The ARHGAP32 isoform PX-RICS is specifically targeted to inhibitory synapses by binding to gephyrin

Precise regulation of excitatory–inhibitory balance is critical for neural circuit function, and its disruption underlies neurodevelopmental disorders such as autism spectrum disorder (ASD) and epilepsy. PX-RICS, a major ARHGAP32 splice variant enriched at inhibitory synapses, has been linked to cognitive dysfunctions; however, the molecular basis of its synaptic targeting and function remains unknown. Here, we identify gephyrin as the primary synaptic anchor for PX-RICS and determine the 2.2 Å crystal structure of their complex. Our structural analysis reveals that the N-terminal gephyrin-binding region (GBR) engages gephyrin E-domain through conserved hydrophobic interactions, explaining the isoform-specific targeting of PX-RICS (but not RICS) to inhibitory synapses. This binding interface overlaps with the neurotransmitter receptor binding site on gephyrin, suggesting a competitive yet dynamic interaction landscape among these inhibitory synaptic proteins. Arhgap32 ΔGBR mice exhibit key features of ARHGAP32 -related disorders, including impaired social novelty recognition and increased seizure susceptibility, indicating that gephyrin-mediated anchoring is critical for PX-RICS to function in inhibitory synapses.