Transmembrane topography of the nicotinic acetylcholine receptor: immunochemical tests contadict theoretical predictions based on hydrophobicity profiles

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Abstract
In our preceding paper [Ratnam, M., Sargent, P.B., Sarin, V., Fox, J.L., Le Nguyen, D., Rivier, J., Criado, M., and Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence .beta.429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the .beta. subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the .alpha. subunit by a more direct approach. We synthesized peptides including the sequences .alpha.330-346, .alpha.349-364, .alpha.360-378, .alpha.379-385, and .alpha.389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to .alpha. subunits between two and six which bound specifically to each of these peptides, except .alpha.389-408. We obtained antibodies specific for .alpha.389-408 both from antisera against the denatured .alpha. subunit and from antisera made against the peptide. These antibodies were specific to .alpha.389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic suface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (.alpha.409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic transmembrane domain M5 (.alpha.364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathy profile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data.

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