Structure and Function of the Acetylcholine Receptor

The acetylcholine-(ACh-)receptor of electric organs of three Torpedo species (marmorata, ocenata and caLifornica) is purified by affinity chromatography. It acts as a single molecular species as judged by electrophoresis, isoelectrofocusing, gel filtration and immunodiffusion tests. It binds 10 nmoles of ACh or a-bungarotoxin per mg protein. Its amino acids are analyzed and only = 180/o of the cysteic acid residues occur as free SH groups. When the receptor is analyzed for the presence of cations by atomic absorption, 4.7°/o of its weight is found to be due to bound Ca*. When Ca++-free solutions are used for purification, bound Ca* still contributes 0.6°/o of its weight. The receptor is an oligomer of an apparent molecular weight of 330 000, with a high tendency to aggregate in the absence of added detergent. It has a doughnut-like appearance with an electron dense core, and consists of 5-6 subunits. Whereas binding of ACh to fresh Torpedo membranes has a Kd of 2 X 10-s JI/I and exhibits positive cooperativity, that to the purified ACh- receptor has similar characteristics, and in addition, has a low affinity component (Kd = 2 X 10-6 M). Antibodies, formed against the ACh-receptor in immunized rabbits or rats, react with the animal\u27s own skeletal neuromuscular ACh-receptors. Incorporation of the ACh-receptor, after mild treatment with trypsin, into oxidized cholesterol bilayers, consistently causes increases in monovalent cation selective conductances (10-over 100 fold), that are triggered by ACh and carbamylcholine and inhibited by curare. The data suggest that the ion conducting carriers or channels of the postjunctional membrane are part of the isolated receptor protein

The effects of conformational constraints in the polyamine moiety of philanthotoxins on AMPAR inhibition

Philanthotoxin‐433 (PhTX‐433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subtype by using two‐electrode voltage‐clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX‐343, with trans‐cyclopropyl‐PhTX‐343 being the most potent (∼28‐fold) and cis‐cyclopropyl‐PhTX‐343 least potent (∼4‐fold). Both cis‐ and trans‐cyclopropyl‐PhTX‐444 had intermediate potency (both ∼12‐fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX‐444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50=12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity