Cloning of the chicken acetylcholinesterase gene and regulation of its expression in skeletal muscle by functional innervation

Abstract

Acetylcholinesterase (AChE) is an essential component of cholinergic synapses where it terminates transmission by hydrolysing acetylcholine. Two catalytic subunits (T and H) are generated from alternative splicing from a single gene in Torpedo and in mammals. The T subunit has a hydrophilic C-terminal region and associates with structural subunits via disulfide bonds. The H subunit has a hydrophobic C-terminus to which a glycophospholipid (GPI) membrane anchor is added posttranslationally. At the neuromuscular junction, synaptic AChE is produced by the muscle and it is under neural control. To begin the study of the mechanisms accounting for the molecular diversity and regulation of AChE, we embarked in the cloning of the avian AChE gene. We confirmed that a single gene encodes all known molecular forms of AChE in the chicken. The structural gene including the complete open reading frame was contained in a {dollar}\sim{dollar}13 kb clone. The coding region is divided into three exons, the first two encoding 95% of the protein and the last one coding for the T form C-terminal region. We found no evidence for alternative splicing in the coding region nor in the 3{dollar}\sp\prime{dollar}-untranslated region. The exon for the H form is absent in the chicken and only one exact consensus polyadenylation addition signal was found. Evidence is provided for at least one 5{dollar}\sp\prime{dollar}-untranslated exon. We show that a 165 aa domain unique to the chicken protein is encoded by exon 2 and, by comparison with the 3-D structure of Torpedo AChE, we predict that this domain is likely to be on the surface of the protein, away from the active site. Transcripts for AChE increased up to the 13-day in ovo in pectoralis muscle; afterwards, AChE mRNA dropped to very low levels in the adult. Surgical denervation of adult chicken skeletal muscle caused an increase in the levels of AChE transcripts that was muscle type specific and paralleled the changes in enzyme activity. Similar changes in nicotinic acetylcholine receptor expression following denervation are reported by others.