MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF A C. ELEGANS NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNIT (ACR-Z). Michael Squire, Camilla Torn0e, Howard Baylis, John Fleming1, Eric Barnard2 and David Sattelle. The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. 1 MGH Cancer Centre, Harvard University, 2Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, UK. As part of an ongoing program to study neurotransmiter receptors in C. elegans we set out to isolate nicotinic acetylcholine receptor (nAChR) subunit clones from C. elegans by screening a C. elegans genomic DNA library with a probe derived from the Drosophila ARD gene (a non-a nAChR subunit clone, kindly supplied by Dr I Hermans-Borgmeyer). A number of putative nAChR subunit clones were isolated. The resulting C elegans clones were mapped to 8 loci on the physical map of the genome (Fleming JT, PhD Thesis, University of Cambridge). We extended our studies on one of these loci (represented by the clone JF#WA56) now called
acr-2 which is located between
svp-7 and
unc-6 on the X chromosome. Iev-9 is also located in this part of the chromosome but cosmids carrying
acr-2 do not rescue
lev-9. A full length
acr-2 cDNA has been isolated and sequenced and the genomic structure is being determined. The cDNA encodes a putative non-a subunit of a nicotinic acetylcholine receptor which shows many of the conserved features of vertebrate and invertebrate non-alpha nAChR subunits, for instance the four putative transmembrane domains and the cysteine delimited extracellular loop. Amongst other nAChR subunits
acr-2 is most similar to the Drosophila ARD subunit to which it shows 49% identity. In order to investigate the function of the subunit
acr-2, cRNA was produced by in vitro transcription and micro-injected into Xenopus oocytes. When expressed alone
acr-2 shows no levamisole gated channel activity. Unusually, such activity is observed when
acr-2 is co-expressed with another C. elegans non-a subunit (
lev-1, Fleming et al, in preparation). Such activity is normally only observed in the presence of an a-subunit. Whether this activity is the result of endogenous a subunit activity or reflects novel behaviour by the C. elegans subunits remains to be determined. When co expressed with a C. elegans a subunit (
unc-38, which is itself unable to form functional homo-oligomers)
acr-2 contributed to the formation of a functional (a, non-a) channel. In both of these cases the levamisole induced current was inhibited by mecamylamine.