| Summary: | De novo mutations affecting the G protein α o subunit (Gαo)-encoding gene (<i>GNAO1</i>) cause childhood-onset developmental delay, hyperkinetic movement disorders, and epilepsy. Recently, we established <i>Caenorhabditis elegans</i> as an informative experimental model for deciphering pathogenic mechanisms associated with <i>GNAO1</i> defects and identifying new therapies. In this study, we generated two additional gene-edited strains that harbor pathogenic variants which affect residues Glu<sup>246</sup> and Arg<sup>209</sup>—two mutational hotspots in Gαo. In line with previous findings, biallelic changes displayed a variable hypomorphic effect on Gαo-mediated signaling that led to the excessive release of neurotransmitters by different classes of neurons, which, in turn, caused hyperactive egg laying and locomotion. Of note, heterozygous variants showed a cell-specific dominant-negative behavior, which was strictly dependent on the affected residue. As with previously generated mutants (S47G and A221D), caffeine was effective in attenuating the hyperkinetic behavior of R209H and E246K animals, indicating that its efficacy is mutation-independent. Conversely, istradefylline, a selective adenosine A<sub>2A</sub> receptor antagonist, was effective in R209H animals but not in E246K worms, suggesting that caffeine acts through both adenosine receptor-dependent and receptor-independent mechanisms. Overall, our findings provide new insights into disease mechanisms and further support the potential efficacy of caffeine in controlling dyskinesia associated with pathogenic <i>GNAO1</i> mutations.
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