Worm Breeder's Gazette 13(4): 78 (October 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Molecular Cloning and Functional Expression of a C. elegans Nicotinic Acetylcholine Receptor Subunit (acr-2).

Michael Squire1, Camilla Tornoe1, Howard Baylis1, John Fleming2, Eric Barnard3, David Sattelle1

1 The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK.
2 MGH Cancer Centre, Harvard University
3 Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, UK.

  As part of an ongoing program to study neurotransmitter 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 sup-7 and unc-6 on the X chromosome. lev-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-alpha 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-alpha
subunit ( lev-1 ,Fleming et al, in preparation). Such activity is normally only observed
in the presence of an alpha-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 coexpressed with a C. elegans alpha subunit ( unc-38 ,which is
itself unable to form functional homo-oligomers) acr-2 contributed to the formation of a
functional (alpha, non-alpha) channel. In both of these cases the levamisole induced
current was inhibited by mecamylamine.