The C. elegans neuromuscular junction (NMJ) contains two pharmacologically distinct nicotinic receptors: a levamisole-sensitive receptor and a levamisole-insensitive receptor. The subunit composition of the levamisole-sensitive receptor has been elucidated, but the levamisole-insensitive acetylcholine (ACh) receptor is still uncharacterized. To identify subunits of the levamisole-insensitive receptor, we examined an Affymetrix microarray data set obtained from FACS-isolated
myo-3::GFP labeled muscle cells (see Fox et al., this meeting). Of seven nicotinic receptor subunit transcripts enriched in muscle, five encode the levamisole receptor subunits (
unc-63,
unc-38,
unc-29,
lev-1 and
lev-8), leaving two candidates for the levamisole-insensitive receptor;
acr-8 and
acr-16. Body muscle expression of both
acr-8 and
acr-16 was confirmed with promoter-GFP fusions.
acr-8(
ok1240) and
acr-16(
ok789) deletion mutants were obtained from the C. elegans Knockout Consortium. Electrophysiological analysis determined that
acr-16(
ok789) mutants exhibit 88% reduction in the ACh response, whereas deletion of
acr-8 had no effect. This electrophysiological defect in
acr-16(
ok789) can be fully rescued by expressing
acr-16 under the control of the
myo-3 promoter in muscles. Evoked responses in
acr-16(
ok789) are also dramatically reduced (86%), suggesting that activation of the ACR-16 receptor contributes the majority of the peak-evoked response. Synaptic localization of ACR-16 was confirmed by expressing the GFP-tagged rescuing construct
pmyo-3:ACR-16::GFP in muscles. Although
acr-16(
ok789) mutants do not exhibit obvious locomotion defects, the double mutants
unc-63(
x37);
acr-16(
ok789) and
unc-29(
x29);
acr-16(
ok789) have a more severe uncoordinated phenotype than either
unc-63 or
unc-29 alone. Consistent with the behavioral phenotype, all cholinergic miniature and evoked synaptic currents are abolished in the
unc-63(
x37);
acr-16(
ok789) mutants. These results suggest that both nicotinic receptor types function at the C. elegans NMJ and contribute to locomotion.