Phylogenetic Differences Detected by Snake A-Neurotoxins (Evolution/Reptiles/Snake Venom/A-Bungarotoxin/Cobrotoxin) STEVEN J

Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3245-3249, August 1975 Physiology

Acetylcholine receptors at neuromuscular synapses: Phylogenetic differences detected by snake a-neurotoxins (evolution/reptiles/snake venom/a-bungarotoxin/cobrotoxin) STEVEN J. BURDEN*, H. CRiss HARTZELLt, AND Doju YOSHIKAMIt * Department of Zoology, University of Wisconsin, Madison, Wisc. 53706, and Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; and t Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Stephen W. Kuffler, May 30,1975

ABSTRACT Phylogenetic differences in acetylcholine re- The structures and mode of action of these a-neurotoxins ceptors from skeletal neuromuscular synapses of various have been well characterized (8-10). They are single poly- species of snakes and lizards have been investigated, using peptide chains and fall into two classes that differ in the the snake venom a-neurotoxins a-atratoxin (cobrotoxin) and a-bungarotoxin. The acetylcholine receptors of the phylo- number of amino-acid residues. a-Atratoxin (a-atraTX, also genetically primitive lizards, like those rom all other verte- known as cobrotoxin) isolated from the venom of the Formo- brates previously tested, are blocked by these a-neurotoxins. san cobra, Naja naja atra, is in the class which contains 61 In contrast, receptors from snakes and advanced lizards are to 62 amino acids, and a-bungarotoxin (a-BuTX) isolated insensitive to one or both of the toxins. It is suggested that from the Formosan banded krait, Bungarus multicinctus, is toxin-resistant acetylcholine receptors appeared early in the in the class containing 71 to 74 amino acids. Both classes of evolution of Squamata and preceded the appearance of a- neurotoxins. toxin bind strongly and specifically to ACh receptors of skeletal muscles and homologous tissues and thereby block the All vertebrate skeletal neuromuscular synapses so far exam- action of ACh at the postsynaptic membrane. The blocking ined have several common features: Acetylcholine (ACh), action of a-neurotoxins has previously been demonstrated in the excitatory transmitter, is released from the presynaptic all vertebrates which have been tested: cartilaginous and terminals in multimolecular packets or quanta. The released bony fishes, amphibians, birds, and mammals (9). We are ACh interacts with "nicotinic" ACh receptors in the postsy- not aware, however, of any reports concerning the effect of naptic membrane of the muscle and produces a conductance these toxins on reptiles. increase of the membrane principally to sodium and potassi- We report here that the skeletal neuromuscular synapses um. The response to a quantum of ACh is a miniature exci- of certain closely related lizards and snakes are not blocked tatory postsynaptic potential (min. EPSP). The action of by a-BuTX and a-atraTX. ACh is terminated by acetylcholinesterase (acetylcholine hy- METHODS drolase, EC 3.1.1.7) at the postsynaptic membrane (see re- cent reviews 1 and 2). Muscle Preparations. We have studied the effect of a- We were interested in the types of differences that can be BuTX and a-atraTX on skeletal neuromuscular synapses of discerned in the functional elements of neuromuscular sy- the species listed in Table 1. These animals were identified napses from different species, and whether these differences by the suppliers. In all cases, we used muscles that were one could be correlated with the phylogenetic development of or, at most, only a few fibers thick. With these thin prepara- the species. ACh receptors have received particular attention tions, penetration of drugs into the intercellular spaces is in this regard because their properties can be readily as- rapid. Furthermore, by using Nomarski optics, one can visu- sessed pharmacologically. Although species differences alize the neuromuscular synapses in the living preparations. among ACh receptors have been detected with plant alka- We used the cutaneous pectoris muscle of the frog (11) and loids and synthetic drugs as agonists and antagonists, few the external oblique muscle of the snake (12). For the liz- clear phylogenetic trends have emerged (3). Species differ- ards, the intercostal and occasionally the abdominal, exter- ences in ACh receptors have also been detected with immu- nal oblique muscles were used. Reptilian Ringer solution nological techniques (4), however, correlations with phylog- contained 158 mM NaCl, 2.15 mM KCI, 3.5 mM CaC12, 1.7 eny have not been made. mM MgCl2, and was buffered at pH 7 with 1 mM sodium To probe for differences in ACh receptors, we chose to N-2-hydroxethylpiperazine-N'-ethanesulfonate (Na-Hepes). use a-neurotoxins isolated from the venoms of Elapid The composition of frog Ringer was 115 mM NaCI, 2.0 mM snakes. We reasoned that since animals are often resistant to KCI, 1.8 mM CaCl2, buffered at pH 7 with 1 mM Na-Hepes. the neurotoxins they produce (5-7), phylogenetic differ- a-Neurotoxins. a-BuTX from the venom of Bungarus ences in ACh receptors may be revealed by studying the multicinctus and a-atraTX from Naja naja atra were puri- sensitivity of the neuromuscular synapses of snakes and re- fied as described by others (13, 14). Toxin purity, as deter- lated species to a-neurotoxins. mined by gel electrophoresis in sodium dodecyl sulfate and end-group analysis, was greater than 99%. a-BuTX was ra- Abbreviations: a-atraTX, toxin from Naja naja atra-this toxin has dioactively labeled with 125I and purified (14). been referred to in the past by the rather nonspecific name, cobro- Electrophysiological Assay for Sensitivity to a-Toxins toxin; a-BuTX, a-bungarotoxin; ACh, acetylcholine; Hepes, N-2- All experiments were done at room temperature. The mem hydroxyethylpiperazine-N'-ethanesulfonate; min. EPSP, miniature brane potentials of muscle fibers were monitored with intra excitatory postsynaptic potential. cellular microelectrodes filled with 3 M KCI that had resih 3245 Downloaded by guest on September 26, 2021 3246 Physiology: Burden et al. Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. List of animals tested for sensitivity to neurotoxins Common name Scientific name Infra-order 1. Leopard frog Rana pipiens Anura A. Lizards 2. Tokay gekko Gekko gekko Gekkota 3. American chameleon Anolis carolinensis Iguania 4. Rainbow lizard Lacerta sp. Scincomorpha 5. African plated lizard Cordylus jonesi Scincomorpha 6. Eastern glass lizard Ophisaurus ventralis Anguinomorpha B. Snakes 7. Columbian rainbow boa Epicrates cenchris mauris Henophidea 8. Black rat snake Elaphe obsoleta obsoleta Caenophidea 9. Ribbon snake Thamnophis sauritus Caenophidea

tances of about 10 MO (12). The microelectrode penetrated solution for several hours at room temperature and then the muscle fiber within 50 ,tm of the visually identified end- overnight at 40, fixed in a mixture of 1% paraformaldehyde plate. Resting potentials were between -70 and -100 mV and 1% glutaraldehyde, and washed. Single or, at most, dou- and spontaneous min. EPSP amplitudes were about 0.5 mV. ble muscle fibers were teased out, mounted on microscope The blocking action of a-BuTX and a-atraTX on ACh re- slides with 2% bovine serum albumin as an adhesive, and ceptors was assessed by measuring the effect of toxin on the coated with Kodak NBT-3 liquid emulsion. After exposure amplitudes of spontaneous min. EPSP's. Before addition of for 5 days, the emulsion was developed. Muscle endplates toxin, min. EPSP's were recorded from several muscle fibers were identified both by Nomarski optics and by staining for and averaged. Immediately after addition of toxin, min. endplate acetylcholinesterase (11, 16). EPSP's were recorded from a single muscle fiber for up to 1 hr. Then, min. EPSP's were sampled from several fibers and RESULTS averaged. Whenever toxin had an effect, the amplitudes of Synaptic morphology responses to single quanta of ACh (min. EPSP's) were pro- gressively attenuated with time. Since quantal packets of The skeletal muscle fibers from both snakes and lizards were ACh are not expected to change in size, this approach pro- about 30-40 ,um in diameter. Each fiber had a single en vides a specific index of the postsynaptic action of toxin. plaque innervation usually near its middle. The fibers and Autoradiography. Binding of 125I-labeled a-BuTX to re- their endplates were typical twitch fibers as judged visually ceptors on muscles was determined by autoradiography (15). with Nomarski optics (compare refs. 12, 17, and 18). The ul- Muscles were incubated in 20,ug/ml of 125I-labeled a-BuTX trastructure of the endplates from lizards (19) is similar to (specific activity 2 Ci/mmol) for 90 min, washed in Ringer that from snakes (12, 20).

Table 2. Sensitivity to ca-neurotoxins Time required to reduce min. EPSP amplitude to one-half (hours)* oY-BuTX (jig/ml) a-atraTX (jig/ml) Preparation (N)t 0.1 1.0 20 200 1.0 20 200 1. Rana pipiens (>10) <0.5 <0.5 <0.5 2. Gekho gekko (1) <0.5 < 0.5 3..Anolis carolinensis (1) <0.5 <0.5 4. Lacerta sp. (1) <0.5 <0.5 No effect >1 5. Cordylus jonesi (1) <0.5 No effect > 2.5 6. Ophisaurus ventralis (1) No effect No effect >5 >6 7. Epicrates cenchris maurus (2) <0.5 No effect >15 8. Elaphe obsoleta obsoleta (1) No effect No effect >15 >15 9. Thamnophis sauritus (>10) No effect No effect No effect >18 > 1 >15 * Time (hours) required to reduce spontaneous min. EPSP's to 50% of their original amplitude in isolated skeletal muscles was determined as described in Methods. t Number of animals tested. Downloaded by guest on September 26, 2021 Physiology: Burden et al. Proc. Nat. Acad. Sci. USA 72 (1975) 3247 BOA RIBBON

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FIG. 1. Autoradiographs of endplates from boa (A) and ribbon snake (B) after exposure to 1251-a-BuTX. External oblique muscles from both animals were treated identically (Methods), and the same endplate of a muscle fiber from each animal is shown in three different ways. A,, B1: Nomarski optics reveals the endplate as an elliptical disk on the muscle surface. The details of the endplate are not seen as clearly as in living preparations (compare 12) due to the air-drying step used to prepare the muscle for autoradiography. A2, B2: Bright field. The plane of focus is on the emulsion at the muscle surface, and therefore the striations of the muscle fiber are not clearly seen. Note the high density of silver grains over the boa endplate (A2) and a very low density over that from the ribbon snake (B2). A3, B3: Bright field after staining for acetylcholinesterase. In the muscle from the boa (A) two fibers are visible, and the endplate of the thinner fiber is not in view. The stain in A3 is partially obscured by silver grains. Fiber in B3 is rotated slightly compared to B1 and B2 due to movement during the staining process.

Electrophysiology lowing test. Ringer solution containing 20 ttg/ml of a-BuTX was removed after 18 hr incubation with ribbon snake mus- Since low concentrations of both a-BuTX and a-atraTX cle, diluted 20-fold with frog Ringer solution, and applied to block min. EPSP's at the frog neuromuscular synapse (21) a fresh frog muscle. The toxin was as effective as fresh toxin this preparation was used as a standard to assess the potency in blocking the min. EPSP's in the frog. of the toxins. For example, 0.1 g/ml of a-BuTX invariably These results raised the question about the sensitivity of reduced the min. EPSP amplitude in the frog by 50% in less other reptiles to these toxins. Therefore, several other species than 30 min. In contrast, the min. EPSP amplitude of the of lizards and snakes were tested, and the results are summa- ribbon snake was not affected by 200 ,ug/ml of a-BuTX rized in Table 2. Only the Gekcko and Anolis lizard muscles even after 1 hr. Thus, the ribbon snake neuromuscular were blocked by both a-BuTX and a-atraTX. The neuro- synapse is at least 1000 times less sensitive to a-BuTX than muscular synapses of the other species were not affected by the frog synapse. Likewise, the ribbon snake synapse is not a-atraTX at concentrations more than 2 orders of magnitude affected by a-atraTX (Table 2). higher than those which blocked the frog muscle. In addi- It is unlikely that the ineffectiveness of toxin at the ribbon tion, the muscles of three species were insensitive to both a- snake synapse is due to poor access of toxin into the synaptic BuTX and a-atraTX. Whenever toxin had no effect, the cleft for the following reasons. Amplitudes of min. EPSP's used toxin was removed from the test preparation, diluted to are not diminished even after 18 hr incubation in 20 gg/ml 1 ,ug/ml and re-tested on a frog muscle. In every case the of a-BuTX at room temperature. Furthermore, large pro- min. EPSP in the frog was blocked, showing that the toxin teins such as horseradish peroxidase can penetrate into the had not been inactivated. synaptic cleft within 30 sec (U. J. McMahan and A. Yee, per- In all instances where toxin did have an effect, further ex- sonal communication). posure to the toxin reduced the min. EPSP amplitude to the The insensitivity of the ribbon snake muscle to a-BuTX is background noise level. This indicated that all of the ACh not due to inactivation of the toxin, as shown by the fol- receptors of that synapse were blocked by toxin. Downloaded by guest on September 26, 2021 3248 Physiology: Burden et al. Proc. Nat. Acad. Sci. USA 72 (1975)

Binding of a-BuTX to ACh receptors Lizards Snakes To determine whether the inability of a-BuTX to block the ribbon snake ACh receptor was due to a corresponding inability of the receptor to bind toxin, we compared the bind- w c 0 ing of 125I-a-BuTX to the endplates of the ribbon snake and the boa under identical conditions (Methods). w *0 o U 0 M 0C o0 Fig. 1A is an autoradiograph of an endplate from the boa, U f C 0 Epictates cenchris, labeled with 125I-a-BuTX. The radioac- C U C 0 6 tivity bound to the muscle after 18 hr of washing is localized to the endplate region of the muscle fiber. This shows that 1251-a-BuTX binds to the ACh receptors of the boa with a high affinity. This was also demonstrated physiologically on the same muscle since we failed to detect min. EPSP's in the labeled muscle after the preparation had been washed for 18 hr in toxin-free Ringer. In contrast, Fig. 1B shows that 125I- a-BuTX does not bind to the endplate of the ribbon snake, T. sauritus, since the silver grains over the endplate were at Squamata the background level. Identification of endplates by Nomar- I Sensitive to: ski optics (Fig. 1A1 and 1B1) and by staining for acetylcholi- atraTX & BuTX - nesterase (Fig. 1A.3 and 1B3) showed that none of the end- BuTX -- plates of the ribbon snake were labeled, while all. those of the neither boa were labeled. FIG. 2. Hypothetical phylogenetic relationships between infra-orders of species, based on their sensitivity to a-neurotoxins. Numbers accompanying infra-orders refer to the species given in DISCUSSION Tables 1 and 2. It was assumed that species which are insensitive to one or both toxins are related by a common ancestor, and that The difference in sensitivity of neuromuscular synapses to the species tested are representative of the infra-order. Solid line a-neurotoxins most likely involves differences in the ACh re- ), blocked by both a-BuTX and a-atraTX; dashed lines --- - ceptor molecules themselves. Toxin resistance is not caused --), blocked only by a-BuTX; dotted lines (.), resistant to by toxin inactivation. Furthermore, since some species (C. both a-BuTX and a-atraTX. Infra-orders Rhiptoglossa of lizards jonesi, Epicrates cenchris, and Lacerta sp.) are blocked by and Scolecophidea of snakes were not studied and are not shown. a-BuTX but not by the smaller molecule, a-atraTX, it is unlikely that a-atraTX binding is prevented at resistant synapses by poor access of toxin to the receptor. which have been proposed for the evolution of Squamata on The ACh receptors from toxin-resistant neuromuscular sy- the basis of comparative anatomy (26, 27). The main feature napses have at least a 1000-fold lower affinity for a-BuTX common to the relationships determined by both methods is and a-atraTX than frog receptors do. The equilibrium disso- that Gekkota and Iguania are the lizards most distantly re- ciation constant of a-BuTX binding to solubilized rat muscle lated to snakes, i.e., that snakes evolved from lizards after receptor has been estimated to be less than 10-I0M (22). the Gekkota and Iguania branched off. Since we see no evidence of neuromuscular block at the rib- Although we did not have the opportunity to test muscles bon snake synapse after 1 hr incubation with 2.5 X 10-5 M from any Elapids that produce the a-neurotoxins, one ex- a-BuTX (200 ,g/ml), the difference in affinity of a-BuTX pects that they would possess toxin-resistant ACh receptors for ribbon snake receptors and solubilized rat receptors is since they belong to the infra-order Caenophidea. The only probably greater than 5 orders of magnitude. reptiles that are known to produce a-neurotoxins are mem- In respects other than toxin sensitivity, the ACh responses bers of this highly evolved infra-order. Since species in sev- at neuromuscular synapses of toxin-resistant reptiles and of eral of the infra-orders less phylogenetically advanced than other vertebrates are quite similar. The reversal potential for Caenophidea exhibit toxin resistance, it is likely that toxin the ACh response is about -15 mV for both frog and ribbon resistance evolved before the appearance of a-neurotoxins. snake (ref. 23 and Kuffler and Yoshikami, unpublished), a-AtraTX belongs to the class of Elapid toxins that con- suggesting that the changes in ionic conductances evoked by tains about 10 fewer amino acids than the other class, which ACh are very likely the same. The kinetics of the ionic gat- includes a-BuTX. There is considerable homology in the ing mechanism and the elementary conductance change are amino-acid sequences of the toxins within each class as well likely to be the same since the amplitudes and time courses as between the classes (9, 10). On the basis of structural con- of the quantal synaptic currents are similar in frogs and rib- siderations, it has been suggested that smaller toxins are phy- bon snakes (24, 25). Furthermore, receptors both from frog logenetically more primitive than the larger toxins (9, 10, and ribbon snake are blocked by curare at concentrations on 28). We have seen that the smaller cx-atraTX does not affect the order of l0-5 g/ml (unpublished). The sensitivity of the as many snakes and lizards as does a-BuTX. Furthermore, ribbon snake receptor to curare, however, might be slightly the smaller toxins apparently do not bind as strongly as the less than that of the frog receptor, larger toxins to ACh receptors from other classes of verte- Although the number of species we tested is very limited, brates (29). Thus, the larger toxins, because of their greater we have constructed a chart of the phylogenetic relation- potency, may have offered a selective advantage to snakes ships among snakes and lizards which we have inferred from possessing these toxins. It should be kept in mind, however, toxin sensitivity (Fig. 2). The phylogenetic relationships be- that the a-neurotoxin is only one of several toxic constituents tween species that are not blocked by a-atraTX or a-BuTX in snake venom, and one wonders if snakes are less suscepti- suggest that toxin resistance evolved early in the evolution of ble to these other toxic substances as well. snakes and lizards. This relationship is similar to schemes Finally, what were the precursor proteins from which tox- Downloaded by guest on September 26, 2021 Physiology: Burden et al. Proc. Nat. Acad. Sci. USA 72 (1975) 3249

ins evolved? It is conceivable that there may have been a 9. Lee, C. Y. (1972) Annu. Rev. Pharmacol. 12,265-286. molecule that had an affinity for ACh receptors and served 10. Tu, A. T. (1973) Annu. Rev. Biochem. 42,235-258. some other, possibly trophic, function before evolving into a 11. McMahan, U. J., Spitzer, N. C. & Peper, K. (1972) Proc. R. toxic substance. This suggests that substances related to the Soc. London Ser. B. 181, 421-430. a-neurotoxins may be found in salivary of non-ven- 12. Kuffler, S. W. & Yoshikami, D. (1975) J. Physiol. (London) glands 244,703-730. omous reptiles and perhaps even of other vertebrates. 13. Yang, C. C. (1964) J. Formosan Med. Assoc. 63,325-31. 14. Berg, D. K., Kelly, R. B., Sargent, P. B., Williamson, P. & We thank Drs. Stephen W. Kuffler, Zach W. Hall, and A. 0. W. Hall, Z. W. (1972) Proc. Nat. Acad. Sci. USA 69, 147-151. Stretton for providing laboratory facilities; Drs. Jon Lindstrom and 15. Fambrough, D. M. & Hartzell, H. C. (1972) Science 176, Uel J. McMahan for communicating their unpublished observa- 189-191. tions; Robert B. Bosler, William Dragun, Joseph A. Gagliardi, James 16. Karnovsky, M. J. (1964) J. Cell Biol. 23,217-232. LaFratta, and Michael LaFratta for technical support; Eleanor P. 17. Proske, U. & Vaughan, P. (1968) J. Physiol. (London) 199, Livingston and Shirley E. Wilson for assistance with the manu- 495-509. script; and John Rollins for the gift of Anolis lizards. This work was 18. Ridge, R. M. A. P. (1971) J. Phystol. (London) 217,393-418. supported in part by Grant no. NS 02253 from the National Insti- 19. Robertson, T. D. (1956) J. Biophys. Biochem. Cytol. 2, 381- tutes of Health. S.J.B. is a National Institutes of Health predoctoral 394. trainee, Grant no. GM 01874. H.C.H. is a postdoctoral fellow of the 20. Hess, A. (1965) J. Cell Biol. 26,467-476. Muscular Dystrophy Associations of America, Inc. 21. Miledi, R. & Potter, L. T. (1971) Nature 233,599-603. 22. Brockes, J. P. & Hall, Z. W. (1975) Biochemistry 14, 2092- 1. Kmjevic, K. (1974) Physiol. Rev. 54, 419-540. 2099 2. Hubbard, J. I. (1973) Physiol. Rev. 53,674-723. 23. Takeuchi, A. & Takeuchi, N. (1959) J. Neurophysiol. 22, 3. Michelson, M. J. & Zeimal, E. V. (1973) Acetylcholine, An 395-411. Approach to the Molecular Mechanism of Action (Pergamon 24. Gage, P. W. & Armstrong, C. M. (1968) Nature 218,363-365. Press, Oxford). 25. Hartzell, H. C., Kuffler, S. W. & Yoshikami, D. (1975) J. 4. Lindstrom, J. M. (1975) J. Supramol. Struct., in press. Physiol. (London), in press. 5. Narahashi, T. (1974) Physiol. Rev. 54,813-889. 26. Camp, C. L. (1923) Bull. Amer. Mus. Nat. Hist. 48,289-481. 6. Kidokoro, Y., Grinnell, A. D. & Eaton, D. C. (1974) J. Comp. 27. Bellairs, A. d'A. & Underwood, G. (1951) Biol. Rev. 26, 193- Physiol. 89,59-72. 257. 7. Albuquerque, E. X., Warnick, J. E., Sansone, F. M. & Daly, J. 28. Strydom, D. J. (1972) Toxicon 10, 39-45. (1973) J. Pharmacol. Exp. Ther. 184,315-329. 29. Lee, C. Y., Chang, C. C. & Chen, Y. M. (1972) J. Formosan 8. Lee, C. Y. (1970) Clin. Toxicol. 3,451-472. Med. Assoc. 71,344-349. Downloaded by guest on September 26, 2021