Acetylcholine Receptor and Ion Conductance Modulator Sites at the Murine Neuromuscular Junction: Evidence from Specific Toxin Reactions

Abstract

The perhydro, derivative of histrionicotoxin rev ersibly blocks thle excitatory ionic traisduction system in the synaptic anld sarcolemmal membranes of mainnalian. slkeletal mtiscle cells. The efficacy of perliydrohistrionicotoxin as an1 antagonist at the post-synaptic remlbraine is increased by the transienit prese'ice of acetylcholine in the entdplate of ininervated muscles and at extrajunctional rece)Lors in denervated imutiscles. 'The excitatory action of acetylcholine at posts-ynaptic inembranes clearly involves at least two stages: first, the combination of the tramismitter with a receptor site, and second, the induced inreeaze in Na+ and K+ conductances that cause the transient depolarization of the membrane. Little is knownl about thel molecular basis for the coul)ling between these two steps. Hypotheses (1---6) include (a) topographically-separate AICh receptors and ion conductance modulators § (1CM); (b) a cooperative complex consisting of receptor and ICAM subunits; and (c) one macromolecule, serving as both receptor and ICM. a-Bungarotoxin (BuTX) has been proposed as a specific reagent for the acetylcholine receptor site in vertebrate skeletal muscle synapse (7-10). No such specific probe for the postsynaptic ICM has been described. The results reported herein suggest that the perhydroderivative (H12-

Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin.

“…The elementary functional unit that accounts for the regulation by acetylcholine (ACh) of cation translocation through an excitable membrane comprises, a priori, two main categories of sites: (i) the ACh receptor site, which binds cholinergic agonists, antagonists (1), and snake venom a-toxins (2); and (ii) a site involved in the selective permeation of small cations. It was further postulated that these two sites were topographically distinct, although coupled by "allosteric" interactions and most likely carried by different polypeptide entities referred to as the ACh-receptor protein sensu stricto, and the ACh ionophore (3) [or ion conductance modulator (4)]. The ACh-receptor protein has been isolated and purified from fish electric organ and skeletal muscle in several laboratories (see ref.…”

“…It behaves in vivo (4,18,19) and in vitro on ACh-receptor-rich membranes (20) in a manner similar, if not identical, to that of a local anesthetic. This analogy is further supported by the recent observation that local anesthetics displace a radioactive derivative of HTX bound to membrane fragments from T. ocellata (21).…”

Perhydrohistrionicotoxin: a potential ligand for the ion conductance modulator of the acetylcholine receptor.

“…It has been proposed that histrionicotoxin (HTX), an alkaloid isolated from the skin extracts of the Colombian frog, Dendrobates histrionicus (9), modifies responses to ACh in neuromuscular preparations by interaction with the ion conductance modulator rather than with the ACh receptor (1,(10)(11)(12). HTX and its completely saturated analog, perhydrohistrionicotoxin (H,2-HTX), reduce the amplitude of the end-plate current (EPC) and shorten the half-decay time without altering the equilibrium potential in mammalian and amphibian nerve-muscle preparations.…”

“…The excitatory action of acetylcholine (ACh) at the postsynaptic membrane involves its binding to a specific recognition site followed by activation of the ion conductance modulator (1) and a marked increase in ionic conductance. The ion conductance modulator may represent an ionic channel or ionophore comprising part of the same macromolecule as the ACh receptor or it may be separate from, but somehow coupled with, the receptor.…”

Stabilization of the high affinity state of the membrane‐bound acetylcholine receptor from Torpedo marmorata by noncompetitive blockers

“…They include, among others, the aminated local anesthetics proadifen and dimethisoquin [3][4][5][6][7], the frog toxin histrionicotoxin (HTX) and its derivatives [8][9][10][11], the hallucinogenic drug phencyclidine [12][13][14]. These pharmacological agents block the physiological response in the/~M range and their binding to saturable sites on ACh-receptor-rich membrane fragments isolated from Torpedo electric organ has been demonstrated with the radiolabelled derivatives [3H]meproadifen [6], [3H] trimethisoquin [ 15 ], [all] perhydro-HTX [ 11,[16][17][18] and [3H]phencyclidine [12,19,20].…”