Immunochemical demonstration that amino acids 360-377 of the acetylcholine receptor gamma-subunit are cytoplasmic.

Abstract

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...

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies

“…The peptides were synthesised in accordance with standard methodology (Atherton and Sheppard 1985) and were composed of the following amino acid residues: Kir2.1: (C)HNQASVPLEPRPL (residues 409-421), Kir2.2: (C)RLQASSGALERPY (residues 409-421) and Kir2.3: (C)RMQAATLPLDNISY (residues 427-440). N-terminal cysteine residues were added to the peptides to facilitate conjugation to ovalbumin carrier protein (Larochelle et al 1985) and were not part of the channel protein sequences. The peptideovalbumin conjugates were inoculated into New Zealand White rabbits.…”

Clustering of B and T Epitopes Within Short Sequence Regions of the Nieotinic Acetylcholine Receptor

“…in [21]). The sequence 7360-377 has been directly shown by Ab binding to be on the cytoplasmic surface ofthe TAChR [22]. Insufficient information on the TAChR structure is available at this point to allow firm conclusions as to whether the other TAChR sequences found to form 'cryptic epitopes' are or are not exposed on the TAChR surface.…”

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

“…Peptides were synthesized according to Atherton and Sheppard 15 and were composed of the following amino acid residues: Kir6.1; (C)KVQFMTPEGNQCPSES (residues 409-424, Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, UK), Kir6.2; (C)KAKPKFSISPDSLS (residues 377-390, Research Genetics Inc. Huntsville, USA), SUR2A; PNLLQHKNGLFSTLVMTNK(C) (residues 1527-1545, Pepceuticals Ltd., Leicester, UK), SUR2B; ESLLAQEDGVFASFVRADM(C) (residues 1528-1546, Pepceuticals Ltd) 15 . N-or C-terminal cysteine residues were added to the peptides to facilitate conjugation 16 to ovalbumin (Kir6.1) or Keyhole Limpet Haemocyanin carrier protein and were not part of the channel subunit sequences. Antisera were raised against peptide carrier protein conjugates in New Zealand White Rabbits (Kir6.1, University of Leicester; Kir6.2, Research Genetics Inc.; SUR2A and SUR2B, Pepceuticals Ltd.).…”