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Abstract. a-Bungarotoxin, a polypeptide of mol wt 8000 purified from the venom of Bungarus multicinctus, blocks irreversibly and specificallythe excitation by cholinergic agonists on the isolated electroplax and on purified membrane fragments in vitro. The toxin also blocks the in vitro binding of decamethonium to a protein recently isolated from electric tissue. This observation strengthens our earlier conclusion that this protein is the cholinergic receptor macromolecule.At a glance, the most evident and simple manner to characterize and identify the physiological receptor of acetylcholine in an excitable membrane is to use compounds that are structurally analogous to acetylcholine and thus present a high affinity for the cholinergic receptor site. However, it is now well established that in most excitable membranes there exist several-distinct classes of sites, all of which are able to bind cholinergic ligands. Among them are, in addition to the physiological receptor site, the catalytic and allosteric sites of the enzyme acetylcholinesterase (AcChE).l The use of cholinergic ligands thus meets with a difficult problem of specificity.2Interestingly enough, in the course of the past few years it has been shown that certain toxins from snake venoms, although completely unrelated structurally to acetylcholine, nevertheless act much like curare. One of them is a-bungarotoxin (a-Bgt), a basic polypeptide of mol wt 8000, which has been extensively studied by Lee and his associates.3-5 a-Bgt is purified from the venom of an elapid snake from Taiwan (Bungarus multicinctus) and the purified toxin gives irreversible neuromuscular blocking effects. In addition, d-tubocurarine protects against the action of a-Bgt. From these findings Lee and Chang4 have concluded that a-Bgt combines irreversibly with the cholinergic receptor at the motor endplate.We report here some experiments carried out with purified a-Bgt and various preparations derived from the electric organ of Electrophorus electricus. It is shown that in vivo, with the isolated electroplax, a-Bgt irreversibly blocks the depolarization caused by bath application of carbamylcholine, a cholinergic agonist. The same effect is observed with purified electric-organ membrane fragments using the in vitro assay of Kasai and Changeux.6 In addition, in agreement with the early observation of Lee and associates, d-tubocurarine protects against a-Bgt blockade. Finally we tested a-Bgt on the binding of 1241

The volume change of sarcoplasmic retriculum vesicles was followed by measuring the light scattering intensity. When the salt concentration of the suspension of sarcoplasmic reticulum vesicles was increased by using a stopped flow apparatus, the light scattering intensity rapidly increased at the beginning and then decreased. The fast increase in the light scattering intensity is caused by the decrease of the volume of sarcoplasmic reticulum vesicles due to the outflow of water. The following decrease in the light scattering intensity is caused by the increase of the volume due to the inflow of the solutes and water. From the former and the latter rates, the permeation times of water and the solutes could be calculated, respectively. According to the same method, permeation times of various salts were determined. The rate of the inflow of the salts was dependent on the movement of the slower ions, that is, ions move as a pair. In the case of potassium salts, an increase in the permeation rate of the salts was observed when valinomycin was added to the membrane suspensions. From these experiments, as a measure of permeability, half permeation times of various ions and molecules were determined. The following are typical results: water 0.1, Li+ 36, Na+ 26, K+20, Rb+ 16, Cl- 0.4, methanesulfonate 20, phosphate 10.5, oxalate 40 in seconds at room temperature. As a whole, sarcoplasmic reticulum was found to be an anion permeable membrane.

Excitation of membrane fragments by cholinergic agonists is measuredin vitro by a filtration technique. Membrane fragments which contain high levels of the enzyme acetylcholinesterase and presumably originate from the innervated excitable faces of electroplax are first purified from homogenates of electric organ ofElectrophorus electricus by centrifugation in a sucrose gradient. Then the fragments, which make closed vesicles or microsacs, are equilibrated overnight with a medium containing(22)Na(+). After equilibration of the inside of the microsacs with the outside medium, the suspension is diluted into a nonradioactive medium. The(22)Na(+) content of the microsacs as a function of time is then followed by rapid filtration on Millipore filters. In the presence of cholinergic agonists, the time course of(22)Na(+) release changes: the rate of(22)Na(+) release increases. This increase is blocked byd-tubocurarine and is absent with microsacs derived from the non-innervated inexcitable membrane of the electroplax. The response to cholinergic agonists is thus followed on a completely cell-free system, in a well-defined environment. The dose-response curves to cholinergic agents obtainedin vitro agree, quantitatively, with the dose-response curves recordedin vivo by electrophysiological methods. In particular, the dose-response curve to agonists is sigmoid, the antagonism betweend-tubocurarine and carbamylcholine competitive, and the antagonism between tetracaine and carbamylcholine noncompetitive. The effects of two different affinity labeling reagents on the response to agonists and on the catalytic activity of acetylcholinesterase are followed in parallel on the same microsac preparation. The effects of dithiothreitol and of gramicidin A on the microsacs are studied and are found to be similar to those observedin vivo with the isolated electroplax.

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An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100 mM Cl-. (2) The channel does not select among monovalent anions but SO2-4 hardly permeates through the channel. (3) SO2-4 added to the cis side (the side to which SR vesicles were added) inhibits Cl- current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO2-4 is located at about 36% of the way across the channel from the cis entrance.