Mechanisms of bioelectric activity in electric tissue. I. The response to indirect and direct stimulation of electroplaques of Electrophorus electricus

1. A preparation is described consisting of one or several layers of innervated cells of the electric organ of Electrophorus electricus. 2. Each plaque is multiply innervated and only at its caudal face. The nerve fibers may derive from two or more different nerve trunks. 3. During activity the innervated face becomes negative relative to the non-innervated. 4. The first electrical response of the cell to an increasing neural volley is graded and has the character of a prepotential. At a critical size of the prepotential the cell discharges with an all-or-nothing spike. 5. Both responses have durations of about 2 msec. 6. A neural volley which does not cause the spike discharge facilitates the discharge of the cell by a second subsequent volley in the same nerve (temporal facilitation). 7. The period of facilitation lasts ca. 900 msec. During the first 100 msec., the facilitation is large enough to cause a spike. In the later portion only the prepotential is facilitated. No electrical concomitant has been detected. 8. Neural volleys reaching the plaque from different trunks interact at the cell to produce a period of facilitation lasting only about 2 msec. This interaction is interpreted as spatial summation. 9. In a population of cells, simultaneous stimulation of 2 nerves causes a smaller discharge than the sum of the two isolated responses (occlusion). 10. Cells denervated for 7 weeks or more can be excited directly, but only by a current flow outward through the caudal face. 11. Weak direct stimulation causes a prepotential in the denervated plaque. On increasing the stimulus the prepotential increases to a critical size when a spike develops. The duration of both responses is about 2 msec. 12. The absolutely refractory period of the denervated cell is about 1.5 msec. and relative refractoriness lasts about 15 msec. 13. Direct stimulation causes slight facilitation lasting as long as 200 msec. 14. Repetitive stimulation of the nerve at low frequencies (2 to 3 per second) causes rapid "fatigue" of transmission. The denervated plaque, however, responds for several minutes to repetitive direct stimulation at high frequencies (25 per second).     

Chemical modification of electric eel acetylcholinesterase by tetranitromethane

The reaction of acetylcholinesterase (acetylcholinehydrolase, EC 3.1.1.7) with tetranitromethane has been studied. The reaction caused a decrease in enzyme activity as measured with the substrate acetylthiocholine under conditions where hydrolysis of the neutral substrate indophenyl acetate was unaltered. The inactivation of acetylcholinesterase by tetranitromethane was greatly accelerated by the quaternary oximes pyridine-2-aldoxime methyl nitrate or toxogonin, though not by other quaternary inhibitors tested and not by an aliphatic oxime. The enhanced inactivation by tetranitromethane in the presence of pyridine-2-aldoxime methyl nitrate was blocked by the enzyme inhibitor decamethonium.The oxime-induced inactivation of acetylcholinesterase by tetranitromethane was accompanied by significant changes in the immunological properties of the enzyme as demonstrated by complement fixation. The reaction also resulted in the disappearance of tyrosine and appearance of nitrotyrosine.     

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes

The effects of three cationic triarylmethane dyes - pararosaniline (PR), malachite green (MG), methyl green (MetG) - on electric eel AChE (eAChE) activity were tested at 25 degrees C, in 100mM MOPS buffer (pH 8) containing 0.125mM 5-5-dithio-bis(2-nitrobenzoic acid), 20-120muM acetylthiocholine and 0-20muM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K(i)=8.4+/-0.67, 1.9+/-0.51 and 0.27+/-0.017muM; alpha (competitive coefficient)=5.8+/-2.0, 4.8+/-1.8 and 2.7+/-0.32; beta (noncompetitive coefficient)=0, 0 and 0.20+/-0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.     

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.     

Kinetic properties of soluble and membrane-bound acetylcholinesterase from electric eel

Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5.     

Development of electrical excitability in embryonic neurons: Mechanisms and roles

Xenopus spinal neurons serve as a nearly ideal population of excitable cells for study of developmental regulation of electrical excitability. On the one hand, the firing properties of these neurons can be directly examined at early stages of differentiation and membrane excitability changes as neurons mature. Underlying changes in voltage-dependent ion channels have been characterized and the mechanisms that bring about these changes are being defined. On the other hand, these neurons have been shown to be spontaneously active at stages when action potentials provide significant calcium entry. Calcium entry provokes further elevation of intracellular calcium via release from intracellular stores. The resultant transient elevations of intracellular calcium encode differentiation in their frequency. Recent studies have shown that different neuronal subpopulations enlist distinct mechanisms for regulation of excitability and recruit specific programs of differentiation by particular patterns of activity. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 190–197, 1998     

Fine structural localization of acetylcholinesterase in electroplaque of the electric eel

The electroplaques composing the electric organ of the eel, Electrophorus electricus, have been utilized for the dual purpose of demonstrating the subcellular sites of acetylcholinesterase activity and as a model for comparison of the several cytochemical methods available. Fresh tissue and tissue fixed by immersion in formalin, hydroxyadipaldehyde, or glutaraldehyde was reacted with the Cu-thiocholine method, the Cu-ferrocyanide thiocholine method, or the thiolacetic acid (TAA) method using Pb, Ag, or Au as capture reagents. Controls were obtained by omission of substrate, or by addition to complete media of varying concentrations of different cholinesterase inhibitors. Reactions were run at 0–5°C at a pH range of 5.0–7.1 for 0.25 to 120 min. Regardless of the capture metal, the localization obtained with TAA as substrate was identical with that observed with acetylthiocholine, the majority of precipitate being deposited on or near the external innervated surface of the plaque and within the tubulovesicular organelles opening onto the innervated surface. Both of the thiocholine methods and the Pb-TAA method showed reaction product in synaptic vesicles of the nerve endings innervating the plaque which was uninhibitable by 10^-4 M physostigmine. All methods also showed some inhibitor-sensitive deposition of reaction product in the mucoid material forming the immediate extracellular environment of the innervated surface.     

Small molecular products of dealkylation in soman-inhibited electric eel acetylcholinesterase.

Product analysis of dealkylation in P(S)C(S)-soman-inhibited electric eel acetylcholinesterase (AChE) by GC-MS using the selected ion monitoring mode has been carried out. The instrument was calibrated with pure standards of 2,3-dimethyl-1-butene and 2, 3-dimethyl-2-butene in the gas phase and methylene chloride extracts of 2,3-dimethyl-2-butanol and 3,3-dimethyl-2-butanol from the aqueous phase. The dealkylation in soman-inhibited AChE at pH 5.0 +/- 0.2 and 25 degrees C produces close to 40% alkenes and 50-60% 2, 3-dimethyl-2-butanol. No 3,3-dimethyl-2-butanol could be detected to provide direct evidence of the intervention of a secondary carbenium ion in the reaction path. All the products of the reaction originate from a tertiary carbenium ion. These findings are in good agreement with the results of Michel et al. [(1967) Arch. Biochem. Biophys. 121, 29], which were obtained by countercurrent distribution of tritium-labeled products and their identification by scintillation counting. The early experiments were performed with the mixture of the four soman diastereomers, all labeled with tritium in Calpha.