Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: I. Pharmacological properties of the electroplaque

The membrane potential (average = ?52 mV) of a freely exposed electroplaque from a dissected prism of Torpedo marmorata electric organ is recorded with an intracellular glass microelectrode. The resting potential decreases with external potassium concentration. Acetylcholine (in the presence of O,O′-diethyl S-(β-diethylamino)ethyl phosphorothiolate), decamethonium, phenyltrimethyl-ammonium and carbamylcholine added to the bath cause a decrease of membrane potential, i.e. behave as agonists. Their effect is blocked in a competitive manner by d-tubocurarine, gallamine and hexamethonium, and in a non-competitive way by prilocaine; 1 μg Erabutoxin/ml completely abolishes the response to carbamylcholine. The apparent dissociation constants for seven cholinergic ligands are determined from the dose-response curves, and found to be closely related to those previously determined with Electrophorus electricus electroplaque with, however, a few differences. During these experiments it was noticed that potassium ions affect, in a differential manner, the response of T. marmorata electroplaque to carbamylcholine and decamethonium.     

Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents

Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43 000-Mr, 50 000-Mr and 66 000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40 000-Mr and 43 000-Mr chains. The microheterogeneity of the 43 000-Mr chain, but not that of the 40 000-Mr chain, might result from a selective phosphorylation of this particular chain.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state

Quinacrine, like a typical local anaesthetic, blocks the response of Electrophorus electricus electroplaque in vivo in a non-competitive manner and enhances, in vitro, the affinity of the cholinergic receptor present in Torpedo marmorata membrane fragments for acetylcholine. The interaction of quinacrine with T. marmorata membrane fragments can be followed by differential fluorescence spectroscopy either upon direct illumination (λ_Ex = 350 nm) or by energy transfer from membrane proteins (λ_Ex = 290 nm). Carbamylcholine and most of the cholinergic ligands tested cause an increase of the light intensity emitted by membrane-bound quinacrine under conditions of direct excitation; all these effects are blocked by a preincubation of the membrane fragments with the α-toxin from Naja nigricollis. When quinacrine is excited by energy transfer, carbamylcholine, phenyltrimethylammonium and hexamethonium cause an increase of fluorescence but flaxedil, tetraethylammonium and the α-toxin give a much smaller fluorescence increase or none.Local anaesthetics like prilocaine or quotane cause a decrease of fluorescence intensity of membrane-bound quinacrine in both the presence and absence of carbamylcholine. Quantitative studies on quinacrine binding and fluorescence as a function of quinacrine concentration reveal at least two populations (saturable and non-saturable) of binding sites, the saturable one being identical or closely related to the specific site of action of local anaesthetics. It is concluded that binding of cholinergic ligands primarily increases the quantum yield of a fraction of bound quinacrine.The curves of variation of fluorescence intensity with agonist and antagonist concentrations determined under conditions of direct illumination, closely resemble the binding curves determined at equilibrium with radioactive ligands. Under these conditions quinacrine therefore enables us to determine the occupancy of the receptor site by cholinergic ligands. On the other hand, the change of quinacrine fluorescence observed by energy transfer, which takes place with some of the cholinergic ligands but not with others, and does not correlate with any variation of the intrinsic fluorescence of membrane proteins, most likely reflects a change of structure bearing a qualitative relationship to the pharmacological activity of the tested ligands.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: V. Qualitative correlation between pharmacological effects and equilibration processes of the cholinergic receptor protein as revealed by the structural probe quinacrine

When the differential fluorescence emission at 515 nm of receptor-rich membrane fragments from Torpedo marmorata labelled with quinacrine is followed by energy transfer, addition of a high concentration of an agonist such as 0.4 mm-carbamylcholine or 0.4 mm-phenyltrimethylammonium causes a fast (unresolved) increase of fluorescence intensity followed by a slow (minute range) decrease, which leads to a final stable level. On the other hand, a stepwise addition of agonist at low concentrations gives only a slow fluorescence increase. Antagonists, such as flaxedil and d-tubocurarine, at all the concentrations tested, bring about exclusively slow fluorescence increases. Decamethonium at 0.4 mm gives no slow reaction but only a fast (unresolved) increase without transient overshoot.Addition of a local anaesthetic reduces the amplitude of the fluorescence response to carbamylcholine. The α-toxin from Naja nigricollis does not cause any change of fluorescence intensity but blocks the effect of the cholinergic agonists and antagonists.Dissolution of the membrane fragments by a non-ionic detergent abolishes the fast transient reaction triggered by the agonists but preserves the slow ones observed in the presence of agonists or antagonists. The data are interpreted in terms of a three-state model, a revised version of the model of Katz & Thesleff (1957): the fast transient reaction brought about by the agonists being assigned to the “activation” of the receptor-ionophore complex and the slow one to its “desensitization”.     

Isolation of pure cholinergic nerve endings from the electric organ of Torpedo marmorata.

A rapid method for the preparation of highly purified cholinergic nerve endings from the electric organ of Torpedo is described. The endings retain their cytoplasmic components, as shown by biochemical and morphological observations. The homogeneity of these synaptosomes make them a useful tool for further studies.     

Biosynthesis of acetyl-coenzyme A in the electric organ of Torpedo marmorata in relation to acetylcholine metabolism.

Formation of acetyl-CoA through acetyl-CoA synthetase (forward reaction) and through choline acyltransferase (backward reaction) was investigated in tissue extract from the electric organ of Torpedo marmorata. When the tissue extract was submitted to gel filtration on Sephadex G-25, the formation of acetyl-CoA by acetyl-CoA synthetase appeared fully dependent on ATP and CoA and partially dependent on acetate (an endogenous supply of acetate is discussed). Choline acetyltransferase was a potent source of acetyl-CoA, only requiring acetylcholine and CoA, and was much more efficient than acetyl-CoA synthetase for concentrations of acetylcholine likely to be present in nerve endings.     

A Raman spectroscopic investigation of the lipid state in acetylcholine receptor-rich membranes from Torpedo marmorata.

The lipid state in acetylcholine receptor (AcChR)-rich membranes purified from electric organ of Torpedo marmorata was studied in the temperature interval from 0 degrees C to 35 degrees C using the (C-H) stretching and (C-C) skeletal optical vibrations. The Raman spectra of AcChR-rich membranes, recorded immediately after preparation of the samples, indicate that the lipids are in a predominant triclinic crystalline lattice and do not undergo a phase transition when the temperature increases up to 35 degrees C. However, the polar groups of the lipids appear subject to temperature-induced variations. After extraction of 43-kd and other non-receptor proteins, spectra indicate an order-disorder phase transition of lipids at approximately 21 degrees C. This transition appears less cooperative than the transition of the membrane lipid extract. The role of the proteins in preservation of the crystalline state of lipids in AcChR-rich membranes is discussed.     

Single channel gating events in tracer flux experiments. III. acetylcholine receptor-controlled Li+ efflux from sealed Torpedo marmorata membrane fragments

Filter assay measurements of Li+ efflux from acetylcholine receptor-containing vesicular Torpedo marmorata membrane fragments (microsacs) are presented. Techniques are introduced for: (a) inducing a complete emptying of the Li+ content of all microsacs containing one or more functionally intact receptors, and (b) for determining the distribution of internal volumes of the microsacs using filtration with membrane filters of different pore sizes. The flux amplitudes resulting for acetylcholine receptor-controlled Li+ efflux, when receptors are inhibited by alpha-bungarotoxin or inactivated by a neuroactivator-induced desensitization process, were measured. Amplitude analysis was used to determine characteristic parameters of the microsacs that may vary with the technique of preparation (e.g., the distribution in size and receptor content), as well as the mean single channel flux amplitude contribution (e-kt)infinity, which represents the mean reduction of the Li+ content of a microsac due to efflux from a single receptor-controlled channel closing due to inhibition or inactivation of the receptor. The ratio keff/ki was found to lie in the range 0.1 less than keff/ki less than 0.5, where keff and ki are, respectively, the rate constant for Li+-Na+ exchange flux and for the slow inactivation reaction mode of the acetylcholine receptor induced by carbamoylcholine at high concentrations.     

The action of botulinum toxin on cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. Detection of a putative toxin receptor

Botulinum neurotoxin type A (BoNTx) inhibits the release of acetylcholine (ACh) from Torpedo electric organ synaptosomes. We have studied several biochemical and morphological aspects in order to characterize the molecular interactions of BoNTx intoxication in our preparation. 1. We are describing for the first time an electrophoretic band from cholinergic presynaptic plasma membrane (PSPM) that is recognized by 125I-BoNTx as a putative BoNTx receptor. 2. Furthermore we describe direct interaction of botulinum toxin-gold complexes with synaptic vesicles through the three-step model of the BoNTx intoxication.     

Studies on the structure of the acetylcholine receptor from Torpedo marmorata

The purified acetylcholine receptor of Torpedo marmorata has been characterized by sedimentation velocity measurements on dilute solutions using an ultracentrifuge and scanner. Several preparations were studied and all exhibited sedimentation coefficients in the vicinity of 24S. In a number of experiments the receptor could be resolved into two sedimenting boundaries of 18S and 26S, corresponding to minimum molecular weights of about 5 × 10⁵ and 10⁶, respectively. Additions of sodium dodecyl sulfate or Triton X-100 resulted in marked decreases in sedimentation coefficient, while treatment with Lubrol-WX had only a slight effect on the S values. Small changes in S_20,w were produced by guanidine hydrochloride alone, although addition of dithiothreitol with 6 M guanidine hydrochloride resulted in an 8.8S component. Electrophoresis in sodium dodecyl sulfate gave one principal band with a molecular weight of 46,000.     

Interaction of the acetylcholine (nicotinic) receptor protein from Torpedo marmorata electric organ with monolayers of pure lipids

Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmorata. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long-chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentration of sodium cholate lowered by dialysis to 0.01% (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film. Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long-chain phosphatidylcholines than for short-chain ones. The degree of unsaturation and fluidity of the 3-sn-phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non-covalently labelled receptor preparations showed that part of the protein recovered with the film lost its alpha-toxin binding ability during the penetration. Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

Development of the electromotor system in Torpedo marmorata: Cationic staining of the electric organ

Summary The electric organs of embryonic Torpedo marmorala have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neuntes during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.     

Ultrahistochemical localization of adenylate cyclase activity in the electric organ of Torpedo marmorata

The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synaptosomes of Torpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5'-adenylyl imidodiphosphate as substrate and 5'-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of lead pyrophosphate precipitate.