<i>GNAI1</i> missense mutations associated with a neurodevelopmental syndrome modify Gα _i1 function

G protein-coupled receptors (GPCRs) play key roles in neurodevelopment by regulating excitatory and inhibitory neuronal pathways. The G protein subunit Gα_i1 is a downstream effector of inhibitory GPCR signaling, and the gene encoding it (<i>GNAI1</i>) is abundantly expressed in the brain. Rare <i>GNAI1</i> variants are linked to a severe neurodevelopmental disorder called <i>GNAI1</i> syndrome, which is characterized by intellectual disability, a range of movement issues, and epilepsy. Here, we investigated the effect of five <i>GNAI1</i> syndrome-associated missense variants on GPCR signaling. Predictions based on genetic biobank data and in silico modeling suggested that these variants were pathogenic. Compared with the wild-type protein, four Gα_i1 variants (T48K, T48I, C224Y, and V332E) showed marked increases in dopamine potency at the dopamine D2 receptor (D2R) and increased constitutive G protein activity when expressed in <i>Xenopus laevis</i> oocytes. By contrast, the Gα_i1 G40C variant was unresponsive to D2R activation. All Gα_i1 variants displayed reduced GTP-γ-S binding rates and undetectable GTP hydrolysis, except for the T48I variant, which showed more rapid binding and hydrolysis. Thus, four <i>GNAI1</i> syndrome variants caused a net gain-of-function effect on D2R signaling, and all studied variants disrupted GTP exchange. These biochemical effects may underlie <i>GNAI1</i> syndrome, and <i>GNAI1</i> mutations should therefore be considered when screening for rare neurodevelopmental disorders.