Susan M. J. Dunn

Independent sites of low and high affinity for agonists on Torpedocalifornica acetylcholine receptor

Abstract It is demonstrated that two classes of binding site for acetylcholine are present on Torpedo californica acetylcholine receptor. One class is the well documented site on each of the two subunits of 40,000 daltons, which can be covalently modified by bromocetylcholine. Both in the absence and in the presence of bromoacetylcholine another binding site is shown to exist by virtue of acetylcholine dependent fluorescence changes in the receptor covalently modified by 4-[N-(iodoacetoxy)ethyl-N-methyl]-amino-7-Nitrobenz-2-oxa-1,3 diazole (IANBD). This site has a low affinity for acetylcholine (Kd ∼ 80 μM) that corresponds closely with the known concentration dependence of acetylcholine mediated activation of this receptor and we conclude that it may represent a site of association that participates in channel opening in this system.

Structural and functional aspects of the nicotinic acetylcholine receptor

The molecular structure of nicotinic acetylcholine receptors (AcChR) from different peripheral tissues and from different brain areas has been studied. AcChRs from Torpedo and Electrophorus electric organ and from piscine, avian, and mammalian muscle have been shown to be highly conserved proteins composed of four types of homologous subunits (alpha, beta, gamma, and delta) which associate in a stoichiometry alpha 2 beta 1 gamma 1 delta 1 to form a pseudosymmetric pentameric complex molecule. Genealogical analysis suggests that all the subunits of these AcChR derive from a common ancestral gene and that the divergence occurred very early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as their highly conserved structures along the animal evolution, suggests that each of the subunits evolved to perform specific crucial roles in the function of the AcChR complex. In Torpedo the two alpha-subunits present in the AcChR molecule are glycosylated to a different extent. This may be the reason why the binding sites for cholinergic ligands that are located on the alpha-subunits are non-equivalent. The pseudosymmetric pentameric complex has all the properties of a physiological receptor with respect to known parameters leading to postsynaptic depolarization, and therefore contains the molecular structure/structures that constitute a cation selective channel for transport across the membrane. The complex also contains recognition sites for acetylcholine, cholinergic antagonists, polypeptide neurotoxins, small molecule neurotoxins, and local anesthetics. Therefore a multiplicity of receptor-ligand associations are possible. This leads to more complex models of such interactions than previously considered.(ABSTRACT TRUNCATED AT 250 WORDS)

The Nicotinic Acetylcholine Receptor

The acetylcholine receptor (AcChR) from Torpedo electroplax has been isolated both in its native membrane-bound state and by affinity chromatography after solubilization (see Conti-Tronconi and Raftery, 1982). Purified membrane fragments can reseal, forming closed, right-side-out vesicles, which can be used for both functional and structural studies. The major physiochemical properties of T. californica AcChR protein are summarized in Fig. 1.

Nicotinic Receptors from Different Peripheral Organs and from Brain are Highly Homologous, Complex Proteins

The acetylcholine receptor (AChR) from Torpedo electroplax can be isolated in a pure form either in its native membrane-bound state or by affinity chromatography after solubilization. Due to the ease with which this receptor can be purified, both its structure and its function have been investigated to a remarkable degree of sophistication.

Structure and Function of the Nicotinic Acetylcholine Receptor and of the Voltage-dependent Na+-Channel

Nicotinic acetylcholine receptors (AChRs) from electric organs and muscle of different species have been shown to be highly conserved proteins composed of four homologous subunits occurring in the stoichiometry α2βγδ and forming a pseudosymmetric pentameric complex (see Raftery et al. 1983). The availability of large quantities of AChR from Torpedo electric organ has greatly facilitated detailed studies of its structure and function. It has been demonstrated that the α2βγδ complex constitutes the complete physiological receptor for postsynaptic depolarization by ACh and contains both the binding sites for cholinergic ligands and the cation gating unit (Moore and Raftery 1980; Wu et al. 1981). Each subunit spans the postsynaptic membrane and each has been shown to be exposed to the lipid bilayer suggesting that all subunits may interact with the surrounding membrane in a related fashion (see Raftery et al. 1983).