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 skel- etal 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 A1 N 4-u-mm-- - 0 M A R ' 4"i b _ ;.* S B1 ~~~10 K l _~~~~~b,_ -As A2 1-.0- ~~ B AT~~ ~~~:# p,,_ - R NW .St~~~~~~~~Mo ..'V: - F ar 'T, G $ { H E T L D An..ads}~~~~~4- lol2 3Xf_?so_ d A3 A- * .