Worm Breeder's Gazette 14(3): 49 (June 1, 1996)

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.

avr-15 encodes a glutamate-gated chloride channel subunit

Joe Dent, Leon Avery

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas TX 75235-9038 (joe@eatworms.swmed.edu)

We previously showed that avr-15 mutant worms lack neurotransmission by
the M3 pharyngeal motor neuron. Furthermore, we were able to demonstrate
that whereas depolarized pharyngeal muscles from wild type animals
respond to the iontophoretic application of glutamate by
hyperpolarizing, pharyngeal muscles from avr-15 mutants do not (Avery et
al., WBG 13(4),72; J. Dent, 1995 International Worm Meeting, 69). These
results suggested that avr-15 is necessary postsynaptically, in
pharyngeal muscle, for the expression of a glutamate-gated chloride
channel that mediates M3 neurotransmission. One possibility was that
avr-15 codes for a component of the channel itself. Cully et al. (Nature
371,707-711) cloned two subunits of a glutamate-gated chloride channel,
GluCla and GluClb, from C. elegans. GluCla encodes a subunit that, as a
homomer, forms an ivermectin-gated chloride channel. Given avr-15's role
in ivermectin sensitivity (see Avery et al., WBG 13(4),72) and
glutamatergic neurotransmission, one possibility was that avr-15 coded
for GluCla. To test this hypothesis, we probed a YAC grid with GluCla.
Although the map position of GluCla was not consistent with it being
encoded by avr-15, we were able to use GluCla to identify a closely
related gene whose map position is consistent with that of avr-15. We
isolated a cDNA encoding this related gene and found that the predicted
protein was 85% identical to GluCla at the amino acid level and ~45%
identical to GluClb. These homologies indicate that the new subunit and
GluCla define a subclass of channel subunits and therefore we designated
the new subunit GluCla2 and the original GluCla we call GluCla1.

To demonstrate that GluCla2 is encoded by avr-15, we showed that a
cosmid containing GluCla2 restores M3 neurotransmission when transformed
into avr-15 worms. We also found that the ad1051 allele of avr-15
contains a nonsense mutation early in the open reading frame, which indi
cates that ad1051 is likely to be a null allele. Finally, we were able
to restore M3 neurotransmis sion to avr-15 mutants by transforming them
with an avr-15 cDNA driven by the myo-2 promoter, which should drive
expression specifically in the pharyngeal muscle. Thus, expression of
the receptor postsynaptically in pharyngeal muscle is, as we predicted,
sufficient to restore M3 neu rotransmission. In addition, we transformed
the myo-2::avr-15 construct into an ivermectin resis tant avr-14;avr-15
double mutant strain and found that the transformed worms became
sensitive to ivermectin. Thus, ivermectin's effect on the pharyngeal
muscle, mediated by AVR-15/ GluCla2, is sufficient to kill C. elegans.