Euro-Myasthenia III

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THE ISOLATION AND CRARACTERISATION OF cDNA AND GENOMIC CLONES ENCODING THE RUMAN MUSCLE ACETYLCHOLINE RECEPTOR. D. Beeson, M. Brydson, A. Morris, M. Betty, S. Povey, A. Vincent and J. Newsom-Davis. University of Oxford, Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU.

The autoimmune response in Myasthenia gravis is directed against the human muscle acetylcholine receptor (AChR). The study of myasthenia is restricted by the low levels of the AChR present in human muscle and the difficulty of purifying it. Our strategy for overcoming this problem has been to isolate both cDNA and genomic clones encoding the human AChq subunits and then use these clones for expression of the AChR subunits and for studies of hereditary myasthenia (HM).

We have isolated genomic and cDNA clones for the human a, p, y, 6 and E subunits. The isolation of a-subunit cDNA clones revealed two forms of the a-subunit mRNA, one of which contains a novel exon encoding an additional 25 amino acids between amino acids 58 and 59 of the mature a-subunit. We have expressed the cDNAs in E.coli, rabbit reticulocyte lysates, and Xenoous oocytes. And we have localised the a and 6 as sub unit genes to chromosome 2 and the p subunit gene to chromosome 17.

ACETYLCHOLINE RECEPTORS THAT DO NOT BIND a-BUNGAROTOXIN: THE BINDING SITE OF SNAKE AND MONGOOSE ACHR D. Barchan, S. Kachalsky, D. Neumann and S. Fuchs. Department of Chemical Immunology, The Weizmann Institute of Science, Reovot 76100, Israel

We have shown previously that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor (AChR) a-subunit, which includes the adjacent cysteine residues 192 and 193, contains the essential elements of the ligand-binding site. In an attempt to further elucidate the molecular basis for the precise binding properties of AChR we investigated the structure of the binding site of muscle AChR from snake and mongoose, both of which are resistant to a-neurotoxins. Fragments from snake and mongoose AChR a-subunit, encoding a domain corresponding to amino acid residues 122-205, were amplified by the polymerase chain reaction (PCR), and then sequenced and expressed in E.coli, using the expression vector PET 8c. Both fragments are highly homologous to other muscle AChR a-subunits and contain the four known cysteines at positions 128, 142, 192 and 193. Most substitutions in the snake and mongoose fragments are present in the presumed ligand-binding site, in the vicinity of cysteines 192 and 193, thus explaining the lack of their binding to a-BTX. Sequence comparison suggests that substitutions at positions 187, 189 and 194, which occur in both snake and mongoose AChR, are most important in determining their resistance to a-BTX. Binding experiments with the expressed fragments of snake, mongoose and mouse AChR indicated that only the mouse fragment binds a-BTX and as expected, the snake and mongoose fragments do not.