acr-2 encodes a nicotinic receptor subunit. A gain-of-function mutation in
acr-2 results in uncoordinated (Unc) movement and spontaneous shrinking or convulsion (Jospin, 2009). These phenotypes are the result of elevated cholinergic excitation as well as decreased GABAergic inhibition in the locomotor circuit. We have tested the effect of the muscarinic agonist arecoline on
acr-2(gf) convulsion and found that this drug suppressed convulsion and locomotion phenotypes (McCulloch and Jin 2020). Here, we investigated underlying mechanisms and pathways involved in arecoline-induced suppression of
acr-2(gf).
There are two major types of muscarinic receptors. The M1/3/5 types are excitatory and act by elevating intracellular calcium. The M2/4 receptors are inhibitory and act by inhibiting cellular cAMP (Jones et al. 2012). C. elegans has three muscarinic receptors:
gar-1,
gar-2 and
gar-3 (Hwang et al. 1999; Lee et al. 2000). Both
gar-2 and
gar-3 have been shown to respond to arecoline in vivo.
gar-3 is homologous to excitatory M1 muscarinic receptors, and promotes excitatory signaling in pharyngeal and motor circuits (Chanet al. 2013; Hwang et al. 1999; Kozlova et al. 2019; Liu et al. 2007; Steger and Avery 2004).
gar-2 is pharmacologically quite different from most muscarinic receptors, however, functionally, it seems to act similarly to inhibitory M2/4 type receptors in inhibiting cholinergic activity in the motor circuit (Dittman and Kaplan 2008; Lee et al. 2000).
gar-1, unlike
gar-2 and
gar-3, is expressed in the PVM neurons and other neurons in the head and has not been shown to be expressed in the motor circuit (Lee et al. 2000). We therefore focused on
gar-2 and
gar-3. We constructed double and triple mutants of
acr-2(gf) with null mutations in
gar-2 or
gar-3 (or mutations of both, respectively). Without arecoline, these mutants showed convulsion phenotypes indistinguishable from
acr-2(gf) alone. Upon treatment with arecoline, we observed suppression of convulsion, similar to
acr-2(gf) alone (Figure 1A). These genetic data suggested that these known muscarinic receptors in the motor circuit are not involved in suppression of
acr-2(gf) by arecoline.
To further address if suppression of
acr-2(gf) by arecoline involves muscarinic signaling, we tested atropine, which is a highly specific muscarinic antagonist and used to delineate muscarinic from non-muscarinic cholinergic effects (Rang, et al. 2012). It is used intravenously to treat patients with dangerously low heart rate, as inhibitory M2 receptors function in the heart to regulate cardiac activity. We treated
acr-2(gf) animals with 5mM atropine for 2 hours, then transferred the animals to new plates with 5 mM atropine plus 15 mM arecoline for another 3 hrs and score them for convulsion. Atropine only, arecoline only, and untreated
acr-2(gf) mutant animals were also scored as controls. However, atropine treatment did not alter the suppression of
acr-2(gf) convulsion by arecoline (Figure 1B). This further supports a non-muscarinic mechanism for arecoline suppression of
acr-2(gf).
Finally, reverse and forward genetic screens were employed to identify factors that are required for suppression of
acr-2(gf) on arecoline. We tested multiple genes that are required for motor circuit function for their requirement for arecoline suppression. We found that null mutations in genes required for GABA signaling, such as
unc-25/GAD and
unc-49/GABAR, completely blocked arecoline suppression of
acr-2(gf) (Figure 1C) (Bamber et al. 1999; Jin et al. 1999). Moreover, convulsion rates of these double mutants were slightly enhanced on arecoline. Null mutations in GABA genes strongly enhance the Unc phenotype of
acr-2(gf) animals to paralysis, and this phenotype was also not suppressed on arecoline in the double mutants. These observations suggested that GABA signaling may be important for suppression of
acr-2(gf) by arecoline.
In parallel to the reverse genetic screen, we conducted a forward genetic screen to identify factors involved in arecoline suppression of
acr-2(gf) (see Reagents and Methods). We isolated three mutants that showed paralyzed phenotypes resembling GABA mutants, with or without arecoline treatment. Non-complementation analyses confirmed two of these to be
unc-49 mutations. Another mutation,
ju1617, fully blocked arecoline suppression. However,
ju1617;
acr-2(gf) double mutants resembled
acr-2(gf) in the absence of arecoline, in contrast to the
unc-49 null mutations. We isolated
ju1617 on its own, and these animals were slow-moving and exhibited loopy movement, somewhat similar to GABA mutants. After whole-genome sequencing analyses, we found a single nucleotide mutation altering a conserved Arg to His within a putative ligand-binding domain of UNC-49B (Bamber et al. 1999). To confirm the identity of
ju1617, we constructed a
ju1617;
acr-2(gf) strain with an
unc-49 transgene krSi2[Punc-49::
unc-49::rfp] and observed that these animals showed suppression of convulsion after arecoline treatment (Figure 1D). The
unc-49(
ju1617) mutation may represent a reduction-of-function mutation of
unc-49,since it does not shrink in response to touch and fails to enhance
acr-2(gf) Unc behaviors.
In summary, our studies from both forward and reverse genetic screens indicate that GABA signaling is required for suppression of
acr-2(gf) by arecoline. One explanation is that arecoline is activating GABA signaling via an un-identified pathway to restore excitation and inhibition balance to the motor circuit, in the presence of
acr-2(gf). Together, these data suggest that arecoline can act through novel means to inhibit cholinergic activity in specific contexts.