Eel Electroplax Research Articles

The electroplax sodium channel revealed

The recent publication of the complete amino acid sequence of the sodium channel from eel electroplax as inferred from the nucleotide sequence of cDNA clones by Noda et al. 12 is a landmark in studies of the molecular properties of voltage-sensitive ion channels and provides an appropriate occasion for a brief progress report on the emerging molecular view of the sodium channel and an indication of likely future directions. This work can be viewed as inaugurating a new phase of research on the molecular properties of ion channels in which the molecular architecture of their functional components is elucidated at ever finer resolution.

Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier

Monoclonal antibodies were generated against native eel electroplax sodium channels in their natural membrane. These antibodies block nerve conduction in rat central (optic) and peripheral (sciatic) nerve. The antibody binding to eel electroplax membrane fragments and to rat brain synaptosomes can be modulated by neurotoxins. Thus it implies that active sites of the sodium channels are immunogenic in their natural membrane. Unlike the antibodies described in the past, our antibodies recognize antigenic determinants which are associated with the physiological activity of the channel and have been conserved through evolution.

A comparison of EEL electroplax and snake venom acetylcholinesterase

1. The effects of some cholinergic ligands, harmala alkaloids and local anaesthetics on the activity of eel electroplax and Naja naja siamensis venom acetylcholinesterase have been studied. In most cases, eel electroplax was found to be more susceptible towards inhibition than the venom acetylcholinesterase. 2. No major difference was observed with respect to the type of inhibition in both enzymes. 3. The activation of the two enzyme preparations by inorganic cations (Ca 2+, Mg 2+ and Na +) showed a similar pattern. In both preparations, the onset of activation was detectable at much lower concentration with the divalent metal ions than with the monovalent Na + 4. Antagonism between Ca 2+ and decamethonium, tubocurarine and tetracaine in both enzymes approached competitive kinetics. 5. The onset of substrate inhibition is delayed by Ca 2+ (30 mM) in both enzymes. It is suggested that the Ca 2+ binding site overlaps with the substrate inhibitory site. 6. It is concluded that cobra venom acetylcholinesterase has similar allosteric binding sites to those of eel electroplax.

Immunochemical properties and cytochemical localization of the voltage- sensitive sodium channel from the electroplax of the eel (Electrophorus electricus)

Immunochemical methods have been used to investigate questions concerning the relatedness of sodium channels from different sources and their distribution in the eel electroplax. The reagents employed were two monoclonal antibodies, 5D10 (Moore, H. -P. H., L. C. Fritz, M. A. Raftery, and J. P. Brockes (1982) Proc. Natl. Acad. Sci. U.S.A. 79: 1673-1677) and 5F3, and a rabbit antiserum; all three were directed against determinants present on the 250,000-dalton component of the eel sodium channel. In quantitative adsorption assays, the three reagents were effectively adsorbed by eel electroplax membranes but not by brain membranes from rat, frog, or chick. The rabbit antiserum bound to immobilized membranes of rat brain at a level only approximately 0.1% of that seen with electroplax membranes. The reactivity of the three reagents with the eel electroplax was further investigated by indirect immunofluorescence on frozen sections. Whereas 5D10 showed no detectable reactivity, the rabbit antiserum and, especially, 5F3 stained the electrically excitable caudal face of the electrocytes but not the inexcitable rostral face. The reactivity of 5F3 was examined in greater detail and showed occasional abrupt discontinuities where the membrane was not stained. The presence of positive 5F3 immunoreactivity appeared to be correlated with extracellular filamentous material.

Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA.

Poly(A) RNA from eel electroplax was used to construct a full length cDNA complementary to calmodulin (CaM) mRNA which was cloned in the PstI site of pBR322 DNA. Recombinant plasmids containing sequences complementary to CaM mRNA were identified by hybridization using a 32P-labeled CaM cDNA (pCM109). Nucleotide sequence analysis reveals that the clone from the plasmid pCM116 contains a 5' nontranslated region of 26 nucleotides, the entire coding region, and a 3' nontranslated region of 408 nucleotides. The amino acid sequence deduced from the nucleotide sequence is similar to those previously reported for CaM from other species. Comparison between the nucleotide sequence of the functional domains of the protein shows extensive homology between all four domains. pCM116 was utilized to determine and compare the populations of RNA present in different tissues. In eel electroplax, three species of cytoplasmic RNAs at 820, 1100, and 2000 nucleotides hybridize to the cDNA probe. The nucleus contains an additional CaM RNA molecule of 5500 nucleotides which may represent a primary transcript of the calmodulin gene. Sequence analysis of the 3' noncoding region of pCM116 reveals 3 possible polyadenylation sites (AATAAA) at positions 573, 580, and 855. The mRNA of 820 nucleotides was derived by polyadenylation at the first site whereas the mRNA of 1100 nucleotides was derived by poly(A) addition at position 855. These data are compatible with the idea that at least 2 of the 3 CaM mRNAs in eel electroplax tissue are derived from a single nuclear transcript by differential polyadenylation during processing.

Ion-gated channel induced in planar bilayers by incorporation of (Na+,K+)-ATPase.

An ion-gated channel was conferred on a planar lipid bilayer membrane upon incorporation of (Na+,K+)-ATPase. The channel exhibited two conductance states. The high conductance state was only observed when an ion gradient was present across the planar membrane. This state corresponded to an enzyme conformation which was ouabain and vanadate sensitive (i.e. conductance was inhibited by these compounds), while the low conductance state showed no sensitivity to either inhibitor. Single channel conductance behavior was observed when minimal amounts of enzyme were incorporated into the planar bilayer. The observed single channel conductance was 270 +/- 14 picosiemens. Similar transport behavior was observed for enzyme purified from ovine kidney using sodium dodecyl sulfate (anionic), eel electroplax using Lubrol-WX (nonionic), and kidney microsomes. In addition, the data strongly suggest that enzyme from the kidney microsomes was asymmetrically incorporated into the planar bilayer.

Use of a monoclonal antibody to purify the tetrodotoxin binding component from the electroplax of Electrophorus electricus.

The tetrodotoxin binding component of the voltage-sensitive sodium channel from Electrophorus electricus electroplax was purified by using a monoclonal antibody. An impure preparation of tetrodotoxin binding component was mixed with the pure monoclonal antibody, and the immune complex so formed was isolated by affinity chromatography on a protein A-Sepharose column. Excess antibody was removed by ion-exchange chromatography. The purified material has a specific activity of over 1,800 pmol of [3H]tetrodotoxin bound per mg of protein. By assuming that the immune complex has a stoichiometry of 1:1, this specific activity then represents an actual specific activity of 3,000 pmol of [3H]tetrodotoxin bound per mg of eel electroplax protein, or 75% of the theoretical specific activity expected for a pure toxin binding component of Mr 250,000. The peptide composition of the purified material was simple with the predominant species present being of Mr approximately equal to 250,000. Minor components were also present with Mrs of approximately equal to 95,000, approximately equal to 44,000, and approximately equal to 23,000.

Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.

The tetrodotoxin binding protein, a major component of the Na+ channel, has been purified from the electric organ of the South American eel Electrophorus electricus. Antibodies to this protein were raised in rabbits and their specificity was demonstrated by a highly sensitive radioimmunoassay and by immunoprecipitation procedures. These antibodies were used to examine the distribution of the binding protein in the eel electroplax membranes and along myelinated nerve axons. The distribution of the antigen was determined by using the peroxidase-antiperoxidase technique at both the light and electron microscopic levels. In the electrocytes of the electric organ, only the innervated face showed staining in experimental material. The stained regions of electroplax plasmalemma included the caveolae of the innervated surface while caveolae of the non-innervated surface did not stain. Thus, the innervated surface including caveolae exclusively contains the Na+ channels. Along myelinated axons, staining was limited to the nodal zone of the node of Ranvier. The paranodal and internodal zones did not stain for the binding protein. Limited diffusion of primary IgG and subsequent reactants into the paranodal and internodal sites was eliminated as a possible source of focal staining at nodes because mechanically demyelinated preparations also exhibited focal nodal staining. Thus, this tetrodotoxin binding protein component of the Na+ channel is located solely within the nodal zone of the node of Ranvier.

Partial purification and characterization of (Na+ + K+)-ATPase from garfish olfactory nerve axon plasma membrane.

The (Na+ + K+)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na+ + K+)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na+ + K+)-ATPase, such as the Km for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme. The partially purified garfish (Na+ + K+)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent 22Na+ transport. The ratio of 22Na+ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na+ + K+)-ATPase, however, gave a 22Na+ pumped to ATP hydrolyzed ratio of nearly 3.

Molecular weight and stoichiometry of the sodium- and potassium-activated adenosine triphosphatase subunits.

The molecular weight and stoichiometry of the subunits of the sodium- and potassium-activated adenosine triphosphatase. (Na,K)-ATPase, have been examined in four highly purified preparations of this enzyme: dog kidney, eel electroplax, dogfish rectal gland, and brine shrimp nauplius. The molecular weights of the subunits were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10 different total acrylamide concentrations (%T) and by a gradient SDS-PAGE system, standardized with 16 molecular weight marker proteins of known primary structure. The molecular weight of the large (Na,K)-ATPase subunit (alpha subunit) was found to be reliably estimated by SDS-PAGE, whereas the small subunit (beta subunit) was not. The molecular weight of the alpha subunit was 97,000, 97,700, 104,200, and 97,800 for the dog, eel, dogfish, and brine shrimp (Na,K)-ATPase, respectively. The molecular weight of the beta subunit is overestimated by SDS-PAGE. Mass ratio analysis of the alpha and beta subunits separated by SDS-PAGE was determined by three techniques: total amino acid quantitation, gel scanning and dye elution of Coomassie blue-stained gels, and UV gel scanning. The former method, which provides the most reliable estimate of the mass ratio, gives an alpha/beta mass ratio of 2.41, 2.33, 2.91, and 2.44 for the dog, eel, dogfish, and brine shrimp preparations, respectively. Results of the mass ratio studies, the molecular weight analysis, and prior cross-linking studies clearly demonstrate that the quaternary structure of the (Na,K)-ATPase is an alpha 2 beta 2 tetramer in all four species. Based on all of the above data, a more reliable molecular weight estimate of the beta subunit is calculated as 40,200, 43,000, 35,800, and 40,100 for the dog, eel, dogfish, and brine shrimp preparations, respectively. These results now confirm that the molecular weight of the protein portion of the alpha subunit is near 100,000 and that of the beta subunit is near 40,000 and that the (Na,K)-ATPase holoenzyme is an alpha 2 beta 2 tetramer of molecular weight 274,000 to 280,000, regardless of species.

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP.

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.

Studies on (Na + + K +)-activated ATPase XLVII. Chemical composition, molecular weight and molar ratio of the subunits of the enzyme from rabbit kidney outer medulla

1. (1) The subunits of a purified preparation of (Na + + K +)-ATPase from rabbit kidney outer medulla have been completely separated by gel filtration in sodium dodecyl sulfate. During the gel filtration procedure 3–4% of the phospholipids present in the purified enzyme preparation remain bound to the separated subunits even after repeated gel filtration. 2. (2) The composition of the bound phospholipids does not greatly differ from that in the original enzyme preparation, which indicates that there is no specific binding of phospholipids to the subunits. The isolated β-subunit contains twice as much phospholipid per mg protein as the α-subunit. 3. (3) The α-subunit is more hydrophobic (43%) in its amino acid composition than the β-subunit (38%). The α-subunit contains more alanine and histidine and much less tyrosine and lysine than the β-subunit; the other amino acids show little difference. The amino acid composition of each subunit closely resembles that of the corresponding subunit of the enzyme from dog and lamb kidney, duck salt gland, shark rectal gland and electric eel electroplax. 4. (4) Both subunits are glycoproteins, although the β-subunit contains 5-times more carbohydrate per g protein than the α-subunit. Both subunits contain glucosamine, galactose, mannose, sialic acid and also glucose, which appears to be covalently bound. Galactosamine, in small amounts, is only detected in the β-subunit.

Immunocytochemical Localization of (Na+,K+)‐ATPase in the Goldfish Optic Nerve

Antiserum to the catalytic subunit of goldfish brain (Na+, K+)-ATPase has been employed at the electron microscopic level by means of the peroxidase-antiperoxidase immunohistochemical method. In optic nerve, antigenic sites are restricted to the nodes of Ranvier. No reaction product is detected in underlying internodal neurolemma. Outgrowing neurites for cultured retinal explants devoid of glial ensheathment exhibit a continuous distribution of the enzyme subunit. Antibodies against eel electroplax (Na+, K+)-ATPase cross-react with the goldfish brain enzyme and show a similar immunocytochemical distribution pattern.

Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A 2 from Naja nigricollis snake venom and less toxic phospholipases A 2 from Hemachatus haemachatus and Naja naja atra snake venoms

Modification of one histidine residue at the active site in the phospholipases A 2 from N. nigricollis, H. haemachatus and N. naja atra snake venoms reduced the enzymatic activity to 0·–0·8% that of the native species, while the Km values for the first two enzymes listed remained practically unchanged. Also unchanged following modification were the characteristic substrate specificities of the three enzymes. While it appears most likely that the residual activity of the modified enzymes originates from contamination by non-modified species, the modified enzyme might have residual activity of its own. The decrease in enzymatic activity was accompanied by a decrease in all other biochemical and physiological parameters studied such as: direct hemolysis (for N. nigricollis phospholipase), ability to hydrolyze red cell phospholipids, intravenous ld 50 in mice, intraventricular ld 50 and cd 50 in rats, brain phospholipid hydrolysis and ability to block action potentials in eel electroplax. The results support the view that catalytic activity is essential for toxic action.

The large subunit of (sodium + potassium)-activated adenosine triphosphatase from the electroplax of [formula omitted] is a glycoprotein

The large subunit of Na,K-ATPase purified from eel electroplax was found to contain amino sugars, neutral sugars and sialic acid. The concentration of these carbohydrates in the large subunit was 5–10% of that found in the smaller subunit and in total accounts for 2.6% of the mass of the large subunit. The periodic acid-Schiff's stain for glycoproteins on polyacrylamide gels is apparently not sufficiently sensitive to detect glycoproteins with these low levels of carbohydrates.

Modification of the (Na + + K +)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na + + K +)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl ( k 0.5 ca. 0.6 m m) or ATP ( K 0.5 ca. 1 μ m) in the medium protected against inactivation, whereas MgCl 2 ( k 0.5 ca. 1 m m) increased inactivation. K +-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na +-dependent phosphorylation of the enzyme and Na +-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k 0.5 for K +-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k 0.5 for neither K + nor Na + was increased, although the apparent k m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K + at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a p K a for the reactive enzyme groups of 7.5–8; and TNBS affected K +-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K + at these sites.

Biosynthesis and insertion of (Na+ + K+)-adenosine triphosphatase subunits into eel electroplax membranes.

Biosynthesis and insertion of (Na+ + K+)-adenosine triphosphatase subunits into eel electroplax membranes.

The presence of two ( [formula omitted] inhibitors in equine muscle ATP: Vanadate and a dithioerythritol-dependent inhibitor

A potent inhibitor of (Na + + K +)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V 2O 5 inhibit sarcolemma (Na + + K +)-ATPase with an I 50 of 1 μM in the presence of 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N′- tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl 2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg 2+. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second (Na + + K +)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg 2+ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na + + K +)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na + + K +)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg 2+-ATPase, Ca 2+-ATPase and (Ca 2+ + Mg 2+)-ATPase .

Anthroylouabain: a specific fluorescent probe for the cardiac glycoside receptor of the Na-K ATPase.

Anthroylouabain (AO) was synthesized by reaction of anthracene-9-carboxylic chloride with ouabain. Nuclear magnetic resonance spectroscopy of AO suggests that the anthracene is esterfied to the rhamnose in the glycoside. AO inhibits Na-K ATPase from human red cells, eel electroplax and rabbit and dog kidney with a KI less than 1muM. AO bound to rabbit or dog kidney Na-K ATPase shows enhanced fluorescence and characteristic spectral shifts. AO binding requires Mg and is optimum in the presence of Mg + Pi or MgATP + Na; ouabain prevents AO binding and fluorescence enhancement if added before AO or reverses it if added after AO is bound. Na inhibits AO binding in the presence of Mg + Pi and K inhibits it in the presence of MgATP + Na. AO binding and dissociation rate constants measured by fluorescence agree qualitatively with reported measurements for ouabain, using other methods, although AO shows faster kinetics than ouabain. Dissociation constants obtained from kinetic measurements are 1.5 X 10(-7) and 1.8 X 10(-7) M for the MgATP + Na complex and Mg + Pi complex, respectively. KD from fluorescence titrations is 2.3 X 10(-7) M for the latter. The enzyme has 2-2.5 nmol of AO binding sites/mg of protein. No differences in the fluorescence parameters of the Mg + Pi or MgATP + Na complexes were observed, suggesting that the same enzyme conformation binds AO under both ligand conditions. Comparison of the AO fluorescence parameters in the enzyme with those of model systems suggests that the binding site is hydrophobic and/or viscous and shielded from H2O. The results indicate that AO is a specific fluorescent probe of the cardiac glycoside receptor of the Na-K ATPase. Possible applications are discussed.

Experimental autoimmune myasthenia gravis: a sequential and quantitative study of the neuromuscular junction ultrastructure and electrophysiologic correlations.

Neuromuscular junction ultrastructure in rat forelimb digit extensor muscle was sequentially and quantitatively investigated in experimental autoimmune myasthenia gravis (EAMG). Experimental animals were immunized with highly purified eel electroplax acetylcholine receptor protein plus complete Freund's adjuvant and B pertussis vaccine; control animals received only adjuvant and vaccine. During the first 7 days (latent period) after immunization end-plate structure and neuromuscular transmission remained normal in the experimental group. Between day 7 and 11 (acute phase) mononuclear cells infiltrated those regions of muscle where the end-plates were located and there was intense degeneration of the postsynaptic regions with splitting away of abnormal junctional folds from the underlying muscle fibers. Macrophages entered the gaps thus formed and removed the degenerating folds by phagocytosis. The nerve terminals were displaced from their usual location but maintained their structural integrity. Neuromuscular transmission was blocked in many muscle fibers. Miniature end-plate potentias (MEPPs), detectable in only a few fibers, were of abnormally low amplitude. After day 11 (chronic phase) the nerve terminals returned to the highly simplified postsynaptic folds became reconstituted and again degenerated. Immature junctions with poorly differentiated postsynaptic regions and nerve sprouts near end-plates were also observed. In two animals relapsing during the chronic phase degeneration of the postsynaptic folds was more intense than in the other chronic-phase animals. The posysynaptic membrane length and length per unit area and the MEPP amplitudes were significantly decreased in all chronic phase animals and the decreases were greater in the relapsing than in the non-lapsing animals. Minor morphometric alterations were also observed in the nerve terminals. These might have been secondary to the postsynaptic changes. The postsynaptic region is the primary target of the autoimmune reaction in EAMG. The ultrastructural, morphometric and electrophysiological abnormalities of the end-plate in chronic EAMG resemble those which have been observed in human myasthenia gravis.