Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Abstract

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...

Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes.

“…4, lane e), which elutes with the column void volume. The creatine kinase activity is therefore present in a protein having an apparent molecular weight equivalent to that of the v proteins of AcChoR membranes (4,20). This is further shown in Fig.…”

“…Purification of this activity is accompanied by the enrichment of the fractions in polypeptides having electrophoretic properties like those of trout creatine kinase isoenzymes (33) malian muscle (6). The I proteins are membrane-associated proteins, their antigenic determinants being mainly exposed on the cytoplasmic face of the membrane (20). (34), suggesting either that the association with the membrane is trivial or that it obeys rules other than enzymatic facilitation via coupling with the membrane structure.…”

“…26. Immunoblotting on nitrocellulose paper was carried out as described (20), using rabbit antisera against chicken BB (brain) or MM (muscle) types of creatine kinase. The immune replicas were first incubated for 2 hr in 5% horse serum in 50 mM Tris-HCl buffer, pH 8.0, followed by 12-hr incubation in the same buffer containing either the MM or the BB antiserum at a 1:100 dilution.…”

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

“…were prepared from the electric organ of T. californica as previously described ( 6,23 ). The electric tissue was fi rst dissected into small pieces, and then homogenized at 4°C using a Virtis 60 glass (The Virtis Co., Inc.) until an homogeneous suspension was obtained.…”

“…Membranes were blocked with 5% nonfat dry milk in TTBS buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.1% (w/v) Tween 20) for 2 h at room temperature followed by incubation with primary antibodies [anti-AChR ␣ -subunit (mAb 210) (1:5000), anti-AChR ␤ -subunit (mAb124) (1:2000), and antirapsyn (mAb 1234) (1:500)] overnight at 4°C. Membranes were washed fi ve times with TTBS buffer and then exposed to the specifi c activity [in the order of 2.0-2.8 nmol ␣ -bungarotoxin sites/mg protein ( 23 )]. …”