GPB-2 is a g-protein beta subunit that interacts with RGS proteins to regulate signaling through g-protein coupled receptors. GPB-2 acts in both Galphao and Galphaq coupled pathways, influencing behaviors such as egg laying, locomotion and defecation. In addition,
gpb-2 mutants are hypersensitive to the muscarinic agonist arecoline: in the presence of 5mM arecoline,
gpb-2 mutants fail to grow past the L1 stage and their pharynxes pump infrequently. This observation has led to the hypothesis that GPB-2 is necessary to regulate signaling through a muscarinic receptor in the pharynx. However, screens to isolate mutants that suppress the arecoline hypersensitivity of
gpb-2 mutants have not generated muscarinic receptor mutants. We have found that two muscarinic receptors,
gar-1(C15B12.5) and
gar-3(Y40H4A.1), are expressed in the pharyngeal muscle, and might function redundantly in GPB-2-linked pathway. Therefore, we conducted a screen in a
gpb-2;
gar-1 background, in which one muscarinic receptor is disrupted by a deletion. In this screen, we isolated arecoline resistant worms that carry a mutation in
gar-3.
gar-3(
vu78) worms carry a mutation that changes a conserved alanine to a threonine. While over-expression of wild type
gar-3 renders wild type worms hypersensitive to arecoline, over-expression of
gar-3(
vu78) does not, suggesting that the
vu78 mutation reduces the function of GAR-3. We believe this is strong evidence that disruption of two muscarinic receptors,
gar-1 and
gar-3, is necessary to overcome the arecoline hypersensitivity of
gpb-2 mutants. In order to elucidate the function of a muscarinic signaling pathway in the pharynx, we are now comparing
gpb-2 arecoline hypersensitive worms with resistant
gpb-2,
gar-1;
gar-3 worms. We have found that, even in the absence of arecoline, mutations in muscarinic receptors increase the growth rate of
gpb-2 mutants. In the presence of arecoline, triple mutants are able to pump more rapidly than
gpb-2 single mutants. We have also found that the pharynxes of
gpb-2 single mutants tend to be hypercontracted during treatment with arecoline. However, the electrical activity of
gpb-2 mutant pharynxes appears fairly normal, even in the presence of arecoline. We therefore hypothesize that muscarinic signaling in the pharynx alters the relationship between excitation and contraction of the muscle.