Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana. These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level . Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold . The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane . The distributions of the receptor and the 43K protein along the membrane were found to be coextensive . Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 ± 7 nm and 26 to 29 ± 7 to 10 nm, respectively) . These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane . They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule .The major proteins of isolated postsynaptic membranes from electric tissue include the subunits of the acetylcholine receptor and a nonreceptor protein having an apparent molecular weight of approximately 43,000 (43K protein) ' (6, 9, 19, 27). The 43K protein has stimulated considerable interest because it is a cytoplasmic, peripheral membrane protein (17,22,30) that has been localized to postsynaptic regions in electric tissue (6,9,18,27) and the neuromuscular junction (7).z Its functions may include anchoring of the receptor against lateral diffusion (1,5,15,21). However, unlike the several cytoskeletal proteins that accumulate at thejunction and may also participate in receptor anchoring (3, 10), the 43K protein seems to have no known counterpart in other systems, and very little is known concerning the mechanisms by which it functions.The postsynaptic location ofthe 43K protein has prompted wide speculation that it has receptor-specific functions mediated by its association with the receptor. This hypothesis predicts that the 43K protein, like the receptor (26), should ' Abbreviations used in this paper. 43K protein, the major membrane-bound M 43,000 protein ofisolated postsynaptic membranes ; mab, monoclonal antibody; 2-D, 2-dimensional . Z S. C. Froehner, manuscript submitted for publication .THE JOURNAL OF CELL BIOLOGY " VOLUME 98 JUNE 1984 2239-2244 C The Rockefeller University Press -0021-9525/84/06/2239/06 $1 .00 RAPID COMMUNICATIONS occur on the juxtaneural postsynaptic membrane of the electroplax, but should be essentially absent from the membrane in the deep portions ofthe postsynaptic invaginations. Hence, a careful comparison of the postsynaptic distributions of the two proteins constitutes an essential test of the hypothesis. The available immunofluorescence measurements (7)Z have not had the resolution necessary for such a test. The ult...

The relationships between extracellular pH (pH.), intracellular pH (pH,), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pH, was measured in single cells by ratio imaging of 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pH. of 7.4, pH, decreased from 7.36 to 633 within 10 min. pH1 remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pH. produced a slightly greater drop in pH1, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H' exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl-in buffer containing HCO3 abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

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TWO monoctonal antibodies (mabs) previously prepared against Torpedo acetylcholine receptor are shown to recognize a synthetic nonadecapeptide corresponding to lys360-glu377 of the gamma subunit. The reaction was demonstrated by solid-phase enzyme-linked immunoabsorbent assays, by inhibition of binding of the mabs to receptor, and by immunoprecipitation of the peptide conjugated to bovine serum albumin. Immunogold electron microscopy on isolated postsynaptic membranes from Torpedo showed that both mabs bind to intracellular epitopes on the receptor. These results establish that amino acid residues 360-377 of the receptor gamma-subunit, and probably the analogous region of the delta-subunit, reside on the cytoplasmic side of the membrane. Since the primary structures of all four subunits suggest a common transmembrane arrangement, the corresponding domains of the alpha-and beta-subunits are probably also cytoplasmic.The recent identification of the amino acid sequences of all four subunits of the acetylcholine receptor (AChR) 1 from Torpedo electric organ (5,7,24,29,36) has led to models for the transmembrane folding pattern of the subunits and for the structure of the receptor's ion channel (for a review, see reference 27). The results of these studies are likely to be of wide impact, because the receptors from Torpedo and mammalian muscle share extensive homology (6, 21, 25) and because they may provide a theoretical framework for the structure and function of other neurotransmitter receptors. Thus, prompt direct tests of these models are of major importance.The polypeptide chain of each receptor subunit contains a large hydrophilic domain at the N-terminus followed by three closely spaced hydrophobic regions, a second large hydrophilic domain, and a fourth hydrophobic domain (5, 7, 26). The recent identification of cysteine 192 as part of the alphaAbbreviations used in this paper: AChR, nicotinic acetylcholine receptor; ELISA, enzyme-linked immunoabsorbent assay; KLH, keyhole limpet hemocyanin; mab, monoclonal antibody. chain that contributes to the acetylcholine-binding site (41) and evidence from cell-free synthesis of receptor subunits (1) establish that the large N-terminal domain is extracellular. In folding models based on hydrophobicity analyses alone, the hydrophobic domains form the only transmembrane elements, probably as helices. Hence, these models predict that the second hydrophilic domain lies on the cytoplasmic side of the membrane and that the C-terminus is extracellular (5,7,26). Searches of the sequences for repeating arrangements of nonpolar and polar residues, however, have produced evidence for an additional, amphipathic helix just preceeding the fourth hydrophobic helix (9, 17). Thus, in this class of model, the large cytoplasmic domain is smaller and the Cterminus is cytoplasmic. In a third model, the second hydrophilic domain is divided roughly into two halves joined by a transmembrane element (19).The most direct initial tests of the proposed folding patterns require localizat...