Marine Toxins - American Chemical Society

Chapter 21 Sea Anemone Polypeptide Toxins Affecting Sodium Channels Initial Structure—Activity Investigations 1

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William R. Kem , Michael W. Pennington , and Ben M. Dunn

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Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610 Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610

Downloaded by UNIV OF ARIZONA on April 26, 2017 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch021

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Sea anemone polypeptides affecting action potentials are o f considerable interest as probes o f the sodium channel and as models for designing new therapeutic and pesticidal compounds. E l e v e n 5,000 dalton homologous toxin variants have been sequenced. They are classified into two groups because o f differences i n sequence, antigenic determinants, and binding sites o n the sodium channel. Multivariate analysis and chemical modification approaches w i t h the natural toxins, plus solid phase synthesis o f monosubstituted toxin analogs, are being used to determine the receptor b i n d i n g domains o f the two toxin types. O v e r 40 different types o f polypeptide toxins have been found i n marine animals (1). M a n y o f these toxins are exquisitely selective i n their actions, affecting a single process o r receptor at minute concentrations. S o far the sea anemone and gastropod (Conus) toxins have attracted the most attention as molecular probes o f i o n channels. In this chapter, we discuss several approaches which are being used to investigate, at the molecular level, the interactions o f the sea anemone neurotoxic polypeptides w i t h sodium channels. A l l sea anemone neurotoxins which have been investigated electrophysiologically inhibit the process o f sodium channel closing (inactivation), thereby prolonging the action potential. A l t h o u g h this has deleterious (sometimes lethal) consequences for predators and prey o f the sea anemone, l o w concentrations o f certain o f these polypeptides have also been found to stimulate mammalian heart contractility with a more favorable therapeutic index than the most c o m m o n l y used drugs for treating congestive heart failure (2). Catterall and Beress (3) first showed that Ammonia sulcata toxin II ( A s II) binds i n a voltage-dependent manner to the same site o n the sodium channel as scorpion a-toxins. A l t h o u g h this observation has since been corroborated by several laboratories (4\ some uncertainty still exists regarding the equivalence o f the b i n d i n g site for A s II w i t h the scorpion a-toxin binding site (5). M o r e recently, it was found that a group o f scorpion polypeptide toxins homologous with the a-toxins, n o w k n o w n as ^-toxins, binds to a separate site o n the sodium channel i n a voltageindependent fashion and selectively enhances channel o p e n i n g (activation). T h e

0097-6156/90/0418-0279$06.0070 o 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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two sites at which the scorpion polypeptide toxins b i n d to the sodium channel protein have been designated as sites 3 (a-toxins) and 4 (^-toxins). In the past five years, a new group o f sea anemone toxins has been investigated by several laboratories (Figure 1). A l t h o u g h homologous with A s II and other related toxins like the anthopleurins, these toxins differ structurally, i m m u nologically, and pharmacologically from the toxins isolated from the family A c t i n i i d a e (6-8). T h e actiniid toxins have been designated as type 1 toxins and the stichodactylid sea anemone toxins as type 2 (7). Structurally, the type 2 toxins lack a single N - t e r m i n a l residue and the two histidinyl residues found i n the first type, possess three consecutive acidic residues at positions 6 - 8 and four consecutive basic residues at the C-terminus, and usually possess a single tryptophan at position 30. Rabbit polyclonal antibodies prepared w i t h type 1 toxins failed to react with type 2 toxins and vice versa (6—8). These two types o f sea anemone toxin also bind to separate sites o n the sodium channel (6). Schweitz et a l . (6) observed competition between Heteractis paumotensis toxins I and II and Androctonus australis toxin II (scorpion a-toxin) for binding to rat brain synaptosomal sodium channels; they concluded that the same site (Catterall's site 3) binds both the type 2 sea anemone toxins and the scorpion a-toxins, but that the type 1 sea anemone toxins bind to another site. However, we have observed no competition between another type 2 toxin, Stichodactyla helianthus toxin I (Sh I), and the same scorpion a-toxin (Pennington et al., submitted). Thus, future structure-activity investigations u p o n sea anemone neurotoxins must experimentally assess the interactions o f these toxins with these different binding sites. In addition to the multiplicity o f binding sites for sea anemone neurotoxic polypeptides o n a single sodium channel, there is also another pharmacological dimension which must be considered: the multiplicity o f sodium channels within organisms. T h e rat brain has been shown to possess at least three genes for the major subunit o f the sodium channel; cardiac and skeletal muscles probably possess additional gene variants (9). Catterall and C o p p e r s m i t h (10) have shown that rat cardiac muscle sodium channels possess an exceptionally high affinity for A s II relative to the scorpion a-toxins, whereas w i t h rat brain sodium channels the affinity for scorpion a-toxin is highest. There are also major pharmacological differences between the neuronal sodium channels o f arthropods, vertebrates, and molluscs, particularly with respect to their sensitivity to the polypeptide toxins. F o r instance, the squid giant axon has been found insensitive to at least two sea anemone toxins type 1 toxins (11,12), and displays a different response (less effect o n inactivation and a significant decrease i n peak sodium conductance) to many scorpion a-toxins (13-14). Thus, differences between sodium channels i n various tissues and organisms must also be considered during structure-activity investigations. Bioassay data obtained for o n e sodium channel must be considered separately from data obtained from a different sodium channel. Sea Anemone Polypeptide Structure A m i n o acid sequences o f eleven homologous sea anemone polypeptides have been elucidated. A l l possess three disulfide bonds. T h e six half-cysteine residues always occur i n the same positions (7,8). Initial studies concerning the toxin secondary and tertiary structures relied u p o n circular dichroism, laser R a m a n , and, to a lesser extent, fluorescence spectral measurements (15—18). T h e circular dichroism spectra o f the four toxins so far examined are essentially superimposable and thus indicate a c o m m o n secondary structure. T h e only peak observed, a negative ellipticity at 203 n m , largely results from a non-regular ("random")



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N G A Y T Y W S A T A I S A N C K C D D E G P D V R T A P L T G T V D L G Y C N E G W E K C A S Y Y T P I A E C C R K K K S D N I S F F S V D

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Figure 1. M o s t c o m m o n (consensus) sequences o f the two types o f sea anemone toxins. B o l d letters represent residues which b o t h toxin types have i n c o m m o n . Letters above each sequence are nonconservative substitutions, w h i l e letters below each sequence are conservative substitutions. A nonconservative substitution was defined as o n e i n which (a) electronic charge changed, (b) a hydrogen-bonding group was introduced o r removed, (c) the molecular size o f the sidechain was changed by at least 5 0 % , o r (d) the secondary structure propensity was changed drastically from b to h o r vice versa (Ref. 28, Table V I I ) . A total o f seven type 1 toxins and four type 2 toxins were compared. C o m p l e t e sequences o f the toxins considered here are cited i n reference 7.

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structure a n d from £ - t u r n residues (16). A small plateau at 2 1 5 - 2 2 5 n m probably results from ^-pleated sheet residues. Analysis o f the R a m a n amide I vibrations o f A s II, Anthopleura xanthogrammica I ( A x I, o r anthopleurin A ) , and Heteractis macrodactylus III ( H m ni) indicated the occurrence o f small proportions o f a-helix and £ - s h e e t i n these toxins. D u r i n g the mid- 1970's, when these studies were being done, computer methods for analyzing the C D and R a m a n spectra to obtain reliable quantitative estimates o f secondary structure h a d not yet been developed. R a m a n analysis also permitted certain inferences about the geometry o f the disulfide bonds and the environmental exposure o f certain aromatic amino acid residues (15,16). M o r e recent studies o n the folded toxin structure by N o r t o n and colleagues have utilized H - and C - N M R techniques (19,20). B y using 2 D - F T - N M R , it was possible to localize a four stranded, antiparallel ^-pleated sheet "backbone" structure i n A s II, A x I, and S h I (21,22). I n addition, W e m m e r et a l . (23) have observed an identical ^-pleated structure i n H p II. N o a-helix was observed i n these four variants. I n the near future, calculated s o l u t i o n conformations o f these toxins, utilizing distance measurements from extracted Nuclear Overhauser Enhancement ( N O E ) effects should greatly stimulate structure-activity investigations.


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Sea Anemone Toxin Activity Ultimately, comparison o f the equilibrium dissociation constants ( K ' s ) for binding o f the sea anemone toxins to the same sodium channel receptor site is the best means o f assessing the structural requirements for toxin action. S o m e data o f this type already exist (6,24, Pennington et al., submitted). These measurements are n o t easy and are complicated by voltage-dependent b i n d i n g (i.e., sealed membrane vesicles must be prepared capable o f maintaining a considerable resting potential). Consequently, w h o l e animal toxicity ( L D ) data are mostly available at this time. D a t a for the actiniid type 1 toxins are presented i n Figure 2. It s h o u l d be kept i n m i n d that L D values are not necessarily p r o p o r t i o n a l to K estimates (25), particularly when considering toxins with l o w K ' s (high affinity). A l s o , it is likely that other factors such as susceptibility to endogenous proteases affect the observed L D estimates. Nevertheless, the LDc0 data are i n semi-quantitative agreement with the K estimates available (6,24, Pennington et al., submitted). N o t e that the mammalian toxicity varies m o r e greatly than the crustacean toxicity i n this group o f toxins. D

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Comparison of Natural Variants T o x i n variants occurring i n the same species often resemble each other rather closely i n structure. Occasionally (the Anemonia sulcata toxins I and II are an example), a large toxicity difference nevertheless exists between two structurally similar variants. O n the other hand, toxins from taxonomically distant species are m o r e likely to display large differences i n sequence. It seems that pharmacological comparisons between toxins varying only slightly i n sequence are most illuminating, at least i n terms o f deciphering the influence o f a particular sidechain u p o n toxicity. Since natural toxins differing i n only a single amino acid residue o f interest w o u l d be difficult to find, o n e must utilize an approach capable o f using toxins with m u l t i p l e differences to provide inferences regarding the importance o f single sidechains. A multivariate analytical approach has recently been applied to smaller molecules, including peptides (26,27). Recently, we have applied this approach to the sea anemone type 1 toxins. A b o u t 9 0 % o f the differences observed i n


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Sea Anemone Polypeptide Toxins Affecting Sodium Channels

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Figure 2. Relative toxicity ( L D ™ and L D ) estimates for actiniid sea anemone toxins u p o n crabs (Carcinus maenas) and mice. Values for Anemonia sulcata (As) and Anthopleura xanthogrammica (Ax) toxins are from ref. 24; data for Condylactis gigantea and Phyllactis flosculifera toxins are unpublished (Kern). T h e arrows indicate that the real mouse L D values for C g II and P f II must exceed the values indicated i n the figure. A l t h o u g h insufficient data are presently available to quantitatively define a relationship between mammalian and crustacean toxicity, it seems that there is usually an inverse relationship, w h i c h may be approximately defined by the stipple zone. 1 0 0

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toxicity between the different type 1 toxins could be accounted for (Hellberg and K e m , unpublished results). Certain amino acid residues seem important for explaining toxicity differences between the variant toxins. Since the method ignores contributions o f sidechains which do n o t vary i n the sample group, it cannot predict the contribution o f non-variant residues towards toxicity. A large number o f toxin variants w o u l d have to be considered i n order for this method to confidently predict the importance o f most residues. Since N a t u r e is unlikely to provide a randomly substituted group o f toxins, this method seems limited as l o n g as it is only applied to naturally occurring toxin variants. Comparisons between these toxins allow delineation o f the variability o f each position i n the sequence. F o r instance, the residues which are extremely invariant (conservative) for b o t h types o f sea anemone toxin are the half-cystines, certain glycyl residues which are expected to be involved i n £ - t u r n s , and only a few other residues - A s p 5 o r 6, A r g 13 o r 14, and Tryp 30 o r 31 (the numbering depends u p o n the toxin type) — expected to be important for folding o r receptor binding. R a t h e r surprising is the variation i n the residues which N M R studies (22,23) have shown are involved i n formation o f the four stranded ^-pleated sheet. Figure 1 summarizes the degree o f variation found along the sequences o f the type 1 and 2 toxins. It should be noted that since there are fewer type 2 toxin sequences currently available, the variability profile for this type is particularly likely to change as new sequences appear. Since H P L C resolves many sea anemone toxins into a much greater number o f variants than was previously expected, many m o r e natural toxin variants should become available for analysis i n the future. Unfortunately, the naturally occurring polypeptide sequences are expected to be those with high toxicity; substitutions which are greatly deleterious for toxicity w o u l d be eliminated by natural selection. Thus, examination o f the natural polypeptides is expected to only reveal what is successful. Sidechain conservatism may be split u p into at least two kinds: 1) substitutions w h i c h conserve sidechain bonding forces — providing similar electrostatic, hydrophilic, o r hydrogen bonding interactions, and 2) substitutions conserving secondary structure propensity. F o r instance, substitution o f glutamic acid with aspartic acid conserves charge, but this could have a considerable effect u p o n the secondary structure propensity o f the peptide.

Chemical Modification This approach is primarily limited by the group rather than residue selectivity o f modifications and by the relatively small number o f selective reagents available for use. M o d i f i c a t i o n reactions carried out with large polypeptides o r proteins generally lead to a complex mixture o f products which cannot be completely resolved and individually analyzed. I n this respect, the sea anemone toxins are relatively promising since i n many cases only 1 o r a few similar residues are present. Often the reaction can be directed towards the p r o d u c t i o n o f a very few products by manipulating p H , time, reagent concentration, o r other conditions. Unfortunately, the importance o f non-reactive residues (about half o f the a m i n o acids) cannot be investigated with this method. S o m e chemical modification studies o n the sea anemone toxins have unfortunately been less than rigorous i n analyzing the reaction products. Consequently, results from many o f these studies can only provide suggestions, rather than firm conclusions, regarding the importance o f particular sidechains. M a n y such studies also have failed to determine i f the secondary and tertiary structures o f the toxin products were affected by chemical modification.


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T h e actiniid toxins A s II and A x II b o t h possess o n l y three carboxyl moieties. O n e aspartyl carboxyl (either 7 o r 9) has an abnormally l o w p K o f 2 and is thought to participate i n a buried salt-bridge w i t h a cationic group. T h e other two carboxyls have n o r m a l p K ' s , the higher o n e (3.5) presumably being at the C-terminus. T h e only chemical modification reported for these carboxyls has been by carbodiimide-catalyzed reaction w i t h glycine methyl ester. M o d i f i c a t i o n o f all three carboxyls destroyed toxicity (29,30). However, B a r h a n i n et al. (30) have reported that their A s II product behaved as a competitive antagonist. Several partially modified A s II products were resolved, but they unfortunately were not chemically analyzed. G r u e n and N o r t o n (57) also modified A x II i n the same manner and found that when two glycine groups were added that this polypeptide lost its native conformation (measured by b o t h * H - N M R and C D spectroscopy) as well as its i n o t r o p i c activity o n guinea p i g heart. a


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T h e most rigorous chemical modification study done o n a sea anemone toxin was that o f B a r h a n i n et al. (30). Besides the above-mentioned carboxyl modifications, they also studied the consequences o f modifying the various basic amino acid sidechains. R e a c t i o n o f A r g 14 i n A s II with cyclohexanedione abolished its toxicity for crabs as well as mice. This contrasts with an earlier report (29) that reaction o f the same residue i n A x II with phenylglyoxal d i d not significantly affect its i n o t r o p i c activity. Several possible explanations for this apparent discrepancy can be considered, including the possibility that the cardiac Na channel receptor is less affected by altering the guanidinyl group. Since only o n e arginyl group resides o n most type 1 toxins it s h o u l d be possible to further assess the importance o f this group by additional chemical modification investigations. +

Carbethoxylation o f the two histidinyl sidechains at positions 32 and 37 o f A s II resulted i n at least a 20-fold loss i n mouse toxicity and a 5-fold loss o f crab toxicity (30). It is not yet clear i f b o t h residues equally contribute to this effect. B o t h residues are conserved i n all except one o f the reported actiniid toxin sequences, but are absent i n the stichodactylid toxins (8). N e w c o m b et al. (29) also reacted A x I with diethyl pyrocarbonate i n essentially the same manner. W h e n both histidinyl groups were modified, the resulting toxin derivative retained full i n o t r o p i c activity u p o n the guinea p i g heart. I n apparent contradict i o n with the results o f B a r h a n i n et al. (30), K o l k e n b r o c k et al. (32) found that modification o f the two histidinyl residues o f A s II w i t h this reagent actually increased i n o t r o p i c activity o n the guinea pig heart. F u r t h e r experiments are needed to clarify the importance o f the histidinyl residues for activity. T h e contributions o f individual primary amino groups are not yet k n o w n i n m u c h detail. Acetylation o r reaction with fluorescamine o f all three groups i n A s II reduced toxicity i n crabs about 8- to 10-fold, but i n o t r o p i c activity was reduced more than 20-fold by both o f these modifications (32). G u a n i d a t i o n o f the two £-amino groups with o-methyl isourea and then acetylation o f the a amino group reduced both activities about 2-fold, suggesting that acetylation o f the a-amino group by itself had little effect o n toxicity. Stengelin et al. (57) utilized Schiff base formation o f the amino groups with pyridoxal phosphate, followed by reduction o f the i m i n e bond with sodium borohydride. They isolated two monosubstituted derivations o f A s II. T h e G l y 1 derivative possessed only 2% o f the native toxin activity, whereas the Lys 35 adduct displayed only 1% o f the activity. Since this modification, besides introducing an aromatic group, also substitutes a negative charge for a positive charge, a greater loss o f toxicity could be expected, compared to the acetyl derivatives. This investigation thus adds further support for the idea that several amino acid sidechains near the N terminus are critical for toxicity.



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O n l y a single paper has so far appeared i n which a type 2 polypeptide toxin was chemically modified (34). M o d i f i c a t i o n o f Heteractis macrodactylus I ( H m I) at Tryp 30 (and possibly the tyrosyl residues) caused a 4.5-fold loss o f mouse toxicity. A l k y l a t i o n w i t h Koshland's reagent (55), a more selective modification o f T r y 30, reduced toxicity only 2-fold. It was concluded that this sidechain is not essential for mouse toxicity. M o d i f i c a t i o n o f 2.7 lysyl residues with 2,4pentanedione led to a 10-fold loss o f mouse toxicity. T h e C D spectrum o f the modified toxin was not significantly different from that o f the natural toxin (34). E x p o s u r e o f this modified toxin derivative w i t h 0.1 M hydroxylamine at p H 6.0 fully restored the toxicity o f H m I; since any modified arginyl sidechains w o u l d not have been affected by this nucleophile, it was concluded that the original decrease i n toxicity was entirely due to amino group modification. R e a c t i o n o f H m I w i t h malonic dialdehyde at A r g 13 resulted i n a 20-fold loss i n toxicity. H m III arginyl modification by phenylglyoxal o r cyclohexanedione resulted i n a 5-fold loss i n mouse toxicity. T h e guanidyl group o f A r g 45 failed to react w i t h either reagent under the conditions employed. It was concluded that the A r g 13 sidechain is not essential for toxicity i n these two ( H m I and H m ni) type 2 toxins (34). C h e m i c a l Synthesis In order to avoid some o f the ambiguities mentioned above for extracting structure-activity relations from a series o f natural o r chemically modified polypeptide variants, we decided to use a synthetic approach to obtain monosubstituted Stichodactyia neurotoxin variants. W e chose to investigate the influence o f single substitutions near the N-terminus, since the acidic residues there had been previously implicated as essential for toxicity. W e originally intended to use semi-synthesis, c o u p l i n g a variety o f N-terminus tridecapeptide analogs to the natural toxin 1 4 - 4 8 fragment, to be obtained by tryptic o r clostripain cleavage. However, we were unsuccessful i n obtaining a selective cleavage o f the native toxin at A r g 13. A t this time, a solid phase automatic peptide synthesizer was acquired, so we attempted total synthesis o f S h I. A solid phase synthesis o f A x I (anthopleurin A ) had previously been reported i n an abstract (36). T h e synthetic A x I possessed only 1 1 % o f the toxicity o f the natural toxin. First, w e investigated whether S h I could be reduced under denaturing c o n ditions, exposed to the hydrogen fluoride cleavage procedure w e intended to use, then reoxidized and refolded successfully. T h e toxicity o f the resulting polypeptide (50% yield) was the same as that o f the untreated natural toxin. F o r the synthesis, a stable phenylacetamidomethyl ( P A M ) linkage was selected to anchor the peptide chain to the resin. A double-coupling p r o t o c o l was also adopted for amino acids which either were ^-branched o r possessed large protecting groups (overall c o u p l i n g efficiency was 9 5 % ) . After deprotection and decoupling with the low-high hydrogen fluoride procedure (57), t h i o l and thioether scavenge compounds were removed from the synthetic peptide extract i o n w i t h ether-ethyl acetate (1:1, w/w). L o w molecular weight impurities were further eliminated by passage o f the toxin through a G - 5 0 Sephadex c o l u m n equilibrated with 10% acetic acid. After dialysis at 4 ° C , the synthetic peptide was reoxidized by the glutathione method employed by A h m e d et a l . (38) with scorpion toxins. T h e resulting sample was centrifuged to remove insoluble c o n stituents and then subjected to phosphocellulose c o l u m n chromatography. E l u t i o n w i t h a linear gradient o f a m m o n i u m formate at p H 4.0, as for purification o f the natural toxin (5), yielded six sharp 280 n m absorbing peaks; the last peak,


21. KEMETAL.


which eluted at the same conductivity as the native toxin, was the o n l y peak displaying crab toxicity. This peak contained 24% o f the initial mass placed o n the c o l u m n . After final purification with a C . H P L C c o l u m n , the synthetic toxin was found to be equivalent to the purified natural toxin by a wide variety o f criteria. Chemically, its amino acid c o m p o s i t i o n , N - t e r m i n a l sequence (ten residues), and isoelectric point were identical to those o f the natural toxin. T h e C D spectra o f the two samples were superimposable, indicating the same secondary structure. T h e one-dimensional high resolution spectra o f the two samples also coincided, except for some m i n o r impurities. The fluorescence activation and emission spectral properties, attributed mainly to T r y p t o p h a n 30, were the same. W h e n injected into crabs and mice, the two toxin samples were found to be o f equal toxicity (39).


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H a v i n g synthesized the natural S h I sequence, we have n o w embarked u p o n a program o f examining the consequences o f substituting single amino acid residues. O u r initial focus has been u p o n ionizable residues, particularly those w i t h carboxylate-containing sidechains. Substitution o f glutamic acid at position 8 by glutamine decreased S h I toxicity over 10,000-fold. Substitution o f asparagine at either position 6 o r 7 similarly abolished crab toxicity. Replacement o f aspartic acid at position 11 with asparagine reduced toxicity about 300-fold. Substitution o f e-N-acetyl lysine for lysine at position 4 reduced toxicity about 1,000-fold. Since the C D spectra o f these analogs did not differ significantly from that o f the natural toxin, it is unlikely that the substitutions affected polypeptide chain folding (40). T h e secondary and tertiary structures o f these analogs w i l l be further analyzed by * H - N M R when adequate samples become available. A n o t h e r means o f investigating the receptor binding d o m a i n w o u l d be to determine i f antibodies specific to single antigenic regions interfere with binding o f the toxin to its receptor. A y e b et al. (41) have recently utilized this approach to investigate the interaction o f A s II with rat brain sodum channels. They found that A s II when b o u n d to its receptor site remained fully accessible to a rabbit polyclonal antibody which binds to two o f three acidic residues i n this toxin (Asp 7, A s p 9, G i n 47). This result seems surprising i n comparison w i t h o u r toxicity data o n synthetic S h I analogs, which strongly implicate acidic residues at positions 6—8 i n receptor binding. However, A s II and S h I do not b i n d to a c o m m o n receptor site. Therefore, the structural requirements for binding to these two sites are unlikely to be the same.

Concluding Remarks C o m b i n i n g the various approaches (isolation o f a variety o f polypeptide natural variants, chemical modification, chemical synthesis, i m m u n o c h e m i c a l measurements) discussed above with N M R and crystallographic approaches for elucidating the native toxin structure should soon provide molecular models for the receptor binding domains o f the two types o f sea anemone toxins. S u c h models w i l l guide future toxin analog syntheses aimed at delineating i n sufficient detail these two receptor binding domains. O n c e the area and topography o f each d o m a i n is recognized, it should then be possible to design simpler molecules m i m i c k i n g the receptor binding surface o f the sea anemone polypeptide. This could eventually lead to the design o f safer, more selective drugs and pesticides acting u p o n sodium channels.

Acknowledgments O u r contributions to the research discussed i n this paper were supported by N I H G M 3 2 8 4 8 . W e thank M s . J . A d a m s for typing the manuscript.


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