Rachel Aronoff et al. (2001) International C. elegans Meeting "Alternative isoforms and mutants of the glr-2 ionotropic glutamate receptor"

Jerry Mellem, Rolf Sprengel, Andres Villu Maricq, Peter Seeburg, Rachel Aronoff

[International C. elegans Meeting, 2001]

Ionotropic glutamate receptors are essential mediators of excitation in the vertebrate brain. The iGluR family in vertebrates is quite large, with at least 16 genes and complex post-transcriptional alterations in gene structure. In order to initiate complementary studies in a simpler genetic system, we began to examine predicted iGluRs in C. elegans . Among these, the glr-2 gene from the cosmid K04G7 is particularly interesting. Its predicted sequence contains the key M3 gating determinant (the lurcher domain, TANLAAF) and residues critical in forming the M2 pore, including the Q/R site, which limits channel Ca ++ conductance when the genomic glutamine codon is edited to arginine. It also carries a carboxy terminus which could be a PDZ interaction sequence (STLF). These features make the gene, like glr-1 , most analogous to the AMPA iGluRs from vertebrates. We have previously found that, although the Q/R site is not detectably edited in RNA expressed from this gene, alternative splicing of a region encoding a conserved amino terminal domain does occur. Now we present results from our analysis of the expression of these isoforms. We have begun to examine the localization of expressed transgenes in not only wt, but glr-2 knockout animals. Behavioural assays are currently in progress, to determine whether the localization of the GFP-tagged GLR-2 isoforms truly reflects functional activity of these iGluRs in the knockout background. At least nose touch sensitivity can apparently be rescued in the knockout by either isoform. The consequences of mutagenesis of the transgenes and of expression in different mutant backgrounds are also being assessed.

Avery L et al. (2000) West Coast Worm Meeting "A Muscarinic Contribution to the Regulation of Feeding"

Avery L, Steger KA

[West Coast Worm Meeting, 2000]

Acetylcholine (Ach) is essential for worms' survival and can stimulate feeding through nicotinic receptors on the pharyngeal muscle. However, we suspect that Ach affects the pharynx through muscarinic receptors as well. Worms that lack Ach (e.g. cha-1 mutants) have a more severe feeding defect than worms that lack pharyngeal nicotinic transmission (eat-2; eat-18). Atropine, a muscarinic antagonist, causes worms to appear starved, although it increases their pumping rate. Three ORFs in the worm genome closely resemble vertebrate muscarinic receptors: C15B12.5 (acm-1), F47D12.2 (acm-2) and C53A5.12 (acm-3). We have examined the expression of these three genes: all three are expressed in pharyngeal tissue (muscle, neurons or both) and in the extra-pharyngeal nerve ring. We have obtained mutant alleles of two putative muscarinic receptors: acm-1, and acm-2 (a gift from Stefan Eimer and Rolf Baumeister). The two mutations have very different effects on worm physiology. acm-2 worms are hypersensitive to aldicarb (an inhibitor of cholinesterase) and to nicotine. They pump more rapidly than wild type worms in the absence of drug, and decrease their pumping rate in the presence of atropine. We suspect that acm-2 mutants are defective in down-regulating nicotinic transmission. We hypothesize that acm-2, which is expressed in neurons both inside and outside the pharynx, acts as a negative regulator at nicotinic synapses. acm-1 mutants, in contrast, are resistant to aldicarb, and are not hypersensitive to nicotine. In an unc-17 background of reduced cholinergic transmission, acm-1 worms are hypersensitive to atropine. We suspect that acm-1, probably in combination with acm-3, affects pumping efficiency or the adaptation of feeding behavior to particular circumstances. We are currently designing assays and appropriate genetic backgrounds to examine these possibilities