The catalytic function of the gephyrin‐binding protein IQSEC3 regulates neurotransmitter‐specific matching of pre‐ and post‐synaptic structures in primary hippocampal cultures

Abstract

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 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 Ca concentrations. Taken together, our findings identify a post-synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre-synaptic terminals.

The activity-dependent transcription factor Npas4 regulates IQSEC3 expression in somatostatin interneurons to mediate anxiety-like behavior

“…BIG3 (brefeldin A-inhibited guanine nucleotide exchange protein 3) was reported to specifically regulate GABAergic synaptic transmission (Liu et al, 2016). More recently, overexpression of a dominant-negative, GTP binding-defective ARF6-T27N mutant was shown to recapitulate the phenotype of IQSEC3 E749A in cultured neurons, exhibiting mismatched apposition of pre-and postsynaptic structures (Fruh et al, 2018), consistent with our data (Figure S4). These observations suggest the intriguing possibility that ARF signaling pathways are involved in modulating activity-dependent Figure S3).…”

Glycine Receptor Complex Analysis Using Immunoprecipitation‐Blue Native Gel Electrophoresis‐Mass Spectrometry

“…However, IQSEC3 was not identified in a recent GABA A R γ2‐subunit pull down study 5 . Nonetheless, IQSEC3 is clearly involved in the organization of GABAergic synapses, in particular, the correct alignment of the GABAergic pre‐ and postsynaptic compartments 23 . Curiously, we also identified IQSEC2, a known constituent of excitatory postsynaptic densities, 26 in GlyR α1β complexes.…”

“…Previous studies have also revealed the co‐localization of IQSEC3 with GPHN, GABA A Rs, and GlyRs at inhibitory synapses in mouse retina 22 and biochemical interactions with GPHN 21,23 . Labelling with iBioID using Bira‐GPHN demonstrated the presence of IQSEC3 in the vicinity of GPHN within GABAergic synapses 24 .…”

The small GTPase ARF6 regulates GABAergic synapse development

“…We next sought to determine whether loss of ARF6 induces network dysfunctions, which are often associated with impaired GABAergic synapse formation and function and a resulting imbalance in excitation/inhibition (E/I) ratio at synaptic and circuit levels [30,35]. To test the effect of ARF6 KD on seizure susceptibility, we employed an acute kainic acid (KA)-induced epileptic mouse model, which has been extensively used to dissect molecular mechanisms underlying initial epileptogenesis event(s) that transforms normal neural networks into hypersynchronous networks.…”

“…However, it is possible to propose that normal ARF6 function is crucial for GABAergic synapse development, as evidenced by reported actions of ARF6 GEFs and GAPs at GABAergic synapses. GIT1 regulates GABA A R trafficking and GABAergic synaptic transmission [27], whereas IQSEC3/BRAG3 directly interacts with gephyrin to regulate GABAergic synapse formation [17,[28][29][30].…”