Snake Acetylcholine Receptor: Cloning of the Domain Containing the Four Extracellular Cysteines of the α Subunit

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 18; pp. 7255 - 7259
• Main Authors: Neumann, Drorit, Barchan, Dora, Horowitz, Mia, Kochva, Elazar, Fuchs, Sara
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.09.1989; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino acids; Animals; Base Sequence; Binding sites; Biological and medical sciences; Blotting, Southern; Cloning, Molecular; Complementary DNA; Cysteine; DNA; DNA - genetics; DNA - isolation & purification; DNA probes; Fundamental and applied biological sciences. Psychology; Gels; Genes. Genome; Genomics; Lizards - genetics; Macromolecular Substances; Mice; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Polymerase chain reaction; Receptors, Cholinergic - genetics; Restriction Mapping; RNA; Sequence Homology, Nucleic Acid; Snakes; Snakes - genetics; Species Specificity; Molecular hybridization; Nucleotide sequence; Subunit; Ophidia; Northern blotting; Vertebrata; Gene amplification; Cholinergic receptor; Reptilia; Acetylcholine; Molecular cloning; Southern blotting; Naja naja; Comparative study; Aminoacid sequence
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.18.7255

The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind α -bungarotoxin. Numerous studies indicate that the ligandbinding site of the AcChoR includes cysteine residues at positions 192 and 193 of the α subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR α subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR α subunit. First, a mouse AcChoR α -subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR α subunit. The domain of the α subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake α subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR α subunits. Sequence comparison revealed that the cloned region of the snake α subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind α -bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp → Phe), 185 (Lys → Trp), 187 (Trp → Ser), and 194 (Pro → Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of α -bungarotoxin-binding to snake AcChoR.