The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine receptor during the course of reconstitution into lipid vesicles were studied by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into two components: (a) adequate nature and concentration of lipids during detergent solubilization; (b) correct lipid environment during reincorporation into lipid vesicles by the cholate dialysis procedure. Optimal bulk lipid concentrations and lipid:cholate ratios for preserving state transitions during solubilization were studied by using both crude soybean lipids and pure synthetic phospholipids. The latter class of lipids was found to be unsuitable substitutes for the crude soybean lipids, irrespective of their polar head group and/or fatty acyl chain, even when detergent:lipid ratios as high as 1:1 (w/w) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of acetylcholine receptor state transitions at cholate:steroid ratios of about 6:1 (w/w). The presence of steroid decreased the amount of protein solubilized. The correct choice of lipid type was also essential to the reincorporation step, and higher concentrations of lipid were required--about 20 mg/mL for soybean lipids. Pure phospholipids at similar concentrations, however, were unable to maintain the state transitions. Again, steroid (40-46% cholesteryl hemisuccinate/mol of total lipid) provided the adequate conditions at the reincorporation stage and enhanced the amount of protein reincorporated into the vesicles. A large (70-90%) percentage of the receptor was reincorporated with the correct vectorial sidedness. No specificity could be detected for the phospholipid polar head or alkyl chain in relation to any of these findings. The effect of the protein on the physical state of the lipids in the reconstituted vesicles was studied by diphenylhexatriene fluorescence depolarization. The results may be interpreted as a disordering of the acyl chains in the gel state and an ordering in the liquid-crystalline state in the presence of protein, accompanied by shifts in the transition temperatures of the pure phospholipids to lower values.

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from Torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possible by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports .Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a mainaxis diameter of 19 ± 3 nm predominate in these membranes . Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrocytes (Heuser, 1 . E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form . The proportion of doublets increases in membranes alkylated with N-ethylmaleímide . Treatment with 5,5'-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n = oligo) predominate.The nonreceptor v-peptide (doublet of M r 43,000) appears to play a role in the receptor monomer-polymer equilibria . Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N-ethylmaleimide, only the receptor appears to self-aggregate . The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments.Monospecific antibodies against the v-peptide are used in conjunction with rhodaminelabeled anti-antibodies in an indirect immunofluorescence assay to map the vectorial exposure of the v-peptide . When intact membranes, v-peptide depleted and "holey" native membranes (treated with 0.3% saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane .The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.Membrane fractions enriched in the acetylcholine receptor can now be obtained with a high degree of purity from the electric organs of Torpedinidae and other convenient sources of this protein. They contain few protein components aside from the one carrying the recognitio...

Chemical receptors are the mediators of specifi c reactions in a variety of biological processes that range from elementary chemosensory activities in primitive bacteria to the highly sophisticated behavioral mechanisms in man. They have the capacity to specifi cally recognize a given chemical and to generate the requisite signal for evolving a biological response. Strictly speaking, the word "receptor" was used as such for the fi rst time in the early papers of Ehrlich (see 54, 137) (in the discipline now known as immunology), although it was in the context of physiology and pharma cology that it really acquired its "wholistic" connotation. Since then the term has been subjected to considerable use and misuse, often leading to confusion, but above all depriving receptors in many instances of their most superb integrating property. It cannot be over-emphasized that spe cific recognition of a chemical does not suffice to define a receptor [for a clear distinction between receptors and acceptors see (2 1)]. The chemical message encoded in a ligand, say a neurotransmitter or a hormone, is virtually meaningless until it is decoded by its corresponding receptor into purposeful regulatory signals in the target cell. Therefore I adhere throughout this review to the early interpretation of the term, using it to denote both the capacity to specifi cally recognize a ligand (cognitive or discriminatory property) and the capacity to initiate the chain of events leading to the biological effect (gating property).I have not attempted to cover the whole fi eld of chemical receptors, but have preferred instead to restrict the discussion to some endogenous receptors fo r hormones and neurotransmitters. This necessarily implies the omission of several important problems in chemoreception. Arbitrary though it may seem, the review progressively concentrates on a single archetypal system, the receptor fo r the neurotransmitter acetylcholine 287 0084-6589/7910615-0287$0 1.00 Annu. Rev. Biophys. Bioeng. 1979.8:287-321. Downloaded from www.annualreviews.org Access provided by State University of New York -Brooklyn on 03/30/15. For personal use only. Quick links to online content Further ANNUAL REVIEWS

A model of the nicotinic acetylcholine receptor transmembrane region has been constructed which may represent the channel in its open-state. The positions of helices flanking the ion channel match those observed by electron microscopy and previously reported by others. Residues labelled, mutated or by other means known to have a strong influence on ion flux are each accessible from the lumen of the modelled channel. The model provides new insights into our current understanding of the ion channel structure, and suggests some novel explanations for the results of labelling and mutation studies such as those involving ion channel blockers and residue-dependent changes in ion selectivity.

A projection, at 15-to 20A resolution, is resented of the structure of the membrane-bound acetylcholine receptor protein from Torpedo marmorata. The projection has its axis perpendicular to the membrane plane; its main contribution originates from a hydrated portion of the protein, which extends from the membrane into the aqueous medium. The structure is distinctly asymmetric, with individual morphological subunits barely resolvable. These results have been obtained by noncrystallographic averaging, using correlation functions, applied to electron micrographs of receptor-rich membrane fragments. The micrographs had been taken with minimal beam exposure in a scanning transmission electron microscope.The acetylcholine receptor (AcChoR) plays a key role in synaptic transmission because it controls the cation permeability of the postsynaptic membrane. It is an integral membrane glycoprotein of t3 X 105 molecular weight and is composed of several polypeptide chains. Reports on its detailed chemical subunit composition and the number of ligand or neurotoxin binding sites disagree (1,2). Reliable ultrastructural information is also lacking. The numbers of morphological subunits deduced from electron micrographs of negatively stained AcChoR range from three to six (3-5). This is due to morphological variations among individual AcChoR particles resulting from stain granularity (enhanced by electron irradiation) and deformations.