01912nas a2200205   4500000000100000008004100001260001500042100002100057700001700078700002400095700003100119700002000150700002000170700001700190245013800207856005300345300001600398490000800414520128400422       2024                            d  c2024-06-181 aEtta Carricaburu1 aOrion Benner1 aScott R. Burlingham1 aCarolina Dos Santos Passos1 aNatalia Hobaugh1 aCharles H. Karr1 aSoham Chanda00aGephyrin promotes autonomous assembly and synaptic localization of GABAergic postsynaptic components without presynaptic GABA release  uhttps://www.pnas.org/doi/10.1073/pnas.2315100121  ae23151001210 v1213 aSynapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin–GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.