Electroplax

Radioiodinated derivatives have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data). In membrane preparations from Torpedo electroplax, photoactivatable azidosalicylate derivatives of a-conotoxin GIA preferentially label the p and 7 subunits of the acetylcholine receptor. However, when the photoactivatable derivative is cross-linked to detergent solubilized acetylcholine receptor (AChR), only the 7 subunit is labeled. Since snake a-neurotoxins mainly bind to the a subunits of AChR and a-conotoxins compete directly with a-bungarotoxin, the cross-linking results above are both intriguing and problematic. 

In the following the Cl -channels will be subdivided into those of the central nervous system, of muscle and Torpedo electroplax, of apolar non-excitable cells and of epithelia. 

The acetylcholine receptor (AChR) of Torpedo electric organ is also a PCP "receptor." However, this nicotinic AChR has about one-tenth the affinity for PCP than that of the rat brain PCP receptor [K0.5 = 0.3 pM, versus = 4-6 pM for Torpedo (Heidmann et al. 1983 flaring et al. 1984)]. Moreover, the nicotinic AChR has subunits of MR<66 kD, and these are the subunits that are specifically labelled with 3H-Az-PCP in the Torpedo electroplax membranes (Heidmann et al. 1983 Haring and Kloog 1984 Haring et al. 1984). These data indicate that the nicotinic AChR-PCP receptor differs from the rat brain PCP receptor. Furthermore, our findings are... 

Most of the above studies have been made possible because of the high density of nAChRs in the Torpedo electroplax membrane. However, with recent technological developments, it may be hoped that similar approaches will prove useful when applied to receptors of much lower abundance. 

The isolation of the nicotinic acetylcholine receptor glycoprotein was achieved almost simultaneously in several laboratories (those of Changeux, O Brien, Brady, and Eldefrawi) and was helped tremendously by the discovery that the electric organ (elec-troplax) of the electric eel (Electrophorus electricus, an inhabitant of the Amazon River) and related species, as well as the electroplax of the electric ray Torpedo tnar-morata) of the Atlantic Ocean and the Mediterranean Sea, contains acetylcholine receptors (AChR) in a much higher concentration than, for instance, in human neuromuscular endplates or brain tissue. 

W. A. CatteraU (1985). The electroplax sodium channel revealed. Trends Neurosci. 8 39-41. 

Disubstituted piperidines (and piperideines) from ants have insecticidal activity and, as venom constituents, serve the ant in a defensive, repellant function (149). 2,6-Disubstituted piperidines are potent noncompetitive blockers of nicotinic receptor-channels in neuromuscular preparations (162) and in electric eel electroplax (163). Synthetic 241D was a potent noncompetitive blocker of nicotinic receptor-channels in both electric eel electroplax and pheochromocytoma cells (64). 

In any case, it is advisable not to use too many parameters in Equation 3. Unless the number of compounds used in the regression is large so that the degrees of freedom are sufficient, a statistically significant correlation would not be obtained. For example, the depolarizing activity of five quaternary ammonium ions against electric eel electroplax (63) is expressed by Equation 30 with the sum of the substituent constants of four N-substituents (11). 

The availability of purified protein enabled Shosaku Numa and coworkers to clone and sequence the cDNA for the sodium channel from the electroplax cells of the eel electric organ and then from the rat. Subsequently, a large number of sodium channel cDNAs have been cloned from other sources, and sequence comparisons have been made. 

Eldefrawi and Eldefrawi [98] reported the purification of the acetylcholine of Torpedo electroplax on an affinity column consisting of cobra (Naja naja siamensis) toxin coupled to Sepharose 4B. Desorption with 10 mM benzoquinonium produced a protein that bound [ I]a-bungarotoxin but not [ H]acetyl-choline. However, desorption with 1 mM carbamylcholine gave a receptor protein that bound pH]acetylcholine decamethonium, [ H]nicotine [ C]dimethyl-(-l-)-tubocuranrine, and [ I]a bungarotoxin. Schmidt and Raftery [99] also purified acetylcholine receptor, from Narcine, on a N-(e-aminohexanoyl)-3-aminopropyltrimethyl-ammonium bromide-HBr-agarose column. 

Traylor and Singer (1967) have provided a detailed synthesis of the diazonium salts XIX and XX. Compound XIX has been used as an affinity label for acetylcholinesterase on erythrocyte membrane (Wofsy and Michaeli 1967) and for the cholinergic receptor from eel electroplax (Changeux et al. 1967). XX has been used for the modifica-... 

Holothurin" (2x10" M) irreversibly blocks a neural and direct response of stimulated monocellular electroplax preparation of Electrophorus electricus [86]. It also produced irreversible depolarization in a monocellular electroplax preparation of Electrophorus electricus [86]. These effects on the resting potential have been attributed to an initial efflux which then decreased steadily. 

EC 3.1.1.7). Replacement of the acetate oxygen of ACh by sulfur yielding the thiol ester analog acetylthiocholine (ASCh, 4468-05-7) profoundly modifies the biological action of this molecular ion (1,2, 2). The relative ability of these isosteres to incTuce polarization in isolated single cell electroplax preparations is 1.0 16.7 ( ). However, the relative rate at which these isosteres are hydrolyzed by AChE is very similar, 1.0 0.6 (5). 

Mapping studies of acetylcholinesterase have been conducted in the intact electroplax with a series of inhibitory 1-methyl-hydroxyquino-linium compounds. The presence of a hydroxyl group located about 5A. from the quaternary nitrogen (as in l-methyl-7-hydroxyquinolinium iodide), increases the inhibitory strength more than a hundredfold. 

Returning to the topic of ACh receptors, preliminary three-dimensional models are available for this receptor from Torpedo electroplax (S8,j5 ). Figure 5 shows the possible structure of the ACh binding site and the residues likely to be involved in ACh binding. Considerable progress can be expected in developing the structure of the comparable receptor from the locust CNS (60). 

Because altered sodium channels have been implicated in kdr and kdr-like resistance phenomena in insects, basic research on the biochemistry and molecular biology of this molecule, which plays a central role in normal processes of nervous excitation in animals, is of immediate relevance. The results of recent investigations of the voltage-sensitive sodium channels of vertebrate nerves and muscles have provided unprecedented insight into the structure of this large and complex membrane macromolecule. Sodium channel components from electric eel electroplax, mammalian brain, and mammalian skeletal muscle have been solubilized and purified (for a recent review, see Ref. 19). A large a subunit (ca. 2 60 kDa) is a common feature of all purified channels in addition, there is evidence for two smaller subunits ( Jl and J2 37-39 kDa) associated with the mammalian brain sodium channel and for one or two smaller subunits of similar size associated with muscle sodium channels. Reconstitution experiments with rat brain channel components show that incorporation of the a and pi subunits into phospholipid membranes in the presence of brain lipids or brain phosphatidylethanolamine is sufficient to produce all of the functional properties of sodium channels in native membranes (AA). Similar results have been obtained with purified rabbit muscle (45) and eel electroplax (AS.) sodium channels. 

Several studies employing oocytes of the clawed frog, Xenopus laevis, for the in vitro translation of sodium channel encoding mRNAs (53-55) suggest that this experimental system may be particularly useful toward this end. The biophysical properties of sodium channels expressed in oocytes following injection of rat brain mRNA were similar to those of sodium channels in their native membrane environment, and were specifically inhibited by the sodium channel blockers tetrodotoxin and saxitoxin (i5.). Sodium channels encoded by mRNAs from rat skeletal muscle and eel electroplax have also been expressed in Xenopus oocytes (56-57). To date the expression of insect sodium channels in the Xenopus oocyte has not been reported, but the utility of this system for the translation and expression of insect acetylcholine receptor mRNA has recently been demonstrated (58). Successful application of this methodology to the expression of insect mRNAs encoding functional sodium channels offers a novel method to test some of the hypotheses for the molecular basis of the kdr mechanism. 

Calmodulin, by contrast, is distributed throughout most, if not all, eukaryotic cells from both animal and plant sources. It has not been reported to exist in bacteria. Calmodulin varies in concentration from tissue to tissue with mammalian brain (4) and testis (5) and the electroplax of Electrophorus electricus (6) possessing particularly high content. While the protein has been found to be predominantly cytoplasmic in subcellular fractionation studies, substantial amounts are particulate-associated as well. Binding of calmodulin to particulate fractions is increased by Ca2+, appears to occur at specific sites (7, 8), and... 

Cholinergic Systems Direct and Model Observations. Since the background of this subject has recently been covered elsewhere (27), only some of the important new highlights are discussed here. These concern the ACh receptor of electroplax tissue, of the mouse diaphragm and the frog toe muscle, especially in relation to AChE as a model receptor. 

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