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The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californca contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the a subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the y subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up =22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide.How the nicotinic acetylcholine receptor (AcChoR) mediates ion conductance in response to binding acetylcholine at the neuromuscular junction is of critical importance to understanding the function of the family of related neuroreceptors (1). The molecular structure of the AcChoR contains five similar subunits that surround the ion-conducting channel, shown to lie in the center (2, 3). While the primary structures of all four subunit types of the acetylcholine receptor from Torpedo californica are homologous from N to C termini (4), there is as yet no identification of all regions that are transmembranous. However, in each of the five subunits (a2zy8), there are four hydrophobic stretches of 19-26 amino acids that are presumed to lie across the membrane (5). Site-directed mutagenesis shows that residues of the second helix, M2, determine aspects of the conductivity (6). On the basis of our x-ray diffraction evidence for oriented a-helices perpendicular to the membrane we proposed that the ion channel could be formed within a bundle of such a-helices. More polar surfaces could provide the lining of the pore. A fifth amphipathic a-helix, which may also be transmembranous (MA), is apparent from the AcChoR primary sequence. Our hypothesis that these helices could also contribute to the pore led to experiments designed to determine the peptide topography, especially in the region of MA, M4, and the C terminus. Our electron microscopic localization of peptidespecific antibodies on intact native tissue and on subcellular fractions (7) and experiments of Ratnam et al. (8) all indicate that certain regions between M3...

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 ؋ g) but the membrane skeleton requires 100,000 ؋ g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin ␣ IIb ␤ 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following ␣ IIb ␤ 3 -ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a ϳ140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.Duchenne muscular dystrophy is one of the most common inherited human diseases. It is caused by a defective gene that codes for a 427-kDa protein, dystrophin (1-5). The deduced amino acid sequence of dystrophin shows that it consists of four domains and suggests that it is a cytoskeletal protein (6). The major rod-shaped domain contains 24 spectrin-like repeats. This domain is flanked on the amino terminus by a domain that has a high degree of homology to the actin-binding domains of spectrin and ␣-actinin, and on the carboxyl terminus by a cysteine-rich domain that shows some homology to a Ca 2ϩ -binding region in ␣-actinin. The most carboxyl-terminal end of dystrophin consists of a short domain that has no homology to any known protein and appears to play a role in linking the molecule to the plasma membrane (7,8). Recent studies using purified protein or recombinant fragments containing the putative actin-binding domain (9 -11) have shown that the protein can bind to actin filaments in vitro, supporting the idea that this molecule functions as a cytoskeletal protein. The finding that dystrophin exists in a submembranous location (8,12,13) and that the carboxyl-terminal end of the molecule associates tightly with a complex of membrane glycoproteins (termed dystroglycan) (14 -17) suggests that dystrophin is a component of a submembranous cytoskeleton.Although there is now considerable inform...

We report the detection of electrostatic interactions between local anesthetics and membrane phospholipids and proteins. A spin-labeled local anesthetic was used to study how membrane-bound tertiary amine anesthetics interact with major molecular components in the membrane. The nitroxyl reporter group of this spin label is located at the polar end of the amphiphilic local anesthetic; it is therefore a uniquely suitable probe for detecting immobilization of the anesthetic due to binding interactions at the polar regions of the bilayer. The binding properties of this spin-labeled anesthetic to human erythrocyte membranes and to vesicles made from human erythrocyte lipids were studied. Lipid vesicle-bound spin labels give rise to a composite electron spin resonance spectrum from which two subcomponent spectra were resolved. Both components are membrane-bound; the first component has a narrower linewidth, indicating a greater mobility of the nitroxyl moiety of the anesthetic probe. The second component has a broader linewidth, indicating a population of constrained spin labels. We infer from the experimental results that electrostatic binding between cationic anesthetics and anionic phosphate of phospholipids produced the constrained component. In similar studies using erythrocyte ghost membranes, both a mobile (nonelectrostatic) component and a constrained (electrostatic) component were resolved from the composite spectrum. However, the constrained component in this case is much broader than the corresponding constrained component from the vesicles. We interpret this broad component in the erythrocyte membrane as an electrostatic interaction of cationic anesthetic probes with phospholipids and with membrane proteins. We conclude that membrane-bound tertiary amine anesthetics in cationic form do interact selectively with phospholipids and proteins.

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Nicotinic acetylcholine receptor isolated from Torpedo californica electric organ is reconstituted into lipid bilayers of zwitterionic dioleoylphosphatidylcholine. These membranes are labeled with a spin-labeled quaternary amine local anesthetic (C6SLMeI), which has been shown previously to be a noncompetitive blocker of acetylcholine receptor-ion channel function in the micromolar concentration range. The electron spin resonance spectral component corresponding to protein-immobilized anesthetic spin-label can be resolved from the composite data spectrum by using spectral subtraction of lipid components. This protein-immobilized component is shown to represent C6SLMeI bound to a finite number of sites on the receptor. We demonstrate that C6SLMeI binds to the receptor as a function of the surface potential on the protein and suggest that the acetylcholine receptor reconstituted into zwitterionic phospholipid, which has no surface potential of its own, provides an excellent model system with which to study effects of protein surface charge. We hypothesize that the primary pathway of interaction of C6SLMeI with the acetylcholine receptor is via the aqueous medium.