Cloning and heterologous expression of D alpha 4, a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification of an alternative exon influencing the efficiency of subunit assembly

A neuronal nicotinic acetylcholine receptor (nAChR) subunit, D alpha4, has been identified and cloned from the fruit fly Drosophila melanogaster, toge ther with several alternatively spliced transcripts. Intron-exon boundaries within the gene encoding D alpha4 (nAcRalpha-80B) have been identified by comparison of cDNA and genomic sequence data. The influence of amino acids encoded by alternatively spliced exons upon nicotinic radioligand binding a nd subunit-subunit co-assembly has been examined by heterologous expression in Drosophila S2 cells. The efficiency of subunit assembly has been shown to be influenced by amino acids surrounding the highly conserved 15 amino a cid cysteine-loop motif within the N-terminal extracellular domain of the n AChR D alpha4 subunit. Extensive use has been made of publicly available da ta determined by the Berkeley Drosophila Genome Project (BDGP). This includ es expressed sequence tag (EST) data as well as whole-embryo in situ hybrid isation and polytene chromosome in situ hybridisation data. BDGP in situ hy bridisation data suggests that the D alpha4 mRNA is expressed within Drosop hila brain and ventral nerve cord and demonstrates that the gene encoding t his nAChR subunit is located at position 80B on chromosome 3. The relations hip between D alpha4 and other previously cloned nAChR subunits has been ex amined and the implications for the nomenclature of insect nAChRs is discus sed. (C) 2000 Elsevier Science Ltd. All rights reserved.