of the receptor as w e l l as its exact anatomical location i n nematodes and insects. C l o n i n g of homologous ion channel protein genes from parasitic nematodes and arthropods w i l l be facilitated as w i l l studies of the developmental regulation of the expression of these genes. Ultimately, we intend to use the genes i n order to establish new mechanism based screens for novel anthelmintic and insecticidal compounds. If the avermectin gated chloride channels are similar to other ligand-gated chloride channel family members, then it is likely that additional d r u g b i n d i n g sites distinct from the ivermectin binding site, w i l l be present on the receptor molecule. It is our goal to discover and exploit these additional drug binding sites i n our search for the next generation of anthelmintic/insecticidal chemical entities. D u r i n g preparation of this manuscript the expression cloning approach was successful and two c D N A s that encode for subunits of a C. elegans avermectin-sensitive glutamate-gated chloride channel were isolated (60). The subunits, termed G l u C l a and G l u C l p , form homomeric chloride


17. ROHRER & ARENA


281

channels with unique pharmacological and electrophysiological properties. When coexpressed the subunits assemble heteromeric chloride channels with properties that are distinct from the homomeric channels. The characteristics of heteromeric channel responses to glutamate and ivermectin resemble those observed in oocytes injected with C. elegans mRNA.


Acknowledgments Ed Hayes, Ethel Jacobson and Elizabeth Birzin collaborated with Susan Rohrer on the affinity labeling and purification experiments. Doris Cully, Philip Paress and Ken Liu carried out the characterization of the C. elegans receptor expressed in Xenopus oocytes in collaboration with Joe Arena. Helmut Mrozik, Peter Meinke and Tom Shih provided chemistry support for the projects described here and synthesized numerous other avermectin analogs. Kodzo Gbewonyo, Leonard Lister, and Bruce Burgess provided C. elegans tissue from large scale preparations. Scott Costa contributed large quantities of Drosophila flies needed for receptor purification. We would also like to acknowledge the support of Jim Schaeffer, Merv Turner, Roy Smith and Mike Fisher. Literature Cited 1. 2.

3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Burg, R. W.; Stapley, E. O. In Ivermectin and Abamectin. W. C. Campbell, Eds.; Springer-Verlag: New York, 1989; pp 24-32. Burg, R. W.; Miller, B. M.; Baker, E. E.; Birnbaum, J.; Currie, J. A.; Harman, R.; Kong, V. L.; Monaghan, R. L.; Olson, G.; Putter, I.; Tunac, J. P.; Wallick, H . ; Stapley, E. O.; Oiwa, R.; Omura, S. Antimicrob. Agents Chemother. 1979, 15, 361-367. Egerton, J. R.; Ostlind, D. A.; Blair, L. S.; Eary, D. H.; Suhayda, D.; Cifelli, S.; Riek, R. F.; Campbell, W. C. Antimicrob. Agents Chemother. 1979, 15, 372-378. Ostlind, D. A.; Cifelli, S.; Lang, R. Vet. Rec. 1979, 105, 168. Putter, I.; MacConnell, J. G.; Preiser, F. A.; Haidri, A. A.; Ristich, S. S.; Dybas, R. A. Experentia. 1981, 37, 963-964. Chabala, J.; Mrozik, H.; Tolman, R.; Eskola, P.; Lusi, A.; Peterson, L.; Woods, M.; Fisher, M . J. Med. Chem. 1980, 23, 1134-1136. Campbell, W. Parasitology Today. 1985, 1, 10-16. Campbell, W. C. Medicinal Research Reviews. 1993, 13, 61-79. Campbell, W. Sem. Vet. Med. & Surg. 1987, 2, 48-55. Ivermectin and Abamectin.; W. C. Campbell, Eds.; Springer-Verlag: New York, 1989. Aziz, M. A.; Diallo, S.; Diop, I. M.; Lariviere, M.; Porta, M . Lancet. 1982, 2, 171-173. Lasota, J. A.; Dybas, R. A. Annu. Rev. Entomol. 1991, 36, 91-117. Knowles, C. O.; Casida, J. E. J. Agric. Food Chem. 1966, 14, 566-572. Eyre, P. J. Pharm> Pharmacol. 1970, 22, 26-36.



282


15. Forbes, L. Southeast Asian J. Trap. Med. Publ. Health. 1972, 3, 235-241. 16. Lewis, J.; Wu, J.; Levine, J.; Berg, H . Neuroscience. 1980, 5, 967-989. 17. Kass, I. S.; Wang, C. C.; Walrond, J. P.; Stretton, A . O. W. Proc. Natl. Acad. Sci. USA. 1980, 77, 6211-6215. 18. Kass, I. S.; Larsen, D. A.; Wang, C. C.; Stretton, A. O. W. Exp. Parasitol. 1982, 54, 166-174. 19. Turner, M . J.; Schaeffer, J. M . In Ivermectin and Abamectin. W. C. Campbell, Eds.; Springer-Verlag: New York, 1989; pp 73-88. 20. Arena, J. P. Parasitology Today. 1994, 10, 35-37. 21. Fritz, L. C.; Wang, C. C.; Gorio, A. Proc. Natl. Acad. Sci. USA. 1979, 76, 2062-2066. 22. Mellin, T. N.; Busch, R. D.; Wang, C. C. Neuropharm. 1983, 22, 89-96. 23. Duce, I. R.; Scott, R. H . Brit.J.Pharmacol.1985, 85, 395-401. 24. Scott, R. H.; Duce, I. R. Pestic. Sci. 1985, 16, 599-604. 25. Lea, T. J.; Usherwood, P. N . R. Comp. Gen. Pharmacol. 1973, 4, 351-363. 26. Lea, T. J.; Usherwood, P. N . R. Comp. Gen. Pharmacol. 1973, 4, 333-350. 27. Zufall, F.; Franke, C.; Hatt, H . J. Exp. Biol. 1989, 142, 191-205. 28. Simpkin, K. D.; Coles, G. C. J. Chem. Technol. Biotechnol. 1981, 31, 6669. 29. Arena, J. P.; Liu, K. K.; Paress, P. S.; Cully, D. F. Mol. Pharmacol. 1991, 40, 368-374. 30. Arena, J. P.; Liu, K. K.; Paress, P. S.; Schaeffer, J. M.; Cully, D. F. Mol. Brain Res. 1992, 15, 339-348. 31. Cully, D. F.; Paress, P. S. Mol. Pharmacol. 1991, 40, 326-332. 32. Rohrer, S. P.; Meinke, P. T.; Hayes, E. C.; Mrozik, H.; Schaeffer, J. M . Proc. Natl. Acad. Sci. 1992, 89, 4168-4172. 33. Gbewonyo, K.; Rohrer, S. P.; Lister, L.; Burgess, B.; Cully, D.; Buckland, B. Biotechnology. 1994, 12, 51-54. 34. Chalfie, M . ; White, J. In The Nematode Caenorhabdtitis elegans. W. B. Wood, Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, 1988; pp 337-391. 35. White, J. G.; Southgate, J. N . ; Thomson, J. N . ; Brenner, S. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1986, 314, 1-340. 36. Stretton, A . O. W.; Davis, R. E.; Angstadt, J. D.; Donmoyer, J. E.; Johnson, C. D. Trends in Neurosci. 1985, 8, 294-300. 37. Schaeffer, J. M.; Haines, H . W. Biochem. Pharm. 1989, 38, 2329-2338. 38. Schaeffer, J. M . ; Fraizer, E. G.; Bergstrom, A . R.; Williamson, J. M . ; Liesch, J. M.; Goetz, M . A. J. Antibiotics. 1990, 9, 1179-1182. 39. Rohrer, S. P.; Birzin, E. T.; Costa, S. D.; Arena, J. P.; Hayes, E . C.; Schaeffer, J. M . Insect Biochem. and Mol. Biol. 1994, in press, 40. Rohrer, S. P.; Birzin, E.; Eary, C.; Schaeffer, J. M . ; Shoop, W. J. Parasitology. 1994, 80, 493-497. 41. Meinke, P. T.; Rohrer, S. P.; Hayes, E. C.; Schaeffer, J. M.; Fisher, M . H.; Mrozik, H . J. Medicinal Chemistry. 1992, 35, 3879-3884. 42. Rohrer, S. P.; Jacobson, E. B.; Hayes, E. C.; Birzin, E.; Schaeffer, J. M . Biochem. J. 1994, in press, 43. Cully, D. F.; Paress, P. S.; Liu, K. K.; Arena, J. P. J. Cell Biochem. 1993, 17C, 115.



17.

ROHRER & ARENA


283

44. Cull-Candy, S. G. J. Physiol. 1976, 255, 449-464. 45. Horseman, B. G.; Seymour, C.; Bermudez, I.; Beadle, D. J. Neurosci. Lett. 1988, 85, 65-70. 46. Lingle, C.; Marder, E. Brain Res. 1981, 212, 481-488. 47. Wafford, K. A.; Sattelle, D. B. J. Exp. Biol. 1989, 144, 449-462. 48. Sigel, E. J. Membrane Biol. 1990, 227, 201-221. 49. Lester, H . A. Science. 1988, 241, 1057-1063. 50. Wang, C.; Pong, S. Prog. Clin. Biol. Res. 1982, 97, 373-395. 51. Sigel, E.; Baur, R. Mol. Pharmacol. 1987, 32, 749-752. 52. Drexler, G.; Sieghart, W. Neurosci. Letters. 1984, 50, 273-277. 53. Drexler, G.; Sieghart, W. Eur. J. Pharmacol. 1984, 101, 201-207. 54. Arena, J. P.; Whiting, P. J.; Liu, K. K.; McGurk, J. F.; Paress, P. S.; Cully, D. F. Biophys. J. 1993, 64, a325. 55. Schinkel, A . H . ; Smit, J. J. M . ; van Tellingen, O.; Beijnen, J. H . ; Wagenaar, E.; van Deemter, L.; Mol, C. A . A . M.; van der Valk, M . A.; Robanus-Maandag, E. C.; te Riele, H . P. J.; Berns, A. J. M.; Borst, P. Cell. 1994, 77, 491-502. 56. Graham, D.; Pfeiffer, F.; Betz, H . Neurosci. Letters. 1982, 29, 173-176. 57. Bermudez, I.; Hawkins, C. A.; Taylor, A. M.; Beadle, D. J. J. Recept. Res. 1991, 11, 221-232. 58. Holden-Dye, L.; Walker, R. J. Parasitology. 1990, 101, 265-271. 59. Martin, R. J.; Pennington, A. J. Br. J. Pharmacol. 1989, 98, 747-756. 60. Cully, D. F.; Vassilatis, D. K.; Liu, K. K.; Paress, P. S.; Van der Ploeg, L. H . T.; Schaeffer, J. M.; Arena, J. P. Nature. 1994, 371, 707-711. RECEIVED November 1, 1994


Ivermectin Interactions with Invertebrate Ion Channels - American

Recommend Documents