The catalytic function of the gephyrin-binding protein IQSEC3 regulates neurotransmitter-specific matching of pre-...

In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post-synaptic proteins of GABAergic synapses are in part apposed to glutamatergic pre-synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre- and post-synaptic elements. We report here that the IQ motif and Sec7 domain-containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post-synaptic density (PSD) proteins. Over-expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co-expressing eGFP-gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP-gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP-gephyrin, endogenous GABA_A receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over-expression. Conversely, GFP-PSD-95 clusters were unaffected by over-expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant-negative mutant over-expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca²⁺ concentrations. Taken together, our findings identify a post-synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre-synaptic terminals.